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The role of podocalyxin in adhesion and cell morphogenesis Nielsen, Julie Susanne 2006

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T H E R O L E O F P O D O C A L Y X I N IN A D H E S I O N AND CELL MORPHOGENESIS by JULIE SUSANNE NIELSEN B . S c , The University of V i c t o r i a , 2000  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE F A C U L T Y OF G R A D U A T E  STUDIES  (Medical Genetics)  THE UNIVERSITY  OF BRITISH  COLUMBIA  August 2006 © J u l i e Susanne N i e l s e n , 2 0 0 6  OF  ABSTRACT Podocalyxin is a sialomucin expressed on kidney podocytes, vascular endothelia, and hematopoietic progenitors. Although podocalyxin and its close relative, CD34 have been studied for many years, their precise functions have remained elusive, and roles in blocking differentiation, preventing cell adhesion, and establishing cell polarity have all been proposed. Despite this ambiguity, the perinatal lethality of podocalyxin knockout mice (as a result of kidney defects) and podocalyxin's close association with cancer progression highlight its biological importance. I therefore used several strategies to clarify podocalyxin's functions and mechanisms of action.  Podocalyxin was overexpressed in epithelial cells, and I observed a striking decrease in cell adhesion and an induction of microvillus formation. Microvillus formation was then used as the endpoint to assess the activity of podocalyxin mutants: the extracellular domain was essential while most of the cytoplasmic tail could be deleted without loss of this function. These in vitro studies also demonstrated that podocalyxin recruits the scaffolding protein, N H E R F 1 , which may have important implications in the regulation of NHERF-related processes, such as interaction with ion transporters and signalling molecules.  In order to study podocalyxin in vivo,  generation  of conditional p o d o c a l y x i n  overexpressing mice was attempted. The intention was to generate a single floxed podxl transgenic mouse line that could be crossed with numerous Cre mice in order to induce  ii  p o d o c a l y x i n e x p r e s s i o n in selected tissues. F o r e x a m p l e , p o d o c a l y x i n o v e r e x p r e s s i o n in m a m m a r y tissue was intended to f a c i l i t a t e e v a l u a t i o n o f p o d o c a l y x i n ' s role i n breast c a n c e r p r o g r e s s i o n . S i m i l a r l y , these m i c e c o u l d be used to s e l e c t i v e l y rescue defects in p o d o c a l y x i n - d e f i c i e n t m i c e . U n f o r t u n a t e l y , c h r o m o s o m a l a b n o r m a l i t i e s in the parental e m b r y o n i c stem cells t e m p o r a r i l y prevented c o m p l e t i o n o f this study.  A s an alternative strategy, we attempted to selectively rescue the k i d n e y defects observed i n p o d o c a l y x i n - n u l l m i c e by creation o f m i c e w i t h a k i d n e y - s p e c i f i c podxl  transgene.  S u r p r i s i n g l y , although transgenic p o d o c a l y x i n was appropriately expressed, and podocyte m o r p h o l o g y appeared relatively n o r m a l in contrast to p o d o c a l y x i n - n u l l m i c e , transgenic m i c e s t i l l d i e d p e r i n a t a l l y . T h i s suggests the p r e s e n c e o f o t h e r s e r i o u s , as  yet  u n d e t e r m i n e d , a b n o r m a l i t i e s in p o d o c a l y x i n - d e f i c i e n t a n i m a l s . C o n t i n u e d assessment o f these defects and p o d o c a l y x i n ' s role in cancer progression is u n d e r w a y . In s u m m a r y , this thesis  reveals  a new  mechanistic role for  podocalyxin  in the  process  of  cell  m o r p h o g e n e s i s and suggests that in a d d i t i o n to its v i t a l role in k i d n e y d e v e l o p m e n t , p o d o c a l y x i n m a y play an essential role in other aspects o f m a m m a l i a n development.  iii  TABLE OF CONTENTS ABSTRACT  «  TABLE OF CONTENTS  iv  LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ACKNOWLEDGEMENTS CHAPTER 1 : INTRODUCTION 1.1 1.1.1 1.1.2 1.1.3 1.2  Podocalyxin is a Member of the CD34 Family of Sialomucins Protein Structure Genomic Organization and Alternative Splicing Expression Pattern Cloning of Podocalyxin  viii ix xii xvii 1 1 2 5 9 10  1.3 Transcriptional Regulation of Podocalyxin 1.3.1 Positive Regulation by WT 1 1.3.2 Negative Regulation by p53 1.3.3 Positive Regulation by Ets-1  13 13 15 16  1.4  16 17 18 23  1.4.1 1.4.2 1.4.3  Intracellular Binding Partners for Podocalyxin Ezrin NHERF Proteins Other CD34-Family Binding Proteins  1.5 Proposed Functions of CD34 Family Members 1.5.1 Enhancing Proliferation and Blocking Differentiation 1.5.2 Pro-Adhesion 1.5.3 Anti-Adhesion 1.5.4 Establishing Polarity 1.5.5 Podocalyxin and NHERF: A Role in Asymmetric Cell Division?  24 25 28 32 39 41  1.6 Podocalyxin in Kidney Development : 1.6.1 Overview of Glomerular Development 1.6.2 Glomerular Diseases and Model Systems 1.6.3 Early Studies of Podocalyxin 1.6.4 Lessons from the Podocalyxin Knockout 1.6.4.1 Podocalyxin is Essential for Podocyte Morphogenesis 1.6.4.2 The Kidney Defect in Podocalyxin-Null Mice is the Apparent Cause of Perinatal Lethality in these Animals 1.6.5 Other Molecules Involved in Glomerular Development 1.6.5.1 Nephrin 1.6.5.2 WT1  45 46 50 51 56 56  1.7 Podocalyxin in the Hematopoietic System 1.7.1 Overview of Hematopoiesis 1.7.2 Podocalyxin's Role in the Hematopoietic System 1.7.3 Hematopoiesis in the Podocalyxin Knockout 1.7.4 Podocalyxin in the Vasculature  67 67 68 71 74  61 63 64 65  iv  1.7.5  Podocalyxin Expression in Hemangioblasts  75  1.8  Podocalyxin in the Brain  76  1.9  Additional Phenotypes in Podocalyxin-Null Animals  77  1.10 Podocalyxin in Cancer 1.10.1 Podocalyxin in Breast Cancer 1.10.2 Podocalyxin in Prostate Cancer 1.10.3 Podocalyxin in Testicular Cancer 1.10.4 Podocalyxin in Leukemia 1.10.5 Podocalyxin in Hepatocellular Carcinoma 1.10.6 Podocalyxin in Wilms' Tumours  78 78 81 82 83 84 85  1.11  86  Thesis Objectives  CHAPTER 2 : MATERIALS AND METHODS  88  2.1 Cloning and Mutagenesis of Podocalyxin 2.1.1 Conditional Podocalyxin Overexpression Transgenic Construct 2.1.2 Podocyte-Specific Podocalyxin Transgenic Construct 2.1.3 Murine Podocalyxin Expression Vector 2.1.4 Chicken Podocalyxin Expression Vectors 2.1.5 Chicken Podocalyxin Mutants  •  2.2 Cell Culture 2.2.1 Culture Conditions 2.2.2 Transfection Techniques 2.3 Expression Analysis 2.3.1 Antibodies 2.3.2 Flow Cytometry and Cell Sorting 2.3.3 Western blotting 2.3.4 RT-PCR  88 88 88 89 89 90 93 93 94  •.  95 95 99 100 100  2.4 Additional Characterization of E S C Clones for Conditional Podocalyxin Overexpressing Transgenic 2.4.1 (3-galactosidase Assay 2.4.2 PCR to Detect Transgene :  101 101 101  2.5  Adhesion Assays  102  2.6  Confocal Microscopy  102  2.7  Scanning Electron Microscopy (SEM)  104  2.8 Transmission Electron Microscopy (TEM) 2.8.1 T E M of Cell Lines 2.8.2 T E M of Kidneys  104 104 105  2.9 Immunofluorescence of Tissue Sections 2.9.1 Sample Preparation 2.9.2 Tissue Staining  105 105 106  2.10  Immunohistochemistry  106  2.11  Mice  107  CHAPTER 3 : OVEREXPRESSION OF WILDTYPE AND MUTANT PODOCALYXIN IN EPITHELIAL CELLS 3.1  Rationale  108 108  v  Expression of Full-Length Podocalyxin in M C F - 7 and M D C K Cells  3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.3  Ill  Expression Analysis Ill Podocalyxin Decreases Cell Adhesion in Epithelial Cells 113 Podocalyxin Recruits NHERF1 to the Apical Surface of Cells 117 Morphological Changes: Podocalyxin Induces Microvillus Formation in Epithelial Cells... 119 Generation and Analysis of Podocalyxin Mutants  124  3.3.1 Podocalyxin Mutant Expression Analysis 126 3.3.1.1 Podocalyxin-Positive Cells were Continuously Lost from Bulk Populations 126 3.3.1.2 Podocalyxin Mutants were All Expressed and were of Expected Molecular Weights 129 3.3.2 Mutation of Phosphorylation Sites and Analysis of Podocalyxin's Splice Variant did not Provide any Novel Insights into Podocalyxin's Function 131 3.3.3 Interaction of Podocalyxin with NHERF1 134 3.3.3.1 Podocalyxin's C-terminal D T H L Sequence is Required for Interaction with NHERF1 134 3.3.4 Analysis of Essential Sequences Required for Morphological Changes 138 3.3.4.1 Interaction with NHERF1 is Not Required for Formation of Microvilli 138 3.3.4.2 Podocalyxin's Extracellular Domain is Required for Formation of Microvilli 140 3.3.5 Interaction with NHERF is Unnecessary for Colocalization of Podocalyxin with Ezrin 145 3.3.6 Recruitment of f-actin Occurs in the Absence of N H E R F Binding. 148 3.4 3.4.1 3.4.2 3.4.3 3.4.4  Discussion Summary Podocalyxin's Role in Determining Cell Morphology Significance of Recruitment of N H E R F by Podocalyxin Podocalyxin and Microvilli in Adhesion / Anti-Adhesion  CHAPTER 4 : GENERATION OF TRANSGENIC MICE CONDITIONALLY OVEREXPRESSING PODOCALYXIN  152 152 152 157 159  161  4.1  Rationale  161  4.2  The Cre-loxP System  162  4.3  Transgenic Construct  165  4.4  In Vitro Validation of Transgenic Construct  167  4.5  Selection of Transgene Positive ES Cells  169  4.6  Transgene Expression Analysis After Cre-Mediated Recombination in ESCs  172  4.7  Production of Chimeric Mice  176  4.8  Explanation for Lack of Germline Transmission in Transgenic Mice  176  4.9  Summary and Conclusions  178  CHAPTER 5 : REPAIR OF KIDNEY DEFECT IN PODOCALYXIN-NULL MICE 179 5.1  Rationale  179  5.2  Transgenic Construct  180  5.3  In Vitro Expression Analysis  182  5.4  Generation of Transgenic Founders  184  5.5 Transgene Expression Analysis 5.5.1 Transgene Expression in Glomeruli of Founders' Kidneys  184 185  vi  5.5.2 Other Tissues Examined by Immunofluorescence for GFP Expression Demonstrate Lack of Non-Specific Expression 188 :  5.6  Breeding Scheme and Expected Numbers of Rescued Mice  193  5.7  Transgenic Mice Still Die within 24 Hours of Birth  195  5.8  Potential Reasons for Lack of Rescue  198  5.9  Transgenic Podocalyxin Expression Analysis  198  5.10  Morphological Analysis of Kidneys  203  5.11  Summary and Conclusions  206  CHAPTER 6.1  6 : CONCLUDING  REMARKS  208  Summary and Discussion 208 6.1.1 Podocalyxin's Role in Cell Morphogenesis 209 6.1.2 Podocalyxin's Role in Cell Adhesion 213 6.1.3 Podocalyxin-Dependent NHERF Localization 216 6.1.4 Ectopic Podocyte-Specific Podocalyxin Expression Repairs Foot Process Architecture but Fails to Rescue Podocalyxin-Null Mice 217  6.2 6.2.1 6.2.2  Significance of Results Podocalyxin in Normal Development Podocalyxin in Cancer Progression  219 219 220  6.3.1 6.3.2 6.3.3  Future Directions Understanding Podocalyxin Mechanistically Podocalyxin's Role in Development Podocalyxin's Role in Cancer Progression  221 221 222 223  6.3  CHAPTER  7 : REFERENCES  225  vn  LIST OF T A B L E S T a b l e 1-1: F r e q u e n c y o f podxl , podxP ', and podxl ' A n i m a l s d u r i n g D e v e l o p m e n t . . . . 6 2 +l+  1  1  T a b l e 2 - 1 : P r i m a r y A n t i b o d i e s used i n this T h e s i s  97  T a b l e 2 - 2 : S e c o n d a r y A n t i b o d i e s used i n this T h e s i s  98  T a b l e 5 - 1 : S u m m a r y o f Transgene E x p r e s s i o n  192  Table 5 - 2 : Expected Numbers o f A l l Genotypes Resulting from  PodxT'Transgene* ' 1  Crosses  194  T a b l e 5 - 3 : A c t u a l N u m b e r s o f T r a n s g e n e - P o s i t i v e M i c e o f E a c h G e n o t y p e i n the First F i v e Litters R e s u l t i n g f r o m PodxP''Transgene ' +l  C r o s s e s Demonstrated a L a c k o f  R e s c u e o f Podxl' M i c e 196 T a b l e 5 - 4 : A c t u a l N u m b e r s o f T r a n s g e n e - P o s i t i v e and T r a n s g e n e - N e g a t i v e M i c e i n E i g h t 1  Litters R e s u l t i n g f r o m B r e e d i n g o f PodxT''Transgene '~ M i c e w i t h PodxP ' +  Transgene''  1  M i c e Demonstrated that Transgene-Integration was not D e t r i m e n t a l . 197  viii  LIST OF FIGURES Figure 1-1: S c h e m a t i c o f C D 3 4 F a m i l y M e m b e r s  4  Figure 1-2: G e n o m i c O r g a n i z a t i o n o f C D 3 4 F a m i l y M e m b e r s  7  Figure 1-3: A l t e r n a t i v e S p l i c i n g Patterns for C D 3 4 F a m i l y M e m b e r s  8  F i g u r e 1-4: Proposed F u n c t i o n s for C D 3 4 F a m i l y : E n h a n c i n g P r o l i f e r a t i o n and B l o c k i n g Differentiation Figure 1-5: Proposed F u n c t i o n f o r C D 3 4 F a m i l y : L - s e l e c t i n M e d i a t e d A d h e s i o n  26 30  Figure 1-6: Proposed F u n c t i o n f o r C D 3 4 F a m i l y : B l o c k i n g A d h e s i o n Figure 1-7: T w o M o d e l s o f P o d o c a l y x i n - D e p e n d e n t A s y m m e t r i c C e l l D i v i s i o n  33 44  Figure 1-8: S c h e m a t i c o f M o l e c u l e s R e l a t e d to P o d o c y t e A r c h i t e c t u r e 48 Figure 1-9: E l e c t r o n M i c r o g r a p h of a N o r m a l G l o m e r u l a r P o d o c y t e w i t h E x t e n d e d M a j o r Processes and F o o t Processes S u r r o u n d i n g a B l o o d V e s s e l  58  Figure 1-10: T E M ' s of K i d n e y s f r o m W i l d t y p e and P o d o c a l y x i n K n o c k o u t M i c e Figure 1 - 1 1 : P o d o c a l y x i n is E x p r e s s e d in H e m a t o p o i e t i c T i s s u e s throughout  59  Development  69  Figure 1-12: Steady State L e v e l s o f A l l H e m a t o p o i e t i c L i n e a g e s in E 1 5 Fetal L i v e r i n P o d o c a l y x i n ( P o d o ) - K n o c k o u t ( K O ) , C D 3 4 - K O , and D o u b l e - K O ( D K O ) M i c e are S i m i l a r to those in W i l d t y p e ( W T ) M i c e 73 Figure 1-13: S u r v i v a l Rates f o r Patients D i a g n o s e d w i t h Breast T u m o u r s S h o w n to be Expressing V a r y i n g Levels of Podocalyxin 80 Figure 3 - 1 : Endogenous H u m a n P o d o c a l y x i n E x p r e s s i o n i n Breast C a r c i n o m a L i n e s . . 110 Figure 3 - 2 : E c t o p i c M u r i n e P o d o c a l y x i n E x p r e s s i o n in Transfected M C F - 7 Breast Epithelial Cells Figure 3 - 3 : Transfected M C F - 7 C e l l s E c t o p i c a l l y E x p r e s s i n g M u r i n e P o d o c a l y x i n  112  E x h i b i t e d Decreased C e l l - S u b s t r a t u m Interactions  114  Figure 3 - 4 : P o d o c a l y x i n Decreases C e l l Substrate A d h e s i o n  115  Figure 3 - 5 : P o d o c a l y x i n - I n d u c e d A l t e r a t i o n o f C e l l - C e l l Junctions  116  Figure 3 - 6 : C o n f o c a l Images Demonstrated A p i c a l R e c r u i t m e n t o f N H E R F 1 by Podocalyxin Figure 3 - 7 : P o d o c a l y x i n Induced M i c r o v i l l u s F o r m a t i o n in M C F - 7 C e l l s  118 121  Figure 3 - 8 : P o d o c a l y x i n A l s o Induced M i c r o v i l l u s F o r m a t i o n i n M D C K E p i t h e l i a l C e l l s . 122 Figure 3 - 9 : T E M ' s of E p i t h e l i a l C e l l s Transfected w i t h E m p t y V e c t o r or V e c t o r Encoding Murine Podocalyxin  123  Figure 3 - 1 0 : Schematic of P o d o c a l y x i n M u t a n t s  125  Figure 3 - 1 1 : P o d o c a l y x i n E x p r e s s i o n A f t e r M u l t i p l e R o u n d s o f C e l l Sorting  128  Figure 3 - 1 2 : Western B l o t s Demonstrated E x p r e s s i o n o f P o d o c a l y x i n Mutants o f the Expected Molecular Weights in Clonal Populations  130  Figure 3 - 1 3 : Western B l o t s Demonstrated E x p r e s s i o n o f P o d o c a l y x i n in C l o n a l P o p u l a t i o n s U s e d f o r Subsequent E x p e r i m e n t s  132  Figure 3 - 1 4 : P o d o c a l y x i n was E x p r e s s e d at C o m p a r a b l e L e v e l s at the C e l l Surface in C l o n a l Populations  133  F i g u r e 3 - 1 5 : C o n f o c a l A n a l y s i s o f N H E R F 1 and P o d o c a l y x i n at the A p i c a l Surface of Transfected M C F - 7 C e l l s  135  ix  Figure 3 - 1 6 : A n a l y s i s o f N H E R F 1 and P o d o c a l y x i n in V e r t i c a l S l i c e s o f C o n f o c a l Stacks  137  F i g u r e 3 - 1 7 : P o d o c a l y x i n Induced M i c r o v i l l u s F o r m a t i o n i n a N H E R F - I n d e p e n d e n t Manner  139  Figure 3 - 1 8 : D e l e t i o n of the M a j o r i t y of P o d o c a l y x i n ' s E x t r a c e l l u l a r D o m a i n A b o l i s h e d M i c r o v i l l u s Formation  141  F i g u r e 3 - 1 9 : M i c r o v i l l u s C o u n t s f o r Transfected C e l l s  143  F i g u r e 3 - 2 0 : C o n f o c a l A n a l y s i s o f P o d o c a l y x i n and E z r i n at the A p i c a l Surface o f Transfected M C F - 7 C e l l s  146  F i g u r e 3 - 2 1 : V e r t i c a l Sections o f C o n f o c a l Stacks Demonstrated Increased A p i c a l R e c r u i t m e n t o f E z r i n in C e l l s Transfected w i t h P o d o c a l y x i n  147  Figure 3 - 2 2 : C o n f o c a l Images o f the A p i c a l Surface of C e l l s S h o w that f A c t i n was :  R e c r u i t e d in P o d o c a l y x i n - T r a n s f e c t e d C e l l s  149  Figure 3 - 2 3 : V e r t i c a l Sections o f C o n f o c a l Stacks Demonstrated A p i c a l R e c r u i t m e n t o f fA c t i n in P o d o c a l y x i n - T r a n s f e c t e d C e l l s  150  F i g u r e 3 - 2 4 : A c t i n F i l a m e n t s were V i s i b l e in Individual M i c r o v i l l i  151  Figure 3 - 2 5 : T w o M o d e l s of P o d o c a l y x i n - I n d u c e d M i c r o v i l l u s F o r m a t i o n  155  F i g u r e 3 - 2 6 : M o d e l o f P o d o c a l y x i n ' s R o l e in the F u n c t i o n o f N H E R F F a m i l y M e m b e r s . 158 Figure 4 - 1 : S c h e m a t i c o f C r e - M e d i a t e d R e c o m b i n a t i o n  164  Figure 4 - 2 : S c h e m a t i c o f T r a n s g e n i c Construct Before and A f t e r C r e - M e d i a t e d Recombination  166  Figure 4 - 3 : C r e - M e d i a t e d R e c o m b i n a t i o n Induced P o d o c a l y x i n E x p r e s s i o n in N S O C e l l s Transfected w i t h the Construct  168  F i g u r e 4 - 4 : O v e r v i e w o f Selection Process  170  Figure 4 - 5 : P C R - B a s e d Detection of Transgene in G e n o m i c D N A o f E S C s  171  F i g u r e 4 - 6 : E x p r e s s i o n of P o d o c a l y x i n m R N A in E S C s  173  F i g u r e 4 - 7 : A n a l y s i s o f P o d o c a l y x i n E x p r e s s i o n by F l o w C y t o m e t r y  174  F i g u r e 4 - 8 : E x p r e s s i o n of G F P m R N A in transfected E S C s  175  F i g u r e 4 - 9 : K a r y o t y p i n g U n c o v e r e d the Presence o f T r i s o m y E i g h t in Tested C l o n e s . . 177 F i g u r e 5 - 1 : S c h e m a t i c of T r a n s g e n i c Construct  181  F i g u r e 5 - 2 : T r a n s i e n t l y Transfected C H O C e l l s Expressed P o d o c a l y x i n and G F P f r o m the T r a n s g e n i c Construct  183  F i g u r e 5 - 3 : G F P E x p r e s s i o n in G l o m e r u l i o f Transgenic Founders  186  F i g u r e 5 - 4 : G l o m e r u l u s f r o m F o u n d e r w i t h Highest G F P E x p r e s s i o n L e v e l s  187  F i g u r e 5 - 5 : L a c k of N o n - S p e c i f i c E x p r e s s i o n in L i v e r and L u n g  189  F i g u r e 5 - 6 : Transgene E x p r e s s i o n was Undetectable in Peripheral B l o o d  190  F i g u r e 5 - 7 : G F P E x p r e s s i o n was N o t Detected in B r a i n s o f F o u n d e r s  191  F i g u r e 5 - 8 : B r e e d i n g S c h e m e used to Generate M i c e E x p r e s s i n g P o d o c a l y x i n o n l y i n Kidney  194  F i g u r e 5 - 9 : P o d o c a l y x i n m R N A was Expressed f r o m the Transgene d u r i n g D e v e l o p m e n t in K i d n e y s of T r a n s g e n i c M i c e  200  F i g u r e 5 - 1 0 : I m m u n o h i s t o c h e m i s t r y was used to Detect P o d o c a l y x i n E x p r e s s i o n in K i d n e y Glomeruli of Transgenic M i c e during Development  201  F i g u r e 5 - 1 1 : A p i c a l S t a i n i n g o f Podocytes was E v i d e n t i n H i g h M a g n i f i c a t i o n Images o f G l o m e r u l i f r o m Podxl' ' T r a n s g e n e - P o s i t i v e M i c e 1  202  x  Figure 5 - 1 2 : T E M ' s o f P o d o c y t e s f r o m E l 8 W i l d t y p e , P o d o c a l y x i n - N u l l , and Podocalyxin-Null/Transgene-Positive Double Transgenic M i c e  204  Figure 5 - 1 3 : H i g h M a g n i f i c a t i o n T E M ' s o f P o d o c y t e s f r o m E 1 8 W i l d t y p e , P o d o c a l y x i n N u l l , and P o d o c a l y x i n - N u l l / T r a n s g e n e - P o s i t i v e D o u b l e T r a n s g e n i c M i c e  205  F i g u r e 6 - 1 : M o d e l D e m o n s t r a t i n g a P o s s i b l e M e c h a n i s m f o r Decreased C e l l - S u b s t r a t e A d h e s i o n Induced by A p i c a l P o d o c a l y x i n E x p r e s s i o n  215  xi  LIST OF ABBREVIATIONS aa  amino acid  AGM  aorta-gonad-mesonephros  ALL  acute l y m p h o b l a s t i c l e u k e m i a  AML  acute m y e l o i d l e u k e m i a  APC  allophycocyanin  bp  base pair  BM  bone m a r r o w  BSA  b o v i n e serum a l b u m i n  B6  C 5 7 B L / 6 w i l d t y p e mouse strain  P-gal  P-galactosidase  p>AR  (^-adrenergic receptor  CFTR  cystic f i b r o s i s transmembrane regulator  CFU  c o l o n y f o r m i n g unit  ch  chicken  ChIP  chromatin immunoprecipitation  CHO  C h i n e s e hamster o v a r y  CKII  casein kinase II  DAB  diaminobenzidine  DAPI  4', 6'-diamidino-2-phenylindole  DDS  D e n y s - D r a s h syndrome  DKO  p o d o c a l y x i n / C D 3 4 double k n o c k o u t  ADTHL  p o d o c a l y x i n mutant l a c k i n g C - t e r m i n a l D T H L  A EC  p o d o c a l y x i n mutant l a c k i n g most o f the extracellular d o m a i n  Atail  p o d o c a l y x i n mutant l a c k i n g c y t o p l a s m i c tail  E  e m b r y o n i c day  EBP50  ezrin b i n d i n g phosphoprotein o f 5 0 k D a  ECL  enhanced c h e m i l u m i n e s c e n c e  EDTA  ethylenediaminetetraacetic a c i d  EGTA  ethylene-bis(oxyethylenenitrilo)tetraacetic  EGFP  enhanced green fluorescent protein  ELAM  endothelial l e u k o c y t e adhesion m o l e c u l e  ER  estrogen receptor  ERM  ezrin-radixin-moesin  ESC  e m b r y o n i c stem cell  E3KARP  N H E 3 kinase A regulatory protein  FACS  fluorescence activated cell sorting  FBS  fetal bovine serum  FC  f l o w cytometry  FTL/FL  fetal liver  GBM  g l o m e r u l a r basement m e m b r a n e  GFP  green fluorescent protein  GLEPP  g l o m e r u l a r epithelial protein  GlyCAM  g l y c o s y l a t i o n - d e p e n d e n t cell adhesion m o l e c u l e  acid  j'  GRK  G p r o t e i n - c o u p l e d receptor kinase  HCC  hepatocellular c a r c i n o m a  HEV  high endothelial venule  HGEC  human g l o m e r u l a r epithelial cells  HRP  horse radish peroxidase  HSC  hematopoietic stem cell  HUVEC  human u m b i l i c a l v e i n endothelial cell  ICAM  intracellular adhesion m o l e c u l e  IF  immunofluorescence  Ig  immunoglobulin  IHC  immunohistochemistry  IL-6  interleukin 6  ip  immunoprecipitation  IRES  internal r i b o s o m e entry site  kDa  kiloDalton  KO  knockout  LIF  l e u k e m i a inhibitory factor  LSK  Lin S c a - l c - K i t  LTR  l o n g term repopulating  MAdCAM  m u c o s a l addressin c e l l adhesion m o l e c u l e  MDCK  M a d i n - D a r b y canine kidney  MEP  M y b - E t s transformed progenitor  ms  mouse  nd  not determined  NHE  Na7H  +  +  +  exchanger  xiv  NHERF  N a 7 H exchanger regulatory factor  NSGCT  nonseminomatous germ cell tumour  pAb  polyclonal antibody  PAGE  polyacrylamide gel electrophoresis  PAN  puromycin aminonucleoside  PB  peripheral blood  PBS  phosphate buffered saline  PC  post coitum  PCR  polymerase chain reaction  PDGFR  platelet-derived growth factor receptor  PDZ  PSD-95/Dlg/ZO-l  PE  phycoerythrin  PFA  paraformaldehyde  PKC  protein kinase C  PMSF  phenylmethanesulfonyl fluoride  Podo  podocalyxin  PolyA/pA  polyadenylation  PP  post partum  PS  protamine sulfate  P2Y1R  purinergic receptor  rb  rabbit  ,  +  RNAi  R N A interference  RT-PCR  reverse transcriptase-polymerase chain reaction  SA  streptavidin  SCF  stem c e l l factor  SDS  s o d i u m d o d e c y l sulfate  SEM  scanning electron m i c r o s c o p y  siRNA  s m a l l interfering R N A  SPL  spleen  TBS  T r i s buffered saline  TBS-T  T r i s buffered saline plus 0.05 % T w e e n 2 0  TEM  transmission electron m i c r o s c o p y  TER  transepithelial resistance  UTR  untranslated region  WT  wildtype  WT1  W i l m s ' tumour 1  YS  y o l k sac  xvi  ACKNOWLEDGEMENTS It has been quite a j o u r n e y , and I have had the o p p o r t u n i t y to w o r k w i t h m a n y great people o v e r the past f e w years. I a m very grateful to D r . K e l l y M c N a g n y for g i v i n g me the chance to carry out m y studies in his lab and for a l w a y s b e l i e v i n g in m e ; his support has really made all this p o s s i b l e . I want to thank everyone in the M c N a g n y lab, past and present, for the advice and e s p e c i a l l y f o r the laughs. I particularly thank Shierley C h e l l i a h for countless hours of help, a l w a y s w i t h a s m i l e . I also thank H e l e n M e r k e n s , D r . ' s R e g i s D o y o n n a s and Sebastian Furness, J a m i e H a d d o n , Steve M a l t b y , and P o h T a n , w h o have all contributed i n m a n y w a y s and also made the M c N a g n y lab an enjoyable place to be.  The  B i o m e d i c a l Research Centre  is a f a b u l o u s  environment  for  research  and  c o l l a b o r a t i o n , and I a m l u c k y to have been a part of this group. I w o u l d particularly l i k e to thank D r . W i l f Jefferies and M a t t F i n l a y f o r generating m y k i d n e y - s p e c i f i c transgenic m i c e . In a d d i t i o n , I w o u l d l i k e to thank D r . ' s H e r m a n n Z i l t e n e r , F a b i o R o s s i , J o h n S c h r a d e r , M u r i e l D a v i d , D o u g C a r l o w , Pete S c h u b e r t , M a y a K o t t u r i , J a s o n G r a n t , Stephane C o r b e l , B r o c k G r i l l , and G a r y M c L e a n , as w e l l as P h i l O w e n , W o o s e o k S e o , B e r n h a r d L e h n e r t z , J e f f D u e n a s , and M i k e W i l l i a m s for a d v i c e and reagents. I a m also grateful to A n d y J o h n s o n f o r hours o f sorting c e l l s , and to the a m a z i n g core staff at the B R C , particularly N i c o l e V o g l m a i e r , T a k a M u r a k a m i , L e a W o n g , and Samantha H o i u m .  I a m a l s o grateful to m a n y p e o p l e outside o f the B R C . D r . ' s C a r o l y n B r o w n , P a u l i n e J o h n s o n , and R o b K a y o f m y supervisory c o m m i t t e e have a l w a y s p r o v i d e d me w i t h good  xvii  a d v i c e and kept me on track, and I really appreciate their c r i t i c a l r e v i e w s o f m y thesis. O n e o f the most p r o d u c t i v e and e n j o y a b l e aspects o f m y thesis research has been m y c o l l a b o r a t i o n w i t h D r . C a i R o s k e l l e y , D r . W a y n e V o g l , M a r c i a M c C o y , and Jane C i p p o l o n e , and 1 a m truly grateful f o r a l l their c o n t r i b u t i o n s to the m i c r o v i l l i project. I also appreciate the expert electron m i c r o s c o p y assistance f r o m D e r r i c k H o m e , as w e l l as a d v i c e f r o m G a r n e t M a r t e n s , at the U B C B i o l m a g i n g F a c i l i t y , the use o f D r . R o b e r t N a b i ' s c o n f o c a l m i c r o s c o p e , and contributions f r o m the Department o f M e d i c a l G e n e t i c s , especially those o f C h e r y l B i s h o p . I a m also grateful to D r . C o r r i n n e L o b e and R a y m o n d W o n g at S u n n y b r o o k H e a l t h S c i e n c e s C e n t r e and D r . J i l l L a h t i at St. Jude C h i l d r e n ' s R e s e a r c h H o s p i t a l f o r assistance w i t h the i n d u c i b l e p o d o c a l y x i n expression project, and to D r . ' s D a v i d K e r s h a w and A t s u s h i M i y a j i m a f o r i n v a l u a b l e reagents. I a m also t h a n k f u l f o r f u n d i n g f r o m N S E R C and U B C and travel f u n d i n g f r o m the S t e m C e l l N e t w o r k .  N o n e o f this w o u l d have been p o s s i b l e w i t h o u t the tremendous support f r o m m y f a m i l y and f r i e n d s . A t U B C , I e s p e c i a l l y want to thank M a r c i a M c C o y , J a s m e e n M e r z a b a n , N i c o l e V o g l m a i e r , Julie W o n g , and Jane C i p p o l o n e f o r their constant encouragement; the m a n y frustrations o f research are so m u c h easier to deal w i t h w h e n y o u have good friends w h o are in s i m i l a r situations. In a d d i t i o n , m y parents have a l w a y s been an i n c r e d i b l e source of encouragement for m e , and I cannot thank them enough. M y grandparents, w h o c o n t i n u e to l i v e l i f e to the f u l l e s t e v e r y d a y , have a l s o been a c o n s t a n t s o u r c e o f i n s p i r a t i o n f o r m e . L a s t l y , there are no w o r d s to express h o w m u c h I appreciate the u n w a v e r i n g support of m y h u s b a n d , C h r i s t i a n N i e l s e n . I a m e x c e p t i o n a l l y grateful f o r his encouragement, every single day throughout m y doctoral studies.  xviii  CHAPTER 1 : INTRODUCTION 1.1 Podocalyxin is a Member of the CD34 Family of  Sialomucins  P o d o c a l y x i n is a c e l l surface protein essential f o r k i d n e y d e v e l o p m e n t ( D o y o n n a s et a l . , 2 0 0 1 ) and i m p l i c a t e d in a w i d e range of cancers ( C a s e y et a l . , 2 0 0 6 ; C h e n et a l . , 2 0 0 4 ; K e l l e y et a l . , 2 0 0 5 ; S c h o p p e r l e et a l . , 2 0 0 3 ; S o m a s i r i et a l . , 2 0 0 4 ; S t a n h o p e - B a k e r et a l . , 2 0 0 4 ) . It was first i d e n t i f i e d by Dr. M a r i l y n F a r q u h a r ' s group and termed " p o d o c a l y x i n " as it is the m a j o r c o m p o n e n t o f the g l y c o c a l y x  of kidney glomerular  podocytes  ( K e r j a s c h k i et a l . , 1984). It has also been c a l l e d p o d o c a l y x i n - l i k e protein 1 ( P C L P - 1 ) , t h r o m b o m u c i n , M y b - E t s transformed progenitor ( M E P ) - 2 1 , and gp 135, and it is encoded by the podxl  gene ( K e r s h a w et a l . , 1995; M c N a g n y et a l . , 1 9 9 2 ; M c N a g n y et a l . , 1997;  M e d e r et a l . , 2005)*.  •  S o m e figures i n this chapter have been p u b l i s h e d i n the f o l l o w i n g articles:  1) D o y o n n a s , R., N i e l s e n , J . S . , C h e l l i a h , S . , D r e w , E., H a r a , T . , M i y a j i m a , A . and M c N a g n y , K . M . (2005) P o d o c a l y x i n is a C D 3 4 - r e l a t e d marker o f m u r i n e h e m a t o p o i e t i c stem c e l l s and e m b r y o n i c erythroid cells. Blood, 105, 4 1 7 0 - 4 1 7 8 . 2) N i e l s e n , J . S . , D o y o n n a s , R. and M c N a g n y , K . M . (2002) A v i a n m o d e l s to study the t r a n s c r i p t i o n a l c o n t r o l o f h e m a t o p o i e t i c lineage c o m m i t m e n t a n d to i d e n t i f y l i n e a g e s p e c i f i c genes. Cells Tissues Organs,  171, 4 4 - 6 3 .  3) S o m a s i r i , A . , N i e l s e n , J . S . , M a k r e t s o v , N . , M c C o y , M . L . , P r e n t i c e , L., G i l k s , C . B . , Chia, S.K., Gelmon, K . A . , Kershaw, D.B., Huntsman, D.G.,  M c N a g n y , K . M . and  R o s k e l l e y , C D . (2004) O v e r e x p r e s s i o n of the anti-adhesin p o d o c a l y x i n is an independent predictor o f breast cancer progression. Cancer Res, 64,  5068-5073.  1  1.1.1  Protein Structure  P o d o c a l y x i n is a type I transmembrane protein w i t h a predicted mass, based on its protein b a c k b o n e , of a p p r o x i m a t e l y 5 5 k D a ( K e r s h a w et a l . , 1997a; M c N a g n y et a l . , 1997). Its s e r i n e - t h r e o n i n e - p r o l i n e r i c h e x t r a c e l l u l a r d o m a i n is e x t e n s i v e l y  O-glycosylated  and  s i a l y l a t e d , r e s u l t i n g i n an a c t u a l mass o f 1 4 0 - 1 7 0 k D a and c h a r a c t e r i z i n g it as a s i a l o m u c i n ( D o y o n n a s et a l . , 2 0 0 1 ; H i l k e n s et a l . , 1 9 9 2 ; K e r j a s c h k i et a l . , 1984; K e r s h a w et a l . , 1997a; M c N a g n y et a l . , 1 9 9 7 ; M i e t t i n e n et a l . , 1 9 9 9 ; O r l a n d o et a l . , 2 0 0 1 ; Sassetti et a l . , 1 9 9 8 ; T a k e d a et a l . , 2 0 0 0 ) . T h e extracellular d o m a i n also contains several potential sites o f N - l i n k e d g l y c o s y l a t i o n , a g l o b u l a r d o m a i n c o n s i s t i n g o f f o u r cysteine residues, and a j u x t a m e m b r a n e stalk region ( D o y o n n a s et a l . , 2 0 0 1 ; K e r s h a w et a l . , 1997a; O r l a n d o et a l . , 2 0 0 1 ) . A  2 6 a m i n o a c i d (aa)  hydrophobic  t r a n s m e m b r a n e d o m a i n , w h i c h is f o l l o w e d  region  encodes a single  by a w e l l - c o n s e r v e d  pass  c y t o p l a s m i c tail  ( K e r s h a w et a l . , 1997a; M c N a g n y et a l . , 1997; Sassetti et a l . , 1998; T a k e d a et a l . , 2000). T h i s i n t r a c e l l u l a r d o m a i n contains putative p h o s p h o r y l a t i o n sites f o r protein kinase C ( P K C ) and casein kinase II ( C K I I ) as w e l l as the C - t e r m i n a l P D Z - b i n d i n g m o t i f , D T H L ( D o y o n n a s et a l . , 2 0 0 1 ; K e r s h a w et a l . , 1 9 9 7 a ; M c N a g n y et a l . , 1997). L i k e other s i a l o m u c i n s , the extracellular d o m a i n o f p o d o c a l y x i n demonstrates a very l o w degree of sequence c o n s e r v a t i o n ( < 3 3 % ) ( K e r s h a w et a l . , 1 9 9 7 a ; O r l a n d o et a l . , 2 0 0 1 ; T a k e d a et a l . , 2 0 0 0 ) . In contrast, the 7 5 aa c y t o p l a s m i c tail e x h i b i t s a h i g h l e v e l o f aa sequence identity (~95 % between rat, rabbit, and h u m a n , a n d s l i g h t l y less in c o m p a r i s o n to c h i c k e n ) , indicative of an important, conserved f u n c t i o n ( K e r s h a w et a l . , 1997a; L i et a l . , 2 0 0 2 ; M c N a g n y et a l . , 1 9 9 7 ; M i e t t i n e n et a l . , 1999).  2  A m i n o a c i d sequence, protein structure, g e n o m i c o r g a n i z a t i o n , and patterns of alternative s p l i c i n g (described b e l o w ) suggest that p o d o c a l y x i n is most c l o s e l y related to C D 3 4 and e n d o g l y c a n ( M c N a g n y et a l . , 1 9 9 7 ; N i e l s e n et a l . , 2 0 0 2 ; Sassetti et a l . , 2 0 0 0 ) . E a c h o f these p r o t e i n s c o n t a i n s a h i g h l y g l y c o s y l a t e d and s i a l y l a t e d e x t r a c e l l u l a r d o m a i n , a g l o b u l a r d o m a i n , and a stalk f o l l o w e d by a s i n g l e pass m e m b r a n e s p a n n i n g d o m a i n ( F i g u r e 1-1) ( B r o w n et a l . , 1 9 9 1 ; H e et a l . , 1 9 9 2 ; K e r s h a w et a l . , 1997a; K r a u s e et a l . , 1996; K r a u s e et a l . , 1 9 9 4 ; M c N a g n y et a l . , 1 9 9 7 ; N a k a m u r a et a l . , 1 9 9 3 ; N i e l s e n et a l . , 2 0 0 2 ; Sassetti et a l . , 2 0 0 0 ; S i m m o n s et a l . , 1992; Suda et a l . , 1992). T h e c y t o p l a s m i c tails contain the highest degree of s i m i l a r i t y across species and between f a m i l y m e m b e r s , w i t h s e q u e n c e s s u g g e s t i v e o f roles in c e l l s i g n a l i n g or c e l l u l a r l o c a l i z a t i o n : p o d o c a l y x i n , e n d o g l y c a n , and C D 3 4 a l l c o n t a i n c o n s e n s u s p h o s p h o r y l a t i o n sites, as w e l l as a C t e r m i n a l P D Z - b i n d i n g m o t i f ( B r o w n et a l . , 1 9 9 1 ; K r a u s e et a l . , 1 9 9 6 ; M c N a g n y et a l . , 1 9 9 7 ; N i e l s e n et a l . , 2 0 0 2 ; Sassetti et a l . , 1 9 9 8 ; Sassetti et a l . , 2 0 0 0 ; S i m m o n s et a l . , 1992).  3  DTEL  mCD34  mPodocalyxin (mMEP21)  mEndoglycan  Figure 1-1: Schematic of C D 3 4 F a m i l y Members.  B l u e : m u c i n d o m a i n s , b l a c k c i r c l e s : potential N - l i n k e d c a r b o h y d r a t e s , h o r i z o n t a l l i n e s : potential O - l i n k e d carbohydrates, triangles: potential s i a l i c a c i d residues, green: g l o b u l a r m o t i f s , y e l l o w : s t a l k , o r a n g e : t r a n s m e m b r a n e d o m a i n s , r e d : c y t o p l a s m i c t a i l s , large c i r c l e s : potential p h o s p h o r y l a t i o n sites. R e p r o d u c e d w i t h k i n d p e r m i s s i o n o f S . K a r g e r A G , B a s e l ( N i e l s e n et a l . , 2 0 0 2 ) .  T h e r e are, h o w e v e r , several notable differences between these three s i a l o m u c i n s . F i r s t l y , the m u c i n d o m a i n s vary i n length, w i t h C D 3 4 b e i n g the shortest protein o v e r a l l ( K r a u s e et a l . , 1 9 9 4 ; Sassetti et a l . , 1 9 9 8 ; Sassetti et a l . , 2 0 0 0 ) . S e c o n d l y , i n the g l o b u l a r region podocalyxin  and C D 3 4 have four and s i x cysteine residues, respectively,  while  e n d o g l y c a n has t w o , w i t h an additional unpaired j u x t a m e m b r a n e cysteine, l i k e l y i n v o l v e d in d i m e r i z a t i o n ( B r o w n et a l . , 1 9 9 1 ; Fieger et a l . , 2 0 0 3 ; K e r s h a w et a l . , 1997a; Sassetti et a l . , 2 0 0 0 ) . T h i r d l y , w h i l e p o d o c a l y x i n and e n d o g l y c a n share the C - t e r m i n a l D T H L m o t i f , C D 3 4 ' s C - t e r m i n a l sequence is slightly altered: it is D T E L ( H e et a l . , 1 9 9 2 ; K e r s h a w et a l . , 1997a; M c N a g n y et a l . , 1 9 9 7 ; Sassetti et a l . , 2 0 0 0 ; S i m m o n s et a l . , 1992). F i n a l l y , e n d o g l y c a n has a unique N - t e r m i n a l region r i c h i n g l u t a m i c a c i d r e s i d u e s ; w h i l e this m o t i f is f o u n d in many intracellular regulators o f transcription, it is rarely a characteristic o f e x t r a c e l l u l a r d o m a i n s (Sassetti et a l . , 2 0 0 0 ) . T h u s , a l t h o u g h this f a m i l y is c l o s e l y related, the three proteins d o have some u n i q u e , and potentially important, features.  1.1.2 Genomic Organization and Alternative Splicing The  genomic organizations  o f podxl,  cd34,  a n d endoglycan  s t r o n g l y suggest an  evolutionary relationship ( F i g u r e 1-2) ( L i et a l . , 2 0 0 1 ; N i e l s e n et a l . , 2 0 0 2 ) . E a c h protein is e n c o d e d by eight e x o n s , a n d , across the f a m i l y , i n d i v i d u a l exons encode e q u i v a l e n t p r o t e i n m o t i f s a n d are v e r y s i m i l a r i n s i z e ( L i et a l . , 2 0 0 1 ; N i e l s e n et a l . , 2 0 0 2 ; Satterthwaite et a l . , 1992). T h r o u g h o u t the f a m i l y , i n t r o n i c distances are also s t r i k i n g l y s i m i l a r , and s p l i c i n g to an a d d i t i o n a l e x o n between e x o n s seven and eight generates a l o n g e r transcript e n c o d i n g a protein l a c k i n g m u c h o f the c y t o p l a s m i c tail ( F i g u r e 1-3) ( K e r s h a w et a l . , 1 9 9 7 a ; L i et a l . , 2 0 0 1 ; M c N a g n y et a l . , 1 9 9 7 ; N a k a m u r a et a l . , 1 9 9 3 ;  5  N i e l s e n et a l . , 2 0 0 2 ; Sassetti et a l . , 2 0 0 0 ; S u d a et a l . , 1992). T h u s , these three s i a l o m u c i n s have been grouped into a single f a m i l y based on protein structure, g e n o m i c o r g a n i z a t i o n , and patterns of alternative s p l i c i n g .  6  0.5  11.0 1  cd34 podxl |  •  m  • M  3 1.2  1  I 7.5  I  I  I  1  7.9  ! 0.3  0.3  13 4  /\ 5:  A  0.3:  2.2  0.3  1.1 :  7.0  0.5  i  44.0  10.0  endgl  2  |  : 0.8  6 A :7: A . 8 • 0.2  1.3  Vi  1  2.2  0.7  J b 0=1  1  5'UTR &  Mucin  C-C  Stalk  Signal peptide  domain  domain  domain  TM:  Cyt.  3'UTR  tail  Figure 1-2: Genomic Organization of CD34 Family Members. Dark purple: signal peptide, blue: mucin domain, green: globular motif, yellow: stalk, orange: transmembrane domain, red: cytoplasmic tail. Numbers refer to intron sizes in kilobase pairs. Reproduced with kind permission of S. Karger A G , Basel (Nielsen et al.,  2002).  7  stop  Long forms \  Truncated forms  stop  | |TM| [  |^  7  8b  1  '  Figure 1-3: Alternative Splicing Patterns for CD34 Family Members. Splicing produces a longer transcript encoding a shorter cytoplasmic tail. Reproduced with kind permission of S. Karger A G , Basel (Nielsen et al., 2002).  8  1.1.3  Expression Pattern  T h e high l e v e l of p o d o c a l y x i n expression on the surface of renal g l o m e r u l a r e p i t h e l i a l c e l l s (podocytes) enabled its initial isolation and characterization f r o m these c e l l s in 1984 ( K e r j a s c h k i et a l . , 1984). P o d o c a l y x i n is also expressed on the l u m i n a l face o f v a s c u l a r endothelia in a w i d e variety o f vessels, by hematopoietic stem cells and progenitors, and even on hemangioblasts, w h i c h are the precursors o f hematopoietic and endothelial c e l l s ( D e l i a et a l . , 1993; D o y o n n a s et a l . , 2 0 0 5 ; H a r a et a l . , 1999; H o r v a t et a l . , 1 9 8 6 ; K e r s h a w et a l . , 1997a; M c N a g n y et a l . , 1 9 9 7 ; M i e t t i n e n et a l . , 1990; Sassetti et a l . , 1998). In more mature h e m a t o p o i e t i c c e l l s , it is o n l y expressed on platelets, as w e l l as their precursors, megakaryocytes ( M c N a g n y  et a l . , 1 9 9 7 ; M i e t t i n e n et a l . , 1999). O u t s i d e o f  the  h e m a t o p o i e t i c s y s t e m , p o d o c a l y x i n is expressed by mesothelial c e l l s l i n i n g m a n y organs ( D o y o n n a s et a l . , 2 0 0 1 ; M c N a g n y et a l . , 1997), and d u r i n g d e v e l o p m e n t , it is expressed in a l l three g e r m l a y e r s , as w e l l as e m b r y o n i c stem c e l l s and a subset o f n e u r o n s ( D o y o n n a s et a l . , 2 0 0 5 ; V i t u r e i r a et a l . , 2 0 0 5 ) .  Like  p o d o c a l y x i n , C D 3 4 is e x p r e s s e d  on v a s c u l a r e n d o t h e l i a l c e l l s as w e l l  as  hematopoietic progenitors and stem cells ( A n d r e w s et a l . , 1989; B a u m h u e t e r et a l . , 1994; B e r e n s o n et a l . , 1 9 8 8 ; E m a et a l . , 1 9 9 0 ; F i n a et a l . , 1 9 9 0 ; Sato et a l . , 1 9 9 9 ; Y o u n g et a l . , 1995). T h e r e has, h o w e v e r , been considerable controversy about expression of C D 3 4 o n l o n g t e r m r e p o p u l a t i n g h e m a t o p o i e t i c stem c e l l s ( L T R - H S C s ) . It is n o w b e l i e v e d that C D 3 4 e x p r e s s i o n fluctuates d e p e n d i n g on d e v e l o p m e n t a l stage and c e l l a c t i v a t i o n : i n older adult m i c e C D 3 4 is expressed o n l y on activated H S C s , whereas expression is more w i d e s p r e a d d u r i n g d e v e l o p m e n t ( D a o et a l . , 2 0 0 3 ; Ito et a l . , 2 0 0 0 ; N a k a m u r a et a l . , 1 9 9 9 ;  9  O g a w a , 2 0 0 2 ; O s a w a et a l . , 1 9 9 6 ; Sato et a l . , 1 9 9 9 ; T a j i m a et a l . , 2 0 0 0 ; Z a n j a n i et a l . , 2 0 0 3 ) . T h u s , it m a y be that the most p r i m i t i v e and quiescent H S C s are C D 3 4 . T h e n , i n order f o r c e l l s to contribute to engraftment, C D 3 4 m a y be upregulated. T h i s c o u l d be f o l l o w e d by a decrease in expression as the bone m a r r o w a c h i e v e s steady state levels o f hematopoietic c e l l s . A d d i t i o n a l l y , C D 3 4 is expressed o n mature m u r i n e mast c e l l s , but it is not f o u n d on platelets ( D r e w et a l . , 2 0 0 2 ) . T h e r e is therefore c o n s i d e r a b l e o v e r l a p between p o d o c a l y x i n and C D 3 4 expression, but there are some notable differences.  A l t h o u g h m u c h less is k n o w n about e n d o g l y c a n , its e x p r e s s i o n pattern d i s p l a y s some s i m i l a r i t y to those of C D 3 4 and p o d o c a l y x i n . S o m e h e m a t o p o i e t i c c e l l s , i n c l u d i n g bone m a r r o w m a c r o p h a g e s and T o l l - l i k e r e c e p t o r - a c t i v a t e d B c e l l s , as w e l l as a subset o f neuronal cells and vascular s m o o t h m u s c l e c e l l s all express e n d o g l y c a n ( ( N i e l s e n et a l . , 2 0 0 2 ; Sassetti et a l . , 2 0 0 0 ) , and H e l e n M e r k e n s and K e l l y M c N a g n y unpublished). T h e r e is also some e v i d e n c e f o r expression on e n d o t h e l i a l c e l l s l i n i n g vessels (Sassetti et a l . , 2000).  1.2 Cloning of Podocalyxin A l t h o u g h p o d o c a l y x i n was i n i t i a l l y i d e n t i f i e d and c h a r a c t e r i z e d i n rat in the 1 9 8 0 ' s ( K e r j a s c h k i et a l . , 1984), it w a s not until 1995 that it w a s f i r s t c l o n e d u s i n g a rabbit g l o m e r u l a r c D N A l i b r a r y ( K e r s h a w et a l . , 1995). A bacterial c D N A l i b r a r y e x p r e s s i o n s y s t e m was screened w i t h m o n o c l o n a l antibodies generated against a p o d o c a l y x i n - l i k e protein expressed  in glomeruli. Six  positive  clones were  identified, and  these  c o r r e s p o n d e d to a 5.5 k b transcript h i g h l y e x p r e s s e d i n g l o m e r u l i . A n a l y s i s o f the  10  predicted a m i n o a c i d sequence d e m o n s t r a t e d that it contained m a n y characteristics o f p o d o c a l y x i n , s o the m o l e c u l e w a s n a m e d " p o d o c a l y x i n - l i k e  protein  1 (PCLP1)".  S u b s e q u e n t l y , rabbit c D N A e n c o d i n g p o d o c a l y x i n w a s used to screen a h u m a n renal cortex c D N A l i b r a r y , and h u m a n P C L P 1 w a s c l o n e d ( K e r s h a w et a l . , 1997a). c D N A c o r r e s p o n d i n g to the c y t o p l a s m i c tail o f h u m a n P C L P 1 was then used to probe the g e n o m i c D N A of a variety o f species. H o m o l o g o u s sequences were f o u n d in m o n k e y , rat, m o u s e , d o g , c o w , r a b b i t , and c h i c k e n , but not yeast, d e m o n s t r a t i n g the r e m a r k a b l e conservation o f this region of the gene in vertebrates ( K e r s h a w et a l . , 1997a).  Concurrently,  the  chicken  "thrombomucin" (McNagny  homolog  of  podocalyxin  et a l . , 1997). T o  was  cloned  begin with, primitive  and  named  hematopoietic  progenitors d e r i v e d f r o m c h i c k e n e m b r y o y o l k sac were transformed w i t h the M y b - E t s o n c o p r o t e i n - e n c o d i n g acute l e u k e m i a v i r u s , E 2 6 ; these c e l l s were t e r m e d  Myb-Ets-  transformed p r o g e n i t o r s , or M E P s ( G r a f et a l . , 1992). A panel of m o n o c l o n a l antibodies was generated against surface antigens o n these c e l l s by using them to i m m u n i z e m i c e (McNagny  et a l . , 1992). O n e o f the a n t i b o d i e s , M E P 2 1 , s p e c i f i c a l l y r e c o g n i z e d an  antigen present on M E P s and platelets, but not o n other mature hematopoietic c e l l s . T h e M E P 2 1 antigen c o u l d o n l y be p u r i f i e d i n very l o w quantities, so nanoelectrospray mass spectrometry w a s used to sequence the p r o t e i n , and degenerate o l i g o n u c l e o t i d e s were used to c l o n e the M E P 2 1 - e n c o d i n g c D N A s ( M c N a g n y et a l . , 1997). T h r e e distinct types of c l o n e s were o b t a i n e d . T h e f i r s t e n c o d e d a 5 2 4 base pair (bp) 5 ' untranslated region ( U T R ) , a 1347 bp 3 ' U T R , and an open r e a d i n g f r a m e c o d i n g f o r 571 a a , w h i c h i n c l u d e d a putative N - t e r m i n a l s i g n a l p e p t i d e , a m u c i n d o m a i n , a g l o b u l a r d o m a i n , a stalk, a  11  putative transmembrane d o m a i n , and a c y t o p l a s m i c tail c o n t a i n i n g consensus P K C and C K I I p h o s p h o r y l a t i o n sites. T h e second type of c l o n e was very s i m i l a r , but it e n c o d e d an insertion of 18 a d d i t i o n a l a m i n o acids in the j u x t a m e m b r a n e region of the c y t o p l a s m i c t a i l . T h e third type of c l o n e contained a novel sequence b e g i n n i n g at the same site as the insertion in type t w o c l o n e s , and it encoded a shorter c y t o p l a s m i c tail w i t h an earlier stop c o d o n . T h i s w a s the f i r s t e v i d e n c e o f a l t e r n a t i v e s p l i c i n g to generate a t r u n c a t e d c y t o p l a s m i c tail in p o d o c a l y x i n . E x p r e s s i o n of this m u c i n on platelets, or t h r o m b o c y t e s , l e d to it being c a l l e d t h r o m b o m u c i n .  S u b s e q u e n t l y , rat podxl w a s c l o n e d u s i n g c D N A libraries f r o m k i d n e y . It w a s p a r t i a l l y c l o n e d u s i n g rabbit c D N A as a probe ( M i e t t i n e n et a l . , 1999); it w a s later c o m p l e t e l y c l o n e d u s i n g p r ime r s designed f r o m h o m o l o g o u s sequences i n the c y t o p l a s m i c tails o f r a b b i t and h u m a n p o d o c a l y x i n  ( T a k e d a et a l . , 2 0 0 0 ) . I n t e r e s t i n g l y , a l t h o u g h  dog  p o d o c a l y x i n (gp 135) has been used as an apical m a r k e r o f M a d i n - D a r b y c a n i n e k i d n e y ( M D C K ) e p i t h e l i a l c e l l s f o r many years ( O j a k i a n and S c h w i m m e r , 1988), it w a s o n l y recently recognized as p o d o c a l y x i n ( M e d e r et a l . , 2 0 0 5 ) . It was p r e v i o u s l y k n o w n s i m p l y as  "gpl35,"  but  nanoelectrospray  after  purification  and  identification  t a n d e m mass s p e c t r o m e t r y ,  of  tryptic  peptides  by  it b e c a m e c l e a r that it w a s , i n f a c t ,  p o d o c a l y x i n . E S T ' s c o r r e s p o n d i n g to p o d o c a l y x i n have n o w been i d e n t i f i e d i n h u m a n , d o g , rat, m o u s e , sq uir r e l, c h i c k e n , f r o g , and zebrafish.  12  1.3 Transcriptional  Regulation of  Podocalyxin  The process of transcriptional regulation of C D 3 4 has proven difficult to decipher, and podxl's  promoter and untranslated regions are likely complicated as well. In fact,  transfection of 54 kb of human genomic D N A is not sufficient to drive hematopoietic progenitor-specific expression of C D 3 4 , while 160 kb is, suggesting that considerable upstream, downstream, and intronic sequences are required for tissue-specific  expression  of C D 3 4 (Radomska et al., 1998; Yamaguchia et al., 1997). Preliminary experiments with the podxl  locus suggest that it may be regulated in an equally complex manner  (unpublished observations, Jon Frampton and Regis Doyonnas (Nielsen et al., 2002)). However, there are a few clues about podocalyxin's regulation. It is positively regulated by the Wilms' tumour 1 (WT1) zinc finger transcription factor, negatively regulated by p53, and also affected by Ets-1 expression (Guo et al., 2002; Palmer et al., 2001; Stanhope-Baker et al., 2004; Teruyama et al., 2001).  1.3.1 WTl  Positive Regulation by WT1 itself is complex: alternative splicing, R N A editing, and multiple translation  initiation sites allow the production of 24 different proteins from the wtl  gene, and  biochemical experiments suggest that it acts as a transcription factor as well as playing a role in R N A processing, depending on its cellular context and splice variants ((Bruening and Pelletier, 1996; Guo et al., 2002; Haber et al., 1991; Scharnhorst et al., 1999; Sharma et al., 1994), and reviewed in (Discenza and Pelletier, 2004)). Furthermore, there is confusion as to whether it activates or represses transcription (Maheswaran et al., 1993;  13  W a n g et a l . , 2 0 0 1 ) , and although it was i n i t i a l l y i d e n t i f i e d as a t u m o u r suppressor it m a y also f u n c t i o n as an oncogene ( r e v i e w e d in ( L o e b and S u k u m a r , 2 0 0 2 ) ) . W T 1 is n o r m a l l y expressed in the d e v e l o p i n g g l o m e r u l u s , as w e l l as in the urogenital ridge, m e s o t h e l i u m , spleen, b r a i n , and spinal cord d u r i n g embryogenesis and in the uterus, o v i d u c t , granulosa c e l l s of the o v a r y , Sertoli cells of the testes, hematopoietic progenitor or stem c e l l s , and k i d n e y podocytes in adult (reviewed in ( D i s c e n z a and Pelletier, 2004)).  S e v e r a l studies have i m p l i c a t e d W T 1 in regulation o f p o d o c a l y x i n e x p r e s s i o n . S p l i c i n g generates t w o m a j o r variants o f W T 1  whereby a three-amino acid ( K T S )  sequence  b e t w e e n the third and f o u r t h z i n c f i n g e r s is either o m i t t e d or i n c l u d e d ( P a l m e r et a l . , 2 0 0 1 ; W a n g et a l . , 2 0 0 1 ) . T h e ( - K T S ) variant binds D N A and acts as a t r a n s c r i p t i o n a l regulator, w h i l e the ( + K T S ) variant is a poor t r a n s c r i p t i o n a l regulator but can instead interact w i t h s p l i c i n g machinery (Roberts, 2005). W i t h i n hours o f i n d u c i b l e expression of the ( - K T S ) i s o f o r m in rat e m b r y o n i c k i d n e y cell lines, a dramatic increase i n p o d o c a l y x i n is detected at both the m R N A and protein l e v e l s ( P a l m e r et a l . , 2 0 0 1 ) . T h i s effect is reversible upon removal of W T 1 . A d d i t i o n a l experiments identified a W T 1  (-KTS)  responsive d o m a i n in the podxl promoter ( P a l m e r et a l . , 2 0 0 1 ) , and an independent g r o u p used c h r o m a t i n i m m u n o p r e c i p i t a t i o n ( C h I P ) to demonstrate that W T 1 does, in fact, b i n d the p r o m o t e r ( S t a n h o p e - B a k e r et a l . , 2 0 0 4 ) . F u r t h e r m o r e , i n a t r a n s g e n i c  mouse  e x p r e s s i n g a mutant f o r m o f W T 1 , there is a s t a t i s t i c a l l y s i g n i f i c a n t d e c r e a s e i n p o d o c a l y x i n expression i n n e w b o r n k i d n e y s , although n o r m a l l e v e l s are a c h i e v e d w i t h i n several m o n t h s o f birth ( G a o et a l . , 2 0 0 4 ) . M o r e o v e r , p o d o c a l y x i n e x p r e s s i o n i n the d e v e l o p i n g g l o m e r u l u s , and i n other c e l l types, c o r r e s p o n d s to that o f W T 1 . In the  14  g l o m e r u l u s , W T 1 is e x p r e s s e d d u r i n g the renal v e s i c l e and S - s h a p e d body stages o f d e v e l o p m e n t , and it is restricted to visceral epithelial cells (podocytes) o f d e v e l o p i n g and mature g l o m e r u l i ( A r m s t r o n g et a l . , 1 9 9 3 ; P a l m e r et a l . , 2 0 0 1 ) . P o d o c a l y x i n is first evident at a s i m i l a r stage o f g l o m e r u l a r development, and expression is greatest in mature p o d o c y t e s ( P a l m e r et a l . , 2 0 0 1 ; P r i t c h a r d - J o n e s et a l . , 1990). In a d d i t i o n , W T 1  and  p o d o c a l y x i n are c o - e x p r e s s e d in m e s o t h e l i u m and hematopoietic precursors ( D o y o n n a s et a l . , 2 0 0 1 ; D o y o n n a s et a l . , 2 0 0 5 ; E l l i s e n et a l . , 2 0 0 1 ; Pritchard-Jones et a l . , 1990). T h u s , W T 1 is the most c o n c l u s i v e regulator o f p o d o c a l y x i n expression to date.  1.3.2  Negative Regulation by p53  In contrast, there is some evidence to suggest that p53 represses p o d o c a l y x i n e x p r e s s i o n . Using c D N A  m i c r o a r r a y a n a l y s i s , p o d o c a l y x i n was i d e n t i f i e d as a p53 target in the  W i l m s ' t u m o u r m o d e l c e l l l i n e , W i T 4 9 ( S t a n h o p e - B a k e r et a l . , 2 0 0 4 ) . A  luciferase  reporter construct was then used to c o n f i r m the c o n s i d e r a b l e decrease in p o d o c a l y x i n levels upon p53 expression.  Interestingly, p53 and W T 1 have been s h o w n to p h y s i c a l l y interact and m o d u l a t e the f u n c t i o n s o f one another i n s o m e s i t u a t i o n s . W h i l e p53 can alter the t r a n s c r i p t i o n a l regulatory a c t i v i t y o f W T 1 , W T 1 c a n s t a b i l i z e p 5 3 , alter its a c t i v i t y , and prevent p 5 3 i n d u c e d apoptosis ( M a h e s w a r a n et a l . , 1 9 9 5 ; M a h e s w a r a n et a l . , 1993). H o w e v e r , in the W i T 4 9 c e l l l i n e , p 5 3 - i n d u c e d p o d o c a l y x i n repression is not related to p 5 3 ' s effects o n W T 1 : a m i n i m a l podxl promoter c o n t a i n i n g the W T l - r e p o n s i v e portion is not s u f f i c i e n t  15  to decrease p o d o c a l y x i n e x p r e s s i o n upon activation o f p53 ( S t a n h o p e - B a k e r et a l . , 2 0 0 4 ) . T h u s , p53 can modulate p o d o c a l y x i n ' s expression i n a VVT1-independent manner.  1.3.3  Positive Regulation by Ets-1  T h e o n l y a d d i t i o n a l i n f o r m a t i o n regarding control o f p o d o c a l y x i n expression c o m e s f r o m a study based o n c D N A m i c r o a r r a y analysis o f h u m a n u m b i l i c a l v e i n e n d o t h e l i a l c e l l s ( H U V E C ) infected w i t h an adenovirus e n c o d i n g the E t s - 1 transcription factor ( T e r u y a m a et a l . , 2 0 0 1 ) . E t s - 1 e x p r e s s i o n i n endothelial c e l l s is k n o w n to promote angiogenesis. Its expression i n H U V E C s leads to a 3 . 3 - f o l d upregulation o f p o d o c a l y x i n i n c o m p a r i s o n to n u l l - v i r u s i n f e c t e d c e l l s , a l t h o u g h there is n o e v i d e n c e to suggest that p o d o c a l y x i n expression is directly regulated by this factor. T h e r e is therefore considerable w o r k still to be done in u n r a v e l i n g the c o m p l e x transcriptional regulation o f p o d o c a l y x i n .  1.4 Intracellular Binding Partners for  Podocalyxin  C l u e s r e g a r d i n g the f u n c t i o n o f n o v e l proteins c a n often be o b t a i n e d by i d e n t i f y i n g b i n d i n g partners w i t h k n o w n f u n c t i o n s . T h e h i g h degree o f sequence c o n s e r v a t i o n i n p o d o c a l y x i n ' s c y t o p l a s m i c tail i m p l i e s that i n t r a c e l l u l a r b i n d i n g partners m i g h t e x i s t ; these potential interactors are thus the f o c u s o f m u c h research.  16  1.4.1  Ezrin  T h e first i n d i c a t i o n o f an i n t r a c e l l u l a r b i n d i n g partner f o r p o d o c a l y x i n c a m e f r o m D r . M a r i l y n F a r q u h a r ' s group in 2001 ( O r l a n d o et a l . , 2 0 0 1 ) . T h e y had p r e v i o u s l y  noticed  that apical p o d o c a l y x i n l o c a l i z a t i o n in podocytes c o i n c i d e s w i t h that o f e z r i n , a m e m b e r o f the e z r i n - r a d i x i n - m o e s i n ( E R M ) f a m i l y o f cytoskeletal l i n k e r proteins ( K u r i h a r a et a l . , 1995). E R M proteins contain a C - t e r m i n a l actin b i n d i n g m o t i f and an N - t e r m i n a l F E R M p r o t e i n m o d u l e , thought to a n c h o r t h e m to m e m b r a n e proteins ( C h i s h t i et a l . , 1 9 9 8 ; T u r u n e n et a l . , 1994). T h e y regulate c e l l a d h e s i o n and m o r p h o g e n e s i s ,  including  f o r m a t i o n o f m i c r o v i l l i and m e m b r a n e ruffles ( r e v i e w e d in ( M a n g e a t et a l . , 1999)). T h e i m p o r t a n c e of p o d o c a l y x i n i n m a i n t a i n i n g p o d o c y t e f o o t process integrity below),  (discussed  and its c o l o c a l i z a t i o n w i t h e z r i n , suggested a p o s s i b l e l i n k to the  actin  c y t o s k e l e t o n . F o r these r e a s o n s , this g r o u p assessed the p o t e n t i a l a s s o c i a t i o n  of  p o d o c a l y x i n w i t h ezrin and actin ( O r l a n d o et a l . , 2001).  O v e r l a p p i n g l o c a l i z a t i o n o f p o d o c a l y x i n and e z r i n i n p o d o c y t e s w a s c o n f i r m e d  by  i m m u n o f l u o r e s c e n c e and dual i m m u n o g o l d l a b e l l i n g o f u l t r a - t h i n c r y o s e c t i o n s o f rat k i d n e y ( O r l a n d o et a l . , 2 0 0 1 ) . B o t h proteins are concentrated a l o n g the a p i c a l p l a s m a membrane o f podocyte cell bodies and f o o t processes above the level o f slit d i a p h r a g m s , a n d they f o r m a s t a b l e , c o - i m m u n o p r e c i p i t a b l e c o m p l e x  ( O r l a n d o et a l . , 2 0 0 1 ) .  Importantly, selective detergent extraction and c o - s e d i m e n t a t i o n assays in p o d o c a l y x i n transfected M D C K  c e l l s d e m o n s t r a t e that a s i g n i f i c a n t p o r t i o n o f p o d o c a l y x i n  is  associated w i t h actin f i l a m e n t s and that this interaction is dependent o n the interaction o f p o d o c a l y x i n w i t h ezrin.  17  Subsequent experiments i d e n t i f i e d an ezrin b i n d i n g site in the j u x t a m e m b r a n e r e g i o n o f p o d o c a l y x i n ' s c y t o p l a s m i c t a i l ( S c h m i e d e r et a l . , 2 0 0 4 ) . T h e H Q R I S s e q u e n c e  of  p o d o c a l y x i n is s i m i l a r to the H Q R S f o u n d in i n t r a c e l l u l a r adhesion m o l e c u l e ( I C A M ) - 3 (Serrador et a l . , 2002). T h e histidine, arginine, and several serine residues are required for the r e c o g n i t i o n o f I C A M - 3 by e z r i n (Serrador et a l . , 2 0 0 2 ) , so these residues were mutated i n a H i s - t a g g e d p o d o c a l y x i n tail construct in order to assess b i n d i n g to G S T tagged N - t e r m i n a l ezrin ( S c h m i e d e r et a l . , 2 0 0 4 ) . M u t a t i o n of serine to a l a n i n e , histidine and arginine to alanine, or a l l three residues to a l a n i n e , or deletion of the 12 N - t e r m i n a l residues o f the c y t o p l a s m i c tail altogether decreases b i n d i n g by 2 2 , 4 6 , 9 5 , or 100 % , r e s p e c t i v e l y . T h e s e results strongly suggest that e z r i n and p o d o c a l y x i n are c a p a b l e o f direct interaction.  1.4.2  N H E R F Proteins  T h e r e is also increasing e v i d e n c e that p o d o c a l y x i n interacts w i t h members o f the N a 7 H  +  exchanger ( N H E ) regulatory factor ( N H E R F ) f a m i l y o f s c a f f o l d i n g proteins ( r e v i e w e d in ( D o n o w i t z et a l . , 2 0 0 5 ; S h e n o l i k a r et a l . , 2 0 0 4 ; T h e l i n et a l . , 2 0 0 5 ; V o l t z et a l . , 2 0 0 1 ; W e i n m a n , 2 0 0 1 ) ) . N H E R F 1 / E B P 5 0 ( e z r i n b i n d i n g p h o s p h o p r o t e i n o f 5 0 k D a ) and N H E R F 2 / E 3 K A R P ( N H E 3 kinase A regulatory protein) both interact w i t h p o d o c a l y x i n ( d i s c u s s e d b e l o w ) , w h i l e the m o r e d i s t a n t l y related and recently d i s c o v e r e d f a m i l y m e m b e r s N H E R F 3 / P D Z K 1 and N H E R F 4 / I K E P P have not yet been s h o w n to b i n d p o d o c a l y x i n . N H E R F m o l e c u l e s a l l have m u l t i p l e p r o t e i n - p r o t e i n interaction m o d u l e s . N H E R F 1 and N H E R F 2 each have t w o tandem P S D - 9 5 / D l g / Z O - l ( P D Z ) d o m a i n s and a  18  C - t e r m i n a l E R M d o m a i n . In contrast, N H E R F 3 and N H E R F 4 both l a c k E R M d o m a i n s , but each have f o u r P D Z d o m a i n s . E R M d o m a i n s facilitate interaction w i t h E R M f a m i l y m e m b e r s and i n d i r e c t l i n k a g e to the a c t i n c y t o s k e l e t o n , w h i l e P D Z d o m a i n s , w h i c h represent one o f the most c o m m o n m o d u l a r d o m a i n s i n the h u m a n g e n o m e , r e c o g n i z e s p e c i f i c sequences, generally at the C - t e r m i n u s o f proteins. T h e r e are several classes o f P D Z d o m a i n s , but the second P D Z d o m a i n s o f both N H E R F 1 and N H E R F 2 r e c o g n i z e the consensus sequence X - ( S / T ) - X - ( I / V / L / M ) where X represents any a m i n o a c i d .  N H E R F proteins have the capacity to h o m o d i m e r i z e or heterodimerize w i t h each other, and their m u l t i p l e protein-interaction d o m a i n s c o u l d enable the f o r m a t i o n of large protein c o m p l e x e s connected to the actin cytoskeleton. E a r l y studies o f this f a m i l y also suggested that N H E R F proteins were i n v o l v e d i n a p i c a l m e m b r a n e l o c a l i z a t i o n o f other proteins, but recent data has raised doubts about this proposed f u n c t i o n . N H E R F f a m i l y m e m b e r s are thought to be i n v o l v e d in a wide variety of b i o l o g i c a l processes, i n c l u d i n g t r a f f i c k i n g , transport, and s i g n a l l i n g , based on their interaction w i t h over 3 0 target proteins. T h e y can interact w i t h i o n transporters, such as N H E 3 , the cystic f i b r o s i s transmembrane regulator ( C F T R ) , and the N a - p h o s p h a t e transporter ( N p t 2 ) , and G p r o t e i n - c o u p l e d receptors, +  i n c l u d i n g |3 -adrenergic receptors ( f 3 - A R ) , the p u r i n e r g i c receptor P 2 Y 1 R , and the K 2  2  o p i o i d receptor. T h e y can also associate w i t h s i g n a l l i n g proteins, s c a f f o l d s , and nuclear proteins, such as platelet-derived g r o w t h factor receptor ( P D G F R ) , phospholipase C(31, 2 , and 3 , the G p r o t e i n - c o u p l e d receptor k i n a s e ( G R K ) , and |3-catenin. It must be n o t e d , h o w e v e r , that many o f these interactions have not yet been c o n f i r m e d in vivo.  19  S h o r t l y after i d e n t i f y i n g e z r i n as a p o d o c a l y x i n b i n d i n g protein, D r . M a r i l y n F a r q u h a r ' s g r o u p also i d e n t i f i e d N H E R F 2 as a b i n d i n g partner f o r p o d o c a l y x i n ( T a k e d a et a l . , 2 0 0 1 ) ) . U s i n g the c y t o p l a s m i c tail o f p o d o c a l y x i n as bait, N H E R F 2 w a s detected i n a yeast t w o - h y b r i d screen o f a rat g l o m e r u l a r c D N A l i b r a r y . F u r t h e r m o r e , i n G S T p u l l d o w n assays, in vitro translated N H E R F 2 binds strongly to G S T - t a g g e d p o d o c a l y x i n tail. T h i s assay a l s o d e m o n s t r a t e s that the related m o l e c u l e , N H E R F 1 c a n interact  with  p o d o c a l y x i n . W h e n p o d o c a l y x i n and N H E R F f a m i l y members are co-transfected in vitro, p o d o c a l y x i n is c o - i m m u n o p r e c i p i t a t e d w i t h both N H E R F 1 and N H E R F 2 . T h e interaction o f p o d o c a l y x i n w i t h these t w o P D Z - c o n t a i n i n g proteins is not surprising in that it has a c o n s e r v e d P D Z b i n d i n g m o t i f , D T H L , at its C - t e r m i n u s ; deletion o f this m o t i f prevents b i n d i n g i n transfected c e l l s ( T a k e d a et a l . , 2 0 0 1 ) . A d d i t i o n a l yeast t w o - h y b r i d and G S T p u l l - d o w n e x p e r i m e n t s u s i n g in vitro translated f r a g m e n t s o f N H E R F l a n d  NHERF2  s h o w e d that p o d o c a l y x i n interacts w i t h the second P D Z d o m a i n o f both m o l e c u l e s , but not w i t h the first d o m a i n , and that the interaction between p o d o c a l y x i n and N H E R F 2 is m u c h stronger than the interaction w i t h N H E R F 1 .  In k i d n e y , N H E R F 1 is expressed i n p r o x i m a l tubules, but not g l o m e r u l i , w h i l e N H E R F 2 is f o u n d a l o n g the a p i c a l p l a s m a m e m b r a n e o f p o d o c y t e f o o t p r o c e s s e s , w h e r e it c o l o c a l i z e s w i t h both p o d o c a l y x i n a n d e z r i n ( T a k e d a et a l . , 2 0 0 1 ) . In a d d i t i o n , c o i m m u n o p r e c i p i t a t i o n ( c o - i p ) e x p e r i m e n t s f r o m g l o m e r u l a r extracts demonstrate that p o d o c a l y x i n c a n interact w i t h b o t h N H E R F 2 a n d e z r i n , but not w i t h N H E R F 1 , i n g l o m e r u l i ( O r l a n d o et a l . , 2 0 0 1 ; T a k e d a et a l . , 2 0 0 1 ) . S i n c e N H E R F proteins contain an E R M b i n d i n g d o m a i n i n a d d i t i o n to their P D Z d o m a i n s , it is l i k e l y that p o d o c a l y x i n ,  20  NHERF2,  and ezrin form a multimeric complex  in podocytes.  Association of  podocalyxin with the cytoskeleton may anchor it to specific membrane microdomains or determine its residence time at the cell surface (Takeda et al., 2001).  At around the same time, Dr. David Kershaw's group also identified N H E R F 2 as a podocalyxin binding protein by screening a rabbit glomerular c D N A library ( L i et al., 2 0 0 2 ) . In contrast to the previous work, they demonstrated that podocalyxin can interact with both P D Z domains of N H E R F 2 , although the interaction with the second P D Z sequence is much more convincing. Again, disruption of podocalyxin's P D Z binding domain, this time by deletion of only the C-terminal leucine residue, abolishes  binding. Co-immunoprecipitation and immunofluorescence  completely  of glomeruli  provided additional support for an association between podocalyxin and N H E R F 2 .  Confocal microscopy and surface biotinylation experiments with M D C K cells transfected with podocalyxin, or podocalyxin lacking the P D Z binding domain, have been performed in an attempt to address the functional significance of p o d o c a l y x i n / N H E R F family interactions (Li et al., 2 0 0 2 ) . Confocal microscopy demonstrates  that full-length  podocalyxin is detected on the apical surface, while some mutated podocalyxin is found in the cytoplasm. Biotinylation of the apical surface of cells confirms that the majority of podocalyxin is located on this surface, while a portion of the mutant podocalyxin is intracellular; time course experiments indicate that full-length podocalyxin also persists at the cell surface longer than the mutant form does. There is a precedent for a N H E R F dependent role in membrane retention: the C-terminal P D Z binding motif of the y-  21  aminobutyric acid transporter is required for its retention at the basolateral surface of cells (Perego et al.,  1999).  Linkage of podocalyxin to the actin cytoskeleton through  NHERF and ezrin may also serve to maintain podocalyxin's localization in specialized membrane subdomains, and perhaps vascular endothelial podocalyxin would be redistributed according to the direction of blood flow by interaction with cationic molecules if it were not anchored to the actin cytoskeleton (Li et al., 2002). Thus, current data suggest that interaction with NHERF proteins assists in efficient apical localization and stability of podocalyxin.  Podocalyxin is able to interact with ezrin both directly, and indirectly through NHERF proteins (Schmieder et al., 2004). Pull-down assays with GST-tagged podocalyxin tail or a mutant lacking the DTHL sequence confirm that the N-terminal portion of ezrin can interact with both forms, although the absence of any potential NHERF binding leads to a weaker association between podocalyxin and ezrin (Schmieder et al., 2004). Moreover, differential detergent extraction demonstrates that podocalyxin lacking the NHERF binding domain does not strongly associate with actin. Although the rationale for both direct and indirect mechanisms of podocalyxin/ezrin interactions is not known, it may be that direct interaction transiently disrupts binding of podocalyxin to NHERF proteins, thereby enabling regulatory events such as phosphorylation and consequent conformational changes (He et al., 2001).  Until very recently, the search for podocalyxin binding partners had focused on the glomerulus, but podocalyxin is also expressed in the hematopoietic system. In order to  22  f i n d h e m a t o p o i e t i c s p e c i f i c interactors, an early h e m a t o p o i e t i c c D N A e x p r e s s i o n library ( M c N a g n y et a l . , 1996) was screened w i t h a b i o t i n y l a t e d peptide c o r r e s p o n d i n g to the c y t o p l a s m i c tail of p o d o c a l y x i n (Tan et a l . , 2 0 0 6 ) . T h i s screen i d e n t i f i e d N H E R F 1 as a p o d o c a l y x i n i n t e r a c t i n g protein in the h e m a t o p o i e t i c s y s t e m ( T a n et a l . , 2 0 0 6 ) .  The  p o d o c a l y x i n peptide was also used to p u r i f y i n t e r a c t i n g proteins f r o m h e m a t o p o i e t i c progenitor c e l l extracts; mass spectrometry c o n f i r m s the association o f p o d o c a l y x i n and N H E R F 1 . A s expected, this interaction is dependent on the C - t e r m i n a l P D Z r e c o g n i t i o n sequence  of  podocalyxin.  These  immunoprecipitable complex  two  proteins  also colocalize  in hematopoietic  progenitors.  and f o r m  a co-  Interestingly,  strong  c o l o c a l i z a t i o n corresponds to c e l l s in w h i c h p o d o c a l y x i n is c a p p e d to one p o l e ; c e l l s expressing  uniform levels of podocalyxin  o v e r the entire s u r f a c e d o not  c o l o c a l i z a t i o n o f the t w o p r o t e i n s . I m p o r t a n t l y ,  display  essentially all hematopoietic cells,  i n c l u d i n g those d i s p l a y i n g c e l l surface m a r k e r s i n d i c a t i v e o f s t e m c e l l s , e x p r e s s N H E R F 1 , w h i c h suggests that it may be an important l i g a n d f o r p o d o c a l y x i n in these cells (Tan et a l . , 2006).  1.4.3  Other CD34-Family Binding Proteins  T o date, there has been o n l y one study i n v e s t i g a t i n g potential i n t r a c e l l u l a r b i n d i n g partners f o r e n d o g l y c a n . U s i n g the hematopoietic c D N A library screen described a b o v e , the c y t o p l a s m i c tail o f e n d o g l y c a n , l i k e that of p o d o c a l y x i n , was also s h o w n to b i n d NHERF1  (Tan  et a l . , 2 0 0 6 ) . S i m i l a r l y , a f f i n i t y  purification from  hematopoietic  progenitor lysates indicates that N H E R F 1 can associate w i t h a peptide c o r r e s p o n d i n g to the C - t e r m i n u s  o f e n d o g l y c a n . S t r i k i n g l y , the s a m e e x p e r i m e n t s r e v e a l a l a c k o f  23  interaction o f N H E R F 1 w i t h C D 3 4 . A l t h o u g h C D 3 4 does c o n t a i n a C - t e r m i n a l  PDZ  r e c o g n i t i o n s e q u e n c e , it d i f f e r s f r o m both p o d o c a l y x i n and e n d o g l y c a n , w h i c h are i d e n t i c a l : p o d o c a l y x i n c o n t a i n s a D T H L m o t i f , w h i l e the c o r r e s p o n d i n g sequence in C D 3 4 is D T E L ( K e r s h a w et a l . , 1 9 9 5 ; S u d a et a l . , 1992). T h i s difference l i k e l y c o n v e y s the s p e c i f i c i t y for interaction o f p o d o c a l y x i n and e n d o g l y c a n w i t h N H E R F proteins, and suggests that w h i l e the three proteins may have s o m e o v e r l a p p i n g f u n c t i o n s , there are probably some key differences.  Although  CD34  does  not  a p p e a r to i n t e r a c t  with  NHERF  family  members,  in  hematopoietic progenitor c e l l s it has been s h o w n to interact w i t h C r k L , a m e m b e r o f the C r k f a m i l y o f adapter proteins ( F e l s c h o w et a l . , 2 0 0 1 ) . T h e s e proteins l i n k proteins that do not possess kinase a c t i v i t y to i n t r a c e l l u l a r s i g n a l i n g cascades, thereby e n a b l i n g them to indirectly transmit signals. A l t h o u g h the exact b i n d i n g site f o r C r k L on C D 3 4 has not yet been d e t e r m i n e d , it is d e p e n d e n t  on a highly  conserved  intracellular  10 aa  j u x t a m e m b r a n e sequence present i n both i s o f o r m s of C D 3 4 . T h e intracellular sequences o f p o d o c a l y x i n and e n d o g l y c a n are m o r e s i m i l a r to each other than to C D 3 4 , so the existence o f diverse b i n d i n g partners is not s u r p r i s i n g ( H e et a l . , 1 9 9 2 ; K e r s h a w et a l . , 1997a; M c N a g n y et a l . , 1 9 9 7 ; Sassetti et a l . , 2 0 0 0 ; S i m m o n s et a l . , 1992).  1.5 Proposed Functions of CD34 Family Members R e m a r k a b l y , a l t h o u g h there are o v e r 12 0 0 0 C D 3 4 - r e l a t e d p u b l i c a t i o n s to date, the f u n c t i o n s o f C D 3 4 and its f a m i l y m e m b e r s r e m a i n to be a s c e r t a i n e d . A v a r i e t y potential functions  have  been p r o p o s e d , h o w e v e r ,  i n c l u d i n g roles i n  of  enhancing  24  p r o l i f e r a t i o n , b l o c k i n g d i f f e r e n t i a t i o n , a c t i n g as a d h e s i v e l i g a n d s , p r e v e n t i n g c e l l a d h e s i o n , establishing polarity, and regulating a s y m m e t r i c c e l l d i v i s i o n .  1.5.1  Enhancing Proliferation and Blocking Differentiation  T h e r e are t w o reasons f o r C D 3 4 ' s h y p o t h e s i z e d role in e n h a n c i n g p r o l i f e r a t i o n or b l o c k i n g d i f f e r e n t i a t i o n . First, its expression o n m u l t i p o t e n t h e m a t o p o i e t i c progenitors and progressive d o w n r e g u l a t i o n on more mature c e l l s suggests a role in m a i n t e n a n c e of the stem c e l l phenotype ( r e v i e w e d in ( K r a u s e et a l . , 1996)). S e c o n d , in one strain of C D 3 4 k n o c k o u t ( K O ) a n i m a l s , there are less p r o g e n i t o r c e l l s in e m b r y o n i c and adult tissues, and a d u l t - d e r i v e d progenitors appear to have a p r o l i f e r a t i o n defect ( F i g u r e 1 - 4 A ) ( C h e n g et a l . , 1996). In c o m p a r i s o n to w i l d t y p e a n i m a l s , C D 3 4 k n o c k o u t s have a s i g n i f i c a n t decrease in total c o l o n y f o r m i n g unit ( C F U ) progenitor cells in adult as w e l l as a reduction in fetal l i v e r - d e r i v e d erythroid and m y e l o i d progenitors, despite the fact that there is n o clear defect in absolute numbers o f mature c e l l s in the h e m a t o p o i e t i c system o f adult a n i m a l s . A l t h o u g h this phenotype c o u l d also be e x p l a i n e d by decreased s u r v i v a l o f progenitors or a d e c l i n e in their retention in the bone m a r r o w , the fact that there is no increase i n these c e l l s in the p e r i p h e r y , and the o b s e r v a t i o n that a l t h o u g h p r o g e n i t o r s s u r v i v e in vitro, there is little e x p a n s i o n in c o m p a r i s o n to w i l d t y p e - d e r i v e d c e l l s , both f a v o u r the initial idea.  25  wild type  CD34 knockout  B  •  t  IL-6  Macrophage  Myeloblast  |  CD34  f  Ectopic CD34  Figure 1-4: Proposed Functions for CD34 Family: Enhancing Proliferation and Blocking Differentiation. ( A ) Hematopoietic progenitor cells from C D 3 4 - n u l l mice proliferate less in comparison to cells from wildtype mice. (B) The C D 3 4  +  progenitor cell line, M l , (light blue) can be  induced to differentiate into macrophages (dark blue) upon addition of I L - 6 or L I F . Ectopic expression of C D 3 4 in this cell line appears to block differentiation.  26  A  CD34-dependent  block in differentiation  is s u p p o r t e d  b y o n e set o f in  o v e r e x p r e s s i o n e x p e r i m e n t s ( F a c k l e r et a l . , 1995). T h e C D 3 4  +  vitro  murine m y e l o b l a s t s  l e u k e m i a c e l l l i n e , M l can be induced to t e r m i n a l l y differentiate into macrophages upon treatment w i t h i n t e r l e u k i n - 6 ( I L - 6 ) or l e u k e m i a i n h i b i t o r y factor ( L I F ) , at w h i c h point C D 3 4 is d o w n r e g u l a t e d ( S u d a et a l . , 1992). W i t h i n 2 4 hours o f L I F or I L - 6 treatment, there is a m a x i m a l reduction i n C D 3 4 m R N A l e v e l s , f o l l o w e d by d i f f e r e n t i a t i o n into m o r p h o l o g i c a l l y mature, f u n c t i o n a l l y active macrophages w i t h i n three days. W h e n C D 3 4 is e c t o p i c a l l y e x p r e s s e d  i n M l c e l l s , it appears to b l o c k I L - 6 a n d L I F i n d u c e d  d i f f e r e n t i a t i o n : c e l l s are m o r p h o l o g i c a l l y immature as they display m i n i m a l v a c u o l a t i o n , a h i g h nucleus to c y t o p l a s m ratio, open c h r o m a t i n , and p r o m i n e n t n u c l e o l i , and they are v i r t u a l l y n o n - p h a g o c y t i c ( F i g u r e 1 - 4 B ) ( F a c k l e r et a l . , 1995). N o t a b l y , the n a t u r a l l y o c c u r r i n g s p l i c e variant o f C D 3 4 is incapable o f b l o c k i n g d i f f e r e n t i a t i o n . T h u s , there is evidence to suggest that C D 3 4 m a y be i n v o l v e d i n b l o c k i n g differentiation or e n h a n c i n g progenitor proliferation.  H o w e v e r , there is also m o u n t i n g evidence to indicate that C D 3 4 f a m i l y proteins are not i n v o l v e d i n e n h a n c i n g proliferation or b l o c k i n g differentiation. R e g a r d i n g the C D 3 4 - n u l l phenotype, there is a second strain o f m i c e l a c k i n g C D 3 4 , and these m i c e d o not d i s p l a y detectable defects i n progenitor c e l l populations ( S u z u k i et a l . , 1996). T o t a l bone m a r r o w c e l l n u m b e r and relative ratios o f T e r l l 9 , B 2 2 0 , M a c - 1 , G r - 1 , C D 3 , c - K i t , a n d S c a - 1 e x p r e s s i n g c e l l s are a l l n o r m a l , as are numbers o f platelets, red b l o o d c e l l s , white b l o o d c e l l s , a n d w h i t e b l o o d c e l l s u b p o p u l a t i o n s in peripheral b l o o d . I m p o r t a n t l y , culture o f bone m a r r o w cells w i t h stem c e l l factor ( S C F ) , I L - 3 , and erythropoietin in vitro produces  27  n o r m a l numbers o f progenitors, as measured by c o l o n y assays. F u r t h e r m o r e , w h e n mast cells (the o n l y mature C D 3 4 expressing hematopoietic c e l l s ) f r o m these m i c e are cultured in vitro, there is no difference i n kinetics o f p r o l i f e r a t i o n , differentiation, or degranulation ( D r e w et a l . , 2 0 0 2 ; D r e w et a l . , 2 0 0 5 ) . M o r e o v e r , i n contrast to the o v e r e x p r e s s i o n o f C D 3 4 i n M l c e l l s , ectopic expression i n t w o other cell lines f a i l s to b l o c k differentiation ( F a c k l e r et a l . , 1995). A l s o w i t h respect to the M l e x p e r i m e n t s , it s h o u l d be kept i n m i n d that t w o characteristics used to measure differentiation were adhesion and p h a g o c y t o s i s , w h i c h c o u l d both p o t e n t i a l l y be a f f e c t e d by an alternative C D 3 4 f u n c t i o n : b l o c k i n g adhesion ( N i e l s e n et a l . , 2002) (discussed b e l o w ) . T h u s , it is u n l i k e l y that C D 3 4 f a m i l y m e m b e r s play an important role i n e n h a n c i n g p r o l i f e r a t i o n or b l o c k i n g d i f f e r e n t i a t i o n . Instead, the observed phenotypes m a y be the result o f other C D 3 4 - r e l a t e d f u n c t i o n s .  1.5.2  Pro-Adhesion  T h e r e is c o n v i n c i n g evidence s h o w i n g that C D 3 4 f a m i l y m e m b e r s can act i n an adhesive m a n n e r f o r recruitment o f l y m p h o c y t e s . L e u k o c y t e s are c o n s t a n t l y recruited f r o m the b l o o d into secondary l y m p h o i d organs and sites o f c h r o n i c i n f l a m m a t i o n i n a m u l t i - s t e p process i n v o l v i n g l o w a f f i n i t y b i n d i n g ( k n o w n as r o l l i n g ) f o l l o w e d by i n t e g r i n - m e d i a t e d f i r m arrest and transendothelial m i g r a t i o n (reviewed i n ( B u t c h e r and P i c k e r , 1 9 9 6 ; L a s k y , 1992)). T h e initial step, w h i c h requires adhesion o f l e u k o c y t e s to endothelial cells under c o n d i t i o n s o f vascular b l o o d f l o w , depends u p o n a s s o c i a t i o n o f selectin m o l e c u l e s w i t h their l i g a n d s . R e c r u i t m e n t o f l y m p h o c y t e s into peripheral l y m p h nodes requires b i n d i n g o f L - s e l e c t i n o n l y m p h o c y t e s to p a r t i c u l a r c a r b o h y d r a t e m o d i f i c a t i o n s o n proteins expressed o n the s p e c i a l i z e d p o s t c a p i l l a r y venules k n o w n as h i g h e n d o t h e l i a l venules  28  ( H E V ) . T h e s e sulfated and s i a l y l a t e d O - l i n k e d carbohydrates are presented to L - s e l e c t i n by m u c i n - l i k e g l y c o p r o t e i n s , i n c l u d i n g g l y c o s y l a t i o n - d e p e n d e n t c e l l adhesion m o l e c u l e ( G l y C A M ) - l , m u c o s a l addressin c e l l adhesion m o l e c u l e ( M A d C A M ) - l , and potentially e n d o m u c i n ( ( S a m u l o w i t z et a l . , 2 0 0 2 ) , and r e v i e w e d in (Rosen et a l . , 1997)).  T h e s i a l o m u c i n s C D 3 4 , p o d o c a l y x i n , and e n d o g l y c a n can a l l act i n a p r o - a d h e s i v e manner as ligands f o r L - s e l e c t i n o n H E V (Figure 1-5). T h e role o f C D 3 4 as an L - s e l e c t i n l i g a n d was first demonstrated i n 1 9 9 3 , w h e n it was s h o w n that a sulfated, H E V restricted f o r m o f C D 3 4 is r e c o g n i z e d  by  L - s e l e c t i n ( B a u m h u e t e r et a l . , 1993). S i m i l a r l y ,  p o d o c a l y x i n is also m o d i f i e d w i t h the appropriate g l y c o s y l a t i o n s for L - s e l e c t i n b i n d i n g in H E V , and in vitro it supports L - s e l e c t i n dependent tethering and r o l l i n g o f l y m p h o c y t e s under c o n d i t i o n s of f l o w (Sassetti et a l . , 1998). E n d o g l y c a n can also act as a ligand f o r L s e l e c t i n , i f e x p r e s s e d i n c o n j u n c t i o n w i t h the necessary e n z y m e s f o r appropriate postt r a n s l a t i o n a l m o d i f i c a t i o n ( F i e g e r et a l . , 2 0 0 3 ) . E n d o g l y c a n b i n d i n g d o e s ,  however,  i n v o l v e alternative g l y c o s y l a t i o n s f r o m those present in C D 3 4 and p o d o c a l y x i n . T h u s , all three m o l e c u l e s can f u n c t i o n as adhesive ligands for L - s e l e c t i n .  29  f  e  CD34  (-) charge  HEV-specific glycosylation  L-selectin  Figure 1-5: Proposed Function for CD34 Family: L-selectin Mediated Adhesion. C D 3 4 family members expressed on H E V enable L-selectin-mediated adhesion. Adapted from (Nielsen et al., 2002).  30  A l t h o u g h the e v i d e n c e to support an a d h e s i v e f u n c t i o n f o r C D 3 4 f a m i l y proteins is c o n v i n c i n g , it is u n l i k e l y that this is their u n i v e r s a l r o l e . O p t i m a l r e c o g n i t i o n glycoproteins  by L - s e l e c t i n r e q u i r e s e x q u i s i t e l y  of  specific modifications, including  s i a l y l a t i o n , f u c o s y l a t i o n , and s u l f a t i o n ( R o s e n et a l . , 1997). In the case o f C D 3 4 and p o d o c a l y x i n , the epitope r e c o g n i z e d by L - s e l e c t i n is termed M E C A - 7 9 ; this epitope is not f o u n d o n C D 3 4 or p o d o c a l y x i n expressed on other v a s c u l a r e n d o t h e l i a l c e l l s or on p o d o c a l y x i n on g l o m e r u l a r p o d o c y t e s ( B a u m h u e t e r et a l . , 1 9 9 3 ; M i c h i e et a l . , 1 9 9 3 ; Sassetti et a l . , 1 9 9 8 ; S e g a w a et a l . , 1997). M o r e o v e r ,  I have d e m o n s t r a t e d that  p o d o c a l y x i n and C D 3 4 expressed on fetal l i v e r c e l l s d o not interact w i t h L - s e l e c t i n , or any other c o m m o n selectin m o l e c u l e s ( D o y o n n a s et a l . , 2 0 0 5 ) . O f note, it has been s h o w n that t w o s u l f o t r a n s f e r a s e s that c a n m o d i f y C D 3 4 have d i s t i n c t e x p r e s s i o n (Bistrup  et a l . , 1999). In  vitro e x p r e s s i o n o f these t w o  patterns  enzymes, along with  a  fucosyltransferase, facilitates m o d i f i c a t i o n o f C D 3 4 f o r L - s e l e c t i n r e c o g n i t i o n ( B i s t r u p et a l . , 1999). A b s e n c e o f i n d i v i d u a l sulfotransferases does not prevent p r o d u c t i o n o f L selectin l i g a n d s , but w h e n both e n z y m e s are present, they s y n e r g i z e to produce a l i g a n d w i t h m u c h greater a f f i n i t y f o r L - s e l e c t i n . Importantly, w h i l e the G a l - 6 - s u l f o t r a n s f e r a s e d i s p l a y s a w i d e tissue d i s t r i b u t i o n , the G l c N A c - 6 - s u l f o t r a n s f e r a s e is h i g h l y restricted to h i g h endothelial c e l l s . T h u s , the expression pattern o f this, or other m o d i f y i n g e n z y m e s , m a y e x p l a i n the interaction o f L - s e l e c t i n w i t h C D 3 4 f a m i l y m e m b e r s o n l y in H E V .  The  question, then, is what the alternative function is for these s i a l o m u c i n s i n other tissues.  31  1.5.3  Anti-Adhesion  A proposed p o d o c a l y x i n f u n c t i o n that is g a i n i n g considerable support is a contrasting role in b l o c k i n g adhesion. C e l l s regulate adhesion by adjusting levels of a d h e s i o n m o l e c u l e s , such as integrins, as w e l l as anti-adhesins. A n t i - a d h e s i n s are often c e l l surface associated m u c i n s ( W e s s e l i n g et a l . , 1996). M u c i n s are h e a v i l y s i a l y l a t e d , O - l i n k e d  glycoproteins  that can block adhesion either by steric hindrance or by charge r e p u l s i o n , both properties c o n v e y e d by their b u l k y , negatively charged e x t r a c e l l u l a r d o m a i n s . S i n c e p o d o c a l y x i n and the related f a m i l y m e m b e r s have this type o f extracellular d o m a i n , they have been proposed to act in this capacity (Figure 1-6). S i a l o m u c i n s are often f o u n d o n the l u m i n a l surface o f vessels where they prevent b l o c k a g e o f the l u m e n c a u s e d by w e a k , n o n s p e c i f i c interaction o f m o l e c u l e s on o p p o s i n g l u m i n a l membranes ( H i l k e n s et a l . , 1992). S i m i l a r l y , expression by m a l i g n a n t tumours can decrease adhesion and prevent i m m u n e r e c o g n i t i o n . P o d o c a l y x i n e x p r e s s i o n on c i r c u l a t i n g platelets m a y a l s o prevent their inappropriate adhesion to vessel w a l l s ( M c N a g n y et a l . , 1997). T h u s , it seems p l a u s i b l e that the C D 3 4 f a m i l y may have global anti-adhesive functions.  32  ttt  f  podocalyxin  Figure  1-6:  ttt  ,  e  I  (-) charge  integrin  Proposed Function for  CD34  Family: Blocking  ttt  ligand  Adhesion.  CD34 family members can block cell-cell adhesion by charge repulsion or steric hindrance. Adapted from (Nielsen et al., 2002).  33  There are several lines of evidence supporting the role of podocalyxin and C D 3 4 in blocking cell adhesion. Ectopic expression of podocalyxin in Chinese hamster ovary (CHO)  cells completely blocks cell-cell adhesion in aggregation assays (Takeda et al.,  2000). This effect is due to charge repulsion, as treatment with sialidase to remove podocalyxin's negatively charged sialic acid residues abrogates the effect. A similar effect is seen in aggregation assays using podocalyxin-transfected M D C K cells. In addition, podocalyxin expression decreases the strength of tight junctions in M D C K cell monolayers. Localization of junctional proteins is more variable, and transepithelial resistance ( T E R ) decreases slightly. Podocalyxin thus blocks cell-cell adhesion and interferes with cell-cell junctions.  The potential role of signalling in the podocalyxin-related reorganization of cell junctions was recently assessed (Schmieder et al., 2004). Since podocalyxin interacts with ezrin, which is maintained in an open and active conformation in a Rho-dependent manner, this pathway was examined (Chen et al., 1995; Matsui et al., 1999; Schmieder et al., 2004; Shaw et al., 1998). In this pathway, it is thought that RhoA activates ezrin, which in turn binds RhoGDI, a negative regulator of Rho GTPases (Chen et al., 1995; Matsui et al., 1999; Shaw et al., 1998; Takahashi et al., 1997). Sequestration of RhoGDI thereby disrupts the RhoA/RhoGDI complex and permits RhoA activation; this, in turn, maintains ezrin activation. Active RhoA is then able to translocate to the plasma membrane where it interacts with effector proteins to mediate downstream signalling and induce actin reorganization (Schmieder et al., 2004). O f interest, R h o G D I a ' mice exhibit massive  34  proteinuria and disruption of foot process architecture, implying a role for proper regulation of Rho GTPases in podocyte structure (Togawa et al., 1999).  In M D C K cells transfected with full-length podocalyxin or podocalyxin lacking the N H E R F binding site, RhoA activation status and distribution of RhoA, RhoGDI, and actin have all been assessed (Schmieder et al., 2004). In this system, ectopic podocalyxin expression increases RhoA activation in a manner dependent on the interaction of podocalyxin with N H E R F .  Likewise, stimulation of f3 -adrenergic receptors and 2  purinergic receptors, both of which are connected to actin through ezrin and N H E R F 1 , also leads to RhoA activation, suggesting that perhaps N H E R F proteins recruit a molecule responsible for activation of RhoA (Sauzeau et al., 2000; Schmieder et al., 2004; Yamauchi et al., 2001). However, N H E R F is not responsible for redistribution of either RhoA or RhoGDI. In control cells, RhoA is found throughout the cytoplasm and is concentrated in the juxtanuclear region. In contrast, RhoA is partially redistributed toward the plasma membrane in cells ectopically expressing full-length or truncated podocalyxin. Similarly, RhoGDI is relocalized toward the apical membrane. Since activated ezrin binds RhoGDI, this relocalization is likely the result of a direct interaction with ezrin and apically expressed podocalyxin (Schmieder et al., 2004). Importantly, the podocalyxin-dependent activation and relocalization of RhoA, along with the association of RhoA with regulation of the structure and function of tight junctions provide a possible explanation for podocalyxin's effects on junctional proteins (Jou et al., 1998; Schmieder et al., 2004).  35  A n independent group confirmed the effect of podocalyxin overexpression on cell-cell junctions in M D C K cells (Li et al., 2002). Furthermore, they demonstrated that ectopic expression of podocalyxin lacking the C-terminal P D Z binding domain is also sufficient to decrease T E R , albeit to a lesser extent than full-length podocalyxin. It can therefore be concluded that while interaction with N H E R F proteins greatly enhances the weakening of cell-cell junctions, it is not absolutely required for this effect. A similar reorganization of cell-cell junctions is thought to exist in podocalyxin-expressing glomerular podocytes (discussed in section 1.6.3below).  Additionally, immortalized human glomerular epithelial cells ( H G E C ) , which mimic the phenotype of podocytes, have been used to assess podocalyxin function (Economou et al., 2004). When these cells are cultured on laminin or a complex mixture of glomerular basement membrane components, podocalyxin expression is upregulated. Unlike the normal protein expression pattern, however, some podocalyxin is expressed on the basolateral surface of these cells, but this is likely an artifact of the system. Regardless, podocalyxin expression leads to decreases in adhesion to laminin in cell adhesion assays. Thus, in this independent model system, podocalyxin is also able to block cell adhesion.  Moreover, there is some evidence to suggest that C D 3 4 family members interfere with integrin-mediated adhesion. Cell-substrate adhesion in H G E C is mediated by integrins, as addition of saturating concentrations of anti-p , integrin antibodies blocks adhesion. 1  Strikingly, however, when non-saturating concentrations of anti-(3, integrin antibodies are added in combination with increasing concentrations of anti-podocalyxin antibodies, the  36  podocalyxin antibodies increases.  block podocalyxin's anti-adhesive  effects, and adhesion  Similarly, anti-CD34 antibodies trigger integrin-mediated adhesion of  progenitor cells (Majdic et al., 1994). This adhesion has been proposed to be a result of C D 3 4 signalling (Cheng et al., 1996), but capping of C D 3 4 may expose integrins and provide an alternative explanation for the increase in adhesion. Most experiments involving overexpression of C D 3 4 family members therefore suggest an anti-adhesive function for these proteins.  Expression of C D 3 4 by a subset of mature hematopoietic cells (mast cells) and the existence of viable CD34-null animals provides another excellent tool for assessing its function (Cheng et al., 1996; Drew et al., 2002; Drew et al., 2005; Suzuki et al., 1996). Comparison of bone marrow derived mast cells from wildtype and C D 3 4 knockout animals demonstrates that C D 3 4 is necessary and sufficient to prevent cell-cell adhesion in this system (Drew et al., 2005). Wildtype cells form single cell suspensions, whereas cells lacking C D 3 4 form small aggregates. The defect can be reversed by ectopic expression of CD34, and the naturally occurring splice variant is an even more potent anti-adhesin. It is possible that the full-length isoform can be relocalized in the plasma membrane in order to expose adhesion molecules, whereas the isoform lacking much of the cytoplasmic tail may not interact with proteins involved in its membrane localization. A n important observation, however, is that the distantly related sialomucin, C D 4 3 , is a much more effective anti-adhesin: loss of C D 3 4 leads to aggregation of 16 ± 7 % of cells, while 70 ± 20 % of CD43-null cells form aggregates.  37  Further e v i d e n c e f o r the a n t i - a d h e s i v e effects o f p o d o c a l y x i n a n d C D 3 4 have been p r o v i d e d by in vivo r e p o p u l a t i o n studies. In the first set o f e x p e r i m e n t s , s h o r t - t e r m h o m i n g assays were performed w i t h w i l d t y p e , p o d o c a l y x i n - n u l l , C D 3 4 - n u l l , and d o u b l e deficient fetal liver cells ( D o y o n n a s et a l . , 2005). S i n g l e k n o c k o u t cells are less efficient at h o m i n g to bone m a r r o w , and c e l l s l a c k i n g both m o l e c u l e s display an additive defect. A l t h o u g h this c o u l d be e x p l a i n e d by a loss o f s p e c i f i c h o m i n g m o l e c u l e s , there is no decrease i n b i n d i n g to the only k n o w n receptors f o r these m o l e c u l e s , the selectins. T h u s , it is more l i k e l y that the loss o f anti-adhesion m o l e c u l e s results in n o n - s p e c i f i c adhesion to endothelial cells o f vessels en route to the bone m a r r o w .  T h e second experiment i n v o l v e d repopulation o f the peritoneal cavity by mast cells after their a b l a t i o n by injection o f water into w i l d t y p e , C D 3 4 - n u l l , C D 4 3 - n u I I , and d o u b l e d e f i c i e n t m i c e ( D r e w et a l . , 2 0 0 5 ) . D o u b l e - d e f i c i e n t m i c e d i s p l a y d e l a y e d mast c e l l repopulation kinetics. W h i l e i n d i v i d u a l loss o f C D 3 4 also appears to delay repopulation, the results are not statistically s i g n i f i c a n t . In another s y s t e m , w i l d t y p e and C D 3 4 - o r C D 4 3 - d e f i c i e n t bone m a r r o w c e l l s were injected into s u b - l e t h a l l y irradiated mast cell deficient  W / W mice v  in competitive  repopulation  experiments,  a n d mast  cell  reconstitution w a s assessed after 1 1 - 1 2 w e e k s . N o t a b l y , almost a l l mast cells i n m i c e injected w i t h a c o m b i n a t i o n o f w i l d t y p e a n d d o u b l e - d e f i c i e n t c e l l s are d e r i v e d f r o m w i l d t y p e bone marrow c e l l s . A g a i n , although the trend is the same w i t h single k n o c k o u t c e l l s , the results are not statistically significant. S t r i k i n g l y , in the same m i c e , there is a s i g n i f i c a n t decrease i n C D 3 4 - d e f i c i e n t h e m a t o p o i e t i c p r o g e n i t o r  cell  engraftment,  whereas C D 4 3 ' cells engraft n o r m a l l y . A g a i n , loss o f C D 3 4 may lead to increased n o n -  38  specific adhesion to vessels en route to the bone marrow. Thus, in vivo data supports the anti-adhesion hypothesis.  One other interesting observation is that C D 3 4 expression is downregulated upon IL-1 induced upregulation of the adhesion molecules I C A M - 1  and endothelial  leukocyte  adhesion molecule ( E L A M ) - l (Delia et al., 1993). This is further support for the idea that podocalyxin and C D 3 4 may inhibit adhesive functions of vascular endothelial cells, and downregulation (Delia et al., 1993)  or relocalization may enable adhesion  when  necessary. A s discussed, there is therefore mounting evidence supporting an antiadhesive function for CD34 and podocalyxin.  1.5.4  Establishing Polarity  A n alternative and particularly interesting podocalyxin function was recently proposed involving establishing polarity in epithelial cells (Meder et al., 2005). Although many cells polarize transiently, epithelial cells of kidney, intestine, and other organs become terminally polarized upon creation of monolayers. This requires separation of apical and basolateral membrane domains by intracellular sorting or selective retention of membrane components as well as the formation of junctional complexes (Mellman and Warren, 2000; Mostov et al., 2003). In this study, it was hypothesized that epithelial polarization begins with establishment of an apical pole in single cells (Meder et al., 2005).  In order to assess polarization of M D C K cells, individual cells were plated and stained for cell surface markers shortly thereafter, or after four hours upon formation of small  39  clusters o f c e l l s ( M e d e r et a l . , 2 0 0 5 ) . A l t h o u g h l o c a l i z a t i o n o f n u m e r o u s a p i c a l and basolateral markers was assessed, a l l proteins d i s p l a y u n i f o r m expression patterns w i t h the exception of E - c a d h e r i n , w h i c h is enriched at c e l l - c e l l contact sites, and g p l 3 5 , w h i c h is a p i c a l l y l o c a l i z e d on s i n g l e c e l l s w i t h i n one hour. A t the t i m e , the identity o f g p l 3 5 was u n k n o w n , but its unique expression e x c l u s i v e l y on the free surface of cells prompted further s t u d y ; as d i s c u s s e d in section 1.2, it is n o w k n o w n to be c a n i n e p o d o c a l y x i n ( M e d e r et a l . , 2005). T h e early polarized distribution of p o d o c a l y x i n , in contrast to other t y p i c a l m a r k e r s , suggests that it m a y play a role in this process. T h i s was investigated further by d e p l e t i n g endogenous p o d o c a l y x i n e x p r e s s i o n by R N A interference ( R N A i ) . P o l a r i z a t i o n , as assessed by m a r k e r d i s t r i b u t i o n on M D C K m o n o l a y e r s , was d e l a y e d in c e l l s l a c k i n g p o d o c a l y x i n ( M e d e r et a l . , 2005).  A m o d e l often used to assess p o l a r i z a t i o n i n M D C K c e l l s is that o f cyst f o r m a t i o n i n a c o l l a g e n m a t r i x . In this assay, single cells g r o w and f o r m m u l t i c e l l u l a r , p o l a r i z e d cysts each w i t h a central l u m e n . S t r i k i n g l y , m a n y p o d o c a l y x i n - d e p l e t e d M D C K c e l l s f o r m cysts w i t h m u l t i p l e l u m e n s , or cysts c o m p l e t e l y d e v o i d o f l u m e n s ( M e d e r et a l . , 2 0 0 5 ) . A l t h o u g h the 2005 publication by M e d e r et al ( M e d e r et a l . , 2005) was the first to suggest a role for p o d o c a l y x i n in establishing polarity, apical expression of g p l 35 (later identified as p o d o c a l y x i n ) was noted in M D C K c e l l s l a c k i n g c e l l j u n c t i o n s in a paper p u b l i s h e d o v e r a decade earlier ( O j a k i a n and S c h w i m m e r , 1988). In a d d i t i o n , r e m o v a l o f surface expressed g p l 3 5 by t r y p s i n i z a t i o n is f o l l o w e d by rapid reinsertion o f p o d o c a l y x i n into the p l a s m a m e m b r a n e , but o n l y at the a p i c a l surface ( O j a k i a n et a l . , 1990). T h u s , recent  40  results suggest the e x c i t i n g p o s s i b i l i t y that p o d o c a l y x i n in i n v o l v e d i n r e g u l a t i n g c e l l polarity.  The  s a m e g r o u p a d d r e s s e d the m e c h a n i s m i n v o l v e d  in p o d o c a l y x i n - i n d u c e d  cell  p o l a r i z a t i o n ( M e d e r et a l . , 2 0 0 5 ) . S i n c e mutant p o d o c a l y x i n l a c k i n g the C - t e r m i n a l D T H L sequence d i s p l a y s s l i g h t l y less restricted l o c a l i z a t i o n , it w a s h y p o t h e s i z e d that N H E R F proteins c o u l d also play a role in early c e l l p o l a r i z a t i o n . M D C K c e l l s were transfected w i t h a green f l u o r e s c e n t protein ( G F P ) - t a g g e d N H E R F 2 f u s i o n protein i n order to f o l l o w N H E R F 2 l o c a l i z a t i o n d u r i n g M D C K c e l l p o l a r i z a t i o n . L o c a l i z a t i o n o f this protein is s i m i l a r to that of endogenous p o d o c a l y x i n throughout a l l stages o f early p o l a r i z a t i o n , and the two proteins f o r m a c o - i m m u n o p r e c i p i t a b l e c o m p l e x that appears to strengthen as cells b e c o m e more polarized. S i n c e N H E R F proteins contain tandem P D Z d o m a i n s w i t h d i f f e r i n g b i n d i n g s p e c i f i c i t i e s , they are able to d i m e r i z e and c r o s s - l i n k m u l t i p l e ligands ( S h e n o l i k a r et a l . , 2004). In the case o f e p i t h e l i a l p o l a r i z a t i o n , perhaps N H E R F proteins can l i n k m u l t i p l e ligands w i t h the E R M f a m i l y of cytoskeletal adapters i n order to establish a p r e - a p i c a l membrane s c a f f o l d f o r d i r e c t i n g j u n c t i o n f o r m a t i o n and m e m b r a n e t r a f f i c k i n g ( M e d e r et a l . , 2005).  1.5.5  Podocalyxin and NHERF: A Role in Asymmetric Cell Division?  A d m i t t e d l y the potential role o f p o d o c a l y x i n in a s y m m e t r i c c e l l d i v i s i o n is based on very little e v i d e n c e , but it is an e x c i t i n g p o s s i b i l i t y w o r t h c o n s i d e r i n g . A s y m m e t r i c c e l l d i v i s i o n has recently gained m u c h attention in the h e m a t o p o i e t i c s y s t e m , as stem c e l l s must d i v i d e in this manner in order to produce daughter c e l l s w i t h d i f f e r i n g properties.  41  M a i n t e n a n c e o f the stem c e l l p o o l requires that one daughter c e l l r e m a i n a s t e m c e l l , w h i l e the other begins to populate the h e m a t o p o i e t i c s y s t e m by virtue o f its increased progress a l o n g the differentiation pathway ( r e v i e w e d i n ( H o , 2 0 0 5 ; W i l s o n and T r u m p p , 2006)).  T h e r e are t w o m o d e l s that address the m e c h a n i s m s u n d e r l y i n g a s y m m e t r i c d i v i s i o n . T h e first suggests that external s t i m u l i f o l l o w i n g c e l l d i v i s i o n provide cues that determine c e l l fate. T h i s is based o n the idea o f a stem c e l l niche and d i v i s i o n a l o n g a plane i n w h i c h one daughter c e l l remains in contact w i t h the n i c h e , and therefore remains an H S C w h i l e the other d o e s not. T h e  s e c o n d m o d e l suggests that m o l e c u l e s w i t h i n a c e l l are  r e d i s t r i b u t e d u n e q u a l l y before d i v i s i o n , s u c h that those r e q u i r e d f o r m a i n t a i n i n g an i m m a t u r e c e l l are o n one side w h i l e determinants o f a more mature c e l l are o n the opposite side. T h e expression of p o d o c a l y x i n i n early hematopoietic progenitor c e l l s , but perhaps not i n the most i m m a t u r e h e m a t o p o i e t i c stem c e l l s , and its a s s o c i a t i o n w i t h the actin c y t o s k e l e t o n and the free surface of c e l l s suggests that it c o u l d p l a y a role i n either situation ( F i g u r e 1-7). In the first case, p o d o c a l y x i n ' s association w i t h the free surface o f c e l l s m a y e n a b l e an i m m a t u r e H S C to adhere to the n i c h e v i a a d h e s i o n m o l e c u l e s segregated  from  the  podocalyxin-containing  domain. Then,  after d i v i s i o n ,  the  p o d o c a l y x i n - c o n t a i n i n g c e l l w i l l be destined to b e c o m e more mature, w h i l e the adherent c e l l w i l l r e m a i n i n contact w i t h the n i c h e , thereby r e c e i v i n g the s i g n a l s required f o r m a i n t a i n i n g the i m m a t u r e state. O n the other h a n d , perhaps p o d o c a l y x i n ' s interaction w i t h N H E R F 1 facilitates segregation o f a multitude of fate d e t e r m i n i n g proteins o n one side o f the c e l l before c e l l d i v i s i o n . T h e fact that p o d o c a l y x i n and N H E R F 1 c o l o c a l i z e to  42  one side o f c e l l s upon I L - 3 s t i m u l a t i o n o f a hematopoietic progenitor c e l l line provides some evidence for r e l o c a l i z a t i o n prior to c e l l d i v i s i o n (Tan et a l . , 2006).  43  Contact with niche AFTER division retains immature cell state  B  Podocalyxin and NHERFI-dependent relocalization of cell fate determinants BEFORE division  f Podocalyxin  VT Adhesion molecules  Niche-derived factors  NHERF1  Cell-fate determinants  F i g u r e 1-7: T w o M o d e l s of P o d o c a l y x i n - D e p e n d e n t A s y m m e t r i c C e l l D i v i s i o n .  (A) Podocalyxin is segregated from adhesion molecules, which allows the HSC to adhere to the niche. After division, the cell remaining in contact with the niche receives signals to remain immature, while the podocalyxin-containing cell becomes more mature. (B) Prior to division, cell-fate determinants are relocalized to one side of the cell through interactions with N H E R F 1 and podocalyxin. Thus, after division, the cell containing podocalyxin is more mature. Lighter blue cells are less mature.  44  P o d o c a l y x i n , C D 3 4 , and e n d o g l y c a n are related s i a l o m u c i n s w i t h o v e r l a p p i n g expression patterns and l i k e l y o v e r l a p p i n g f u n c t i o n s . A l t h o u g h a role i n e n h a n c i n g p r o l i f e r a t i o n or b l o c k i n g d i f f e r e n t i a t i o n was i n i t i a l l y p r o p o s e d , there is m o r e e v i d e n c e in support o f alternative f u n c t i o n s . T h e y c a n all act as adhesive tethers f o r L - s e l e c t i n in H E V but are not appropriately m o d i f i e d f o r this f u n c t i o n i n other tissues. G l o b a l l y , it is more l i k e l y that p o d o c a l y x i n and C D 3 4 , at least, act as b l o c k e r s of a d h e s i o n . In a d d i t i o n , there is recent e v i d e n c e to suggest that p o d o c a l y x i n m a y regulate c e l l p o l a r i t y , and it is possible that the C D 3 4 f a m i l y c o u l d also be i n v o l v e d in regulation o f a s y m m e t r i c c e l l d i v i s i o n . T h e s e f u n c t i o n s a l l rely on a l i n k to the actin c y t o s k e l e t o n , w h i c h , in the case o f p o d o c a l y x i n , has been e s t a b l i s h e d to o c c u r v i a i n t e r a c t i o n s w i t h e z r i n and  NHERF  proteins.  1.6 Podocalyxin in Kidney Development W h i l e m u c h i n f o r m a t i o n can be gained through o v e r e x p r e s s i o n e x p e r i m e n t s and in vitro a n a l y s i s , it is a l s o n e c e s s a r y to i n v e s t i g a t e f u n c t i o n a l i m p l i c a t i o n s o f a p r o t e i n ' s expression in tissues where it is n o r m a l l y f o u n d . In the case o f p o d o c a l y x i n , it was first i d e n t i f i e d i n the g l o m e r u l u s o f rat k i d n e y , and its e x p r e s s i o n i n this tissue is not o n l y abundant, but also essential f o r podocyte d e v e l o p m e n t ( D o y o n n a s et a l . , 2 0 0 1 ; K e r j a s c h k i et a l . , 1984).  45  1.6.1  Overview of Glomerular Development  G l o m e r u l i are responsible f o r b l o o d f i l t r a t i o n and urine p r o d u c t i o n in the kidney ( G a o et a l . , 2 0 0 4 ) . T h e y are c o m p o s e d o f c a p i l l a r y loops l i n e d with fenestrated endothelial c e l l s , s u p p o r t i n g m e s a n g i a l c e l l s , a g l o m e r u l a r basement m e m b r a n e ( G B M ) , and g l o m e r u l a r e p i t h e l i a l c e l l s c a l l e d p o d o c y t e s ( G a o et a l . , 2 0 0 4 ) . W h i l e v a s c u l a r e n d o t h e l i a l c e l l s p r o v i d e the first barrier i n p r o d u c t i o n o f the g l o m e r u l a r f i l t r a t e , e n d o t h e l i a l fenestrae p r o v i d e an exit route f o r s m a l l m o l e c u l e s ( G e l b e r g et a l . , 1996). T h e G B M consists o f a network  of collagen IV,  l a m i n i n , heparan sulfate proteoglycans,  and  fibronectin  ( E c o n o m o u et a l . , 2 0 0 4 ) ; its s i z e - and c h a r g e - s e l e c t i v i t y p r o v i d e the m a i n s e l e c t i v e barrier e n a b l i n g retention of m a c r o m o l e c u l e s in the c i r c u l a t i o n ( r e v i e w e d in (Farquhar, 1975; K a n w a r , 1984)).  P o d o c y t e s have a unique architecture w i t h three m a i n parts. T h e c e l l body contains the majority o f the c e l l ' s c y t o p l a s m i c organelles, i n c l u d i n g the nucleus, m i t o c h o n d r i a , rough and s m o o t h e n d o p l a s m i c r e t i c u l u m , and w e l l - d e v e l o p e d G o l g i , w h i c h are l i k e l y to be i n v o l v e d i n f o r m a t i o n a n d d e g r a d a t i o n o f the G B M  components (Takeda,  2003).  Projections, termed major processes, extend f r o m the c e l l body and surround the c a p i l l a r y loops ( A n d r e w s , 1979). S m a l l e r projections, c a l l e d foot processes, extend f r o m the m a j o r processes, interdigitate w i t h foot processes f r o m n e i g h b o u r i n g podocytes, and connect the basal surface of p o d o c y t e s to the G B M , m a i n l y t h r o u g h a (3, i n t e g r i n - m e d i a t e d f o c a l 3  contacts ( A d l e r , 1 9 9 2 ; A n d r e w s , 1979). F o o t processes l a c k c y t o p l a s m i c organelles, and instead c o n t a i n a dense n e t w o r k o f actin f i l a m e n t s , w h i c h s t a b i l i z e the f o o t process structure through adhesion to the G B M and by f o r m i n g c o n n e c t i o n s w i t h proteins of the  46  slit d i a p h r a g m c o m p l e x ( F i g u r e 1-8) ( K o b a y a s h i et a l . , 2 0 0 4 ; T a k e d a , 2 0 0 3 ) . S l i t d i a p h r a g m s are t e n u o u s b r i d g e s between adjacent f o o t processes t h r o u g h w h i c h the g l o m e r u l a r filtrate must pass ( K u r i h a r a et a l . , 1992). Recent evidence suggests that, a l o n g w i t h the G B M , slit d i a p h r a g m s also play a role in f i l t r a t i o n ( r e v i e w e d i n (Salant and T o p h a m , 2 0 0 3 ; T r y g g v a s o n , 1999)). T h e a p i c a l surface o f p o d o c y t e s , w h i c h faces the u r i n a r y s p a c e , is c o a t e d by a s i a l i c a c i d - r i c h g l y c o c a l y x  d e s i g n a t e d the e p i t h e l i a l  p o l y a n i o n , and n o w k n o w n to be m a i n l y c o m p o s e d o f p o d o c a l y x i n ( K e r j a s c h k i et a l . , 1984; M i c h a e l et a l . , 1970).  47  GBM  F i g u r e 1-8: Schematic of Molecules Related to Podocyte Architecture. T h i s d i a g r a m s h o w s t w o adjacent p o d o c y t e f o o t processes b r i d g e d b y a c o m p l e x o f proteins that f o r m the slit d i a p h r a g m . P r o t e i n s f o u n d i n the basal d o m a i n , s u c h as a p , 3  i n t e g r i n ( a p , ) , i n t e r a c t w i t h the a c t i n c y t o s k e l e t o n a n d l i n k p o d o c y t e s 3  glomerular  basement  membrane  (GBM).  Podocalyxin  interacts  with  with the  the actin  c y t o s k e l e t o n t h r o u g h N H E R F 2 and e z r i n , and m o l e c u l e s o f the slit d i a p h r a g m c o m p l e x a l s o interact w i t h the c y t o s k e l e t o n . O t h e r a b b r e v i a t i o n s i n c l u d e c t - a c t 4 : a - a c t i n i n - 4 , a D G : a-dystroglycan,  |3-DG: |3-dystroglycan,  P: p a x i l l i n , P - c a d : P - c a d h e r i n ,  Synpo:  s y n a p t o p o d i n , T : t a l i n , and V : v i n c u l i n . R e p r o d u c e d w i t h k i n d p e r m i s s i o n o f L i p p i n c o t t W i l l i a m s & W i l k i n s ( M u n d e l and S h a n k l a n d , 2 0 0 2 ) .  48  D u r i n g g l o m e r u l a r d e v e l o p m e n t , w h i c h proceeds v i a a series o f l o o s e l y d e f i n e d stages, m e s e n c h y m a l c e l l s are i n d u c e d to c o n d e n s e and u n d e r g o m e s e n c h y m a l to e p i t h e l i a l transition ( G u o et a l . , 2 0 0 2 ) . T h e y i n i t i a l l y f o r m a cluster of c e l l s k n o w n as the renal v e s i c l e ( G u o et a l . , 2 0 0 2 ; R e e v e s et a l . , 1978). T h e renal v e s i c l e is i n v a g i n a t e d  by  m e s e n c h y m a l c e l l s , and the cluster o f cells present at this stage rearranges and matures v i a the c o m m a - and S - s h a p e d body stages to f o r m a central l u m e n , w h i c h later becomes B o w m a n ' s space ( G u o et a l . , 2 0 0 2 ; Reeves et a l . , 1978). D u r i n g the S - s h a p e d body stage, m e s a n g i a l c e l l s d e v e l o p f r o m the m e s e n c h y m a l cleft, w h i l e the c e l l s o n one side o f the cleft begin to f o r m the g l o m e r u l a r e p i t h e l i u m and those on the other side differentiate to f o r m the p r o x i m a l tubule and capillary e n d o t h e l i u m (Reeves et a l . , 1978). T h e g l o m e r u l a r e p i t h e l i a l c e l l s i n i t i a l l y have tight j u n c t i o n s at their a p i c e s , but upon e x p r e s s i o n  of  p o d o c a l y x i n the j u n c t i o n s begin to migrate laterally toward the G B M .  T h r o u g h o u t the next stage, k n o w n as the d e v e l o p i n g c a p i l l a r y l o o p stage, g l o m e r u l a r e p i t h e l i a l c e l l s (podocytes) proliferate, and m o v e m e n t o f the c e l l - c e l l j u n c t i o n s enlarges the i n t r a c e l l u l a r spaces, w h i c h are c o n t i n u o u s w i t h B o w m a n ' s space ( R e e v e s et a l . , 1978). E x t e n s i v e m o r p h o l o g i c a l rearrangements occur whereby foot processes extend and j u n c t i o n s r e d i s t r i b u t e b e t w e e n f o o t processes. S l i t d i a p h r a g m s later r e p l a c e these j u n c t i o n s (Reeves et a l . , 1 9 7 8 ; Schnabel et a l . , 1989). P o d o c a l y x i n redistribution f o l l o w s that o f the j u n c t i o n s : it is a l w a y s f o u n d a l o n g the apical surface of p o d o c y t e c e l l bodies and f o o t processes a b o v e the l e v e l o f the slit d i a p h r a g m s ( K e r j a s c h k i et a l . , 1 9 8 4 ; K u r i h a r a et a l . , 1 9 9 2 ; S c h n a b e l et a l . , 1989). T h e m a t u r i n g g l o m e r u l u s stage i n v o l v e s further m a t u r a t i o n o f p o d o c y t e s , maturation of the G B M i n t o . a c o m p l e x structure with  49  several layers, and fenestration o f endothelial c e l l s to f o r m the f i n a l g l o m e r u l a r structure (Reeves e t a l . , 1978).  1.6.2  Glomerular Diseases and Model Systems  M a i n t e n a n c e o f the intricate architecture o f g l o m e r u l a r podocytes is essential f o r optimal kidney f u n c t i o n ; numerous h u m a n diseases and a n i m a l m o d e l s o f g l o m e r u l a r m a l f u n c t i o n i n v o l v e d i s r u p t i o n o f these u n i q u e structures ( E c o n o m o u et a l . , 2 0 0 4 ) . L o s s o f f o o t process architecture is the m a i n m o r p h o l o g i c a b n o r m a l i t y detected in patients w i t h nephrotic s y n d r o m e ( F a r q u h a r et a l . , 1957). D i a b e t i c n e p h r o p a t h y , a l e a d i n g cause o f c h r o n i c k i d n e y f a i l u r e and end-stage renal d i s e a s e , also i n v o l v e s b r o a d e n i n g o f f o o t processes and is a c c o m p a n i e d by a decrease in g l o m e r u l a r s i a l i c a c i d content (Cardenas e t a l . , 1 9 9 1 ; Pagtalunan et a l . , 1997).  P u r o m y c i n a m i n o n u c l e o s i d e ( P A N ) nephrosis and p r o t a m i n e sulfate ( P S ) perfusion are t w o rodent m o d e l s o f g l o m e r u l a r disease ( F a r q u h a r and P a l a d e , 1 9 6 1 ; K u r i h a r a et a l . , 1992). S i a l y l a t i o n o f p o d o c a l y x i n is r e d u c e d i n P A N n e p h r o s i s , and P S neutralizes p o d o c a l y x i n ' s negative charge ( K e r j a s c h k i et a l . , 1 9 8 5 ; S e i l e r et a l . , 1977). T h u s , in both m o d e l s , the negative charge o n p o d o c a l y x i n is d e c r e a s e d , f o o t process architecture is d i s r u p t e d , f i l t r a t i o n slits are r e d u c e d i n n u m b e r , and slit d i a p h r a g m s are d i s p l a c e d or c o m p l e t e l y r e p l a c e d by l e a k y , d i s c o n t i n u o u s tight j u n c t i o n s ; o v e r a l l , the e p i t h e l i u m resembles that o f immature g l o m e r u l i ( G a o et a l . , 2 0 0 4 ; K u r i h a r a et a l . , 1992). Injection o f sialidase intraperitoneally causes s i m i l a r m o r p h o l o g i c a l alterations as a result of loss of sialic a c i d residues f r o m the surface of podocytes ( G e l b e r g et a l . , 1996). Features of these  50  m o d e l s i n c l u d e decreased g l o m e r u l a r filtration and a perhaps counterintuitive increase in protein in the urine (proteinuria) ( B o h r e r et a l . , 1 9 7 7 ; K u r i h a r a et a l . , 1992). R e d u c t i o n i n g l o m e r u l a r charge selectivity results in an increase in G B M p e r m e a b i l i t y and subsequent proteinuria. H o w e v e r , there is an overall d e c l i n e in g l o m e r u l a r f i l t r a t i o n as the c o l l e c t i v e slit pore a r e a , a n d thereby the e x i t path f o r f i l t r a t e , decreases ( B o h r e r et a l . , 1 9 7 7 ; K u r i h a r a et a l . , 1992). B l o c k a g e of the exit path m a y also lead to a reactive e l e v a t i o n i n g l o m e r u l a r b l o o d pressure and a subsequent d r a m a t i c increase in p e r m e a b i l i t y o f the glomerular filter (Gelberg  et a l . , 1996). P o d o c y t e s , their e x t e n s i v e e x p r e s s i o n  of  p o d o c a l y x i n , and alterations in disease states have therefore been the f o c u s o f m u c h research f o r several decades.  1.6.3  Early Studies of Podocalyxin  A l t h o u g h p o d o c a l y x i n was not i d e n t i f i e d until 1984, the i m p o r t a n c e o f the " e p i t h e l i a l p o l y a n i o n " i n m a i n t e n a n c e o f p o d o c y t e m o r p h o l o g y w a s noted m o r e than ten years earlier ( K e r j a s c h k i et a l . , 1984; M i c h a e l et a l . , 1970). A reduction i n foot process n u m b e r in a s s o c i a t i o n w i t h loss of the epithelial p o l y a n i o n was first proposed i n 1970 ( M i c h a e l et a l . , 1970). T h i s w a s c o n f i r m e d by experiments i n w h i c h rat k i d n e y s were perfused w i t h p o l y c a t i o n s to neutralize the e p i t h e l i a l p o l y a n i o n . T h i s leads to d i s t o r t i o n o f p o d o c y t e m o r p h o l o g y : s l i t pores are n a r r o w e r or absent and f o o t processes are f u s e d together ( S e i l e r et a l . , 1977). In contrast, perfusion w i t h p o l y a n i o n s and neutral m o l e c u l e s has n o effect, a l t h o u g h p e r f u s i o n of a n i o n i c m o l e c u l e s after p e r f u s i o n o f c a t i o n i c m o l e c u l e s partially reverses their disruptive effects. T h e phenotypes observed upon cation perfusion are analogous to those observed in proteinuric c o n d i t i o n s , where podocyte m o r p h o l o g y is  51  s i m i l a r to that o b s e r v e d i n i m m a t u r e g l o m e r u l i ( S e i l e r et a l . , 1977). T h u s , it is not s u r p r i s i n g that appearance of the epithelial p o l y a n i o n is associated w i t h extension o f foot processes d u r i n g d e v e l o p m e n t : it was d e m o n s t r a t e d in 1978 by R e e v e s et al that the epithelial p o l y a n i o n , as detected by c o l l o i d a l iron staining, first appears on the surface o f g l o m e r u l a r epithelial cells just before f o r m a t i o n of foot processes and slit pores (Reeves e t a l . , 1978).  A m a j o r contributor to the negative charge on the epithelial p o l y a n i o n is its h i g h s i a l i c a c i d content ( M i c h a e l et a l . , 1 9 7 0 ; M o h o s and S k o z a , 1969). T h i s was demonstrated by the o b s e r v e d r e d u c t i o n in c o l l o i d a l i r o n s t a i n i n g o f p o d o c y t e s after treatment w i t h n e u r a m i n i d a s e , w h i c h r e m o v e s s i a l i c a c i d residues. S i m i l a r to the effects seen w i t h p o l y c a t i o n p e r f u s i o n , n e u r a m i n i d a s e treatment also disrupts f o o t process architecture (Andrews,  1979). F o o t processes fuse together, s l i t d i a p h r a g m s are d i s p l a c e d , and  j u n c t i o n a l c o m p l e x e s , lost d u r i n g d e v e l o p m e n t , reappear. F u r t h e r m o r e , it has been suggested that the defects o b s e r v e d i n P A N m o d e l s o f n e p h r o s i s m a y be c a u s e d by interference w i t h s i a l i c a c i d m e t a b o l i s m i n p o d o c y t e s and a c o r r e s p o n d i n g loss o f the sialic a c i d content in the g l y c o c a l y x .  It was at this stage that the m o l e c u l a r identity o f the e p i t h e l i a l p o l y a n i o n was f i n a l l y d e t e r m i n e d ( K e r j a s c h k i et a l . , 1984). S e v e r a l characteristics o f the e p i t h e l i a l p o l y a n i o n had p r e v i o u s l y been d e m o n s t r a t e d , and these features, a l o n g w i t h it b e i n g the most abundant m e m b r a n e g l y c o p r o t e i n i n the g l o m e r u l u s , enabled its i s o l a t i o n . F i r s t l y , it is h i g h l y negatively charged, as demonstrated by its r e c o g n i t i o n by c a t i o n i c h i s t o c h e m i c a l  52  stains ( M i c h a e l et a l . , 1970; M o h o s and S k o z a , 1969). S e c o n d l y , neuraminidase treatment alters its s t a i n i n g properties, thereby d e m o n s t r a t i n g its exten si v e s i a l i c a c i d content. T h i r d l y , it is r e c o g n i z e d by wheat germ a g g l u t i n i n before neuraminidase treatment and peanut a g g lut in in afterwards ( H o l t h o f e r et a l . , 1981). U s i n g these characteristics, a single 140 k D a p r o t e i n , c o m p o s e d o f a p p r o x i m a t e l y 2 0 % hexose and 4 . 5 % s i a l i c a c i d by w e i g h t , was i s o l a t e d , c h a r a c t e r i z e d , and n a m e d p o d o c a l y x i n ( K e r j a s c h k i et a l . , 1984). T h e s i a l i c a c i d rich nature o f p o d o c a l y x i n can be f u l l y appreciated w h e n it is realized that it contains the majority o f the p r o t e i n - b o u n d s i a l i c acid o f the g l o m e r u l u s . T h e presence o f O - l i n k e d g l y c o s y l a t i o n s was i m p l i e d based on the b i n d i n g o f peanut l e c t i n after n e u r a m i n i d a s e treatment; the s i m u l t a n e o u s presence o f some N - l i n k e d o l i g o s a c c h a r i d e chains was also suggested by the b i n d i n g o f c o n c a n a v a l i n A . C o n s i d e r a b l e a n a l y s i s has been undertaken to begin to characterize the o l i g o s a c c h a r i d e c h a i n s of  podocalyxin's  m u c i n - l i k e d o m a i n , i n c l u d i n g their sialylations and sulfations ( D e k a n et a l . , 1991). M u c i n d o m a i n s are p r e d i c t e d to be r i g i d , extended structures, so p r e s u m a b l y  podocalyxin's  e x t r a c e l l u l a r d o m a i n f u n c t i o n s as an attachment site f o r the m a n y n e g a t i v e l y  charged  moieties that contribute to the a n i o n i c g l y c o c a l y x of podocytes ( ( K e r s h a w et a l . , 1997a), and reviewed  i n ( J e n t o f t , 1990)). T h u s , 15 years after d e t e c t i o n o f the e p i t h e l i a l  p o l y a n i o n , its m o l e c u l a r nature was determined.  T h e i d e n t i f i c a t i o n o f p o d o c a l y x i n and generation o f s p e c i f i c m o n o c l o n a l a n t i b o d i e s f a c i l i t a t e d a d e t a i l e d assessment of its e x p r e s s i o n pattern ( K e r j a s c h k i et a l . , 1984). In adult, p o d o c a l y x i n is h i g h l y expressed on the apical surface of podocytes and at 5 - 1 0 f o l d l o w e r levels on endothelial cells ( D e k a n et a l . , 1 9 9 0 ; K e r j a s c h k i et a l . , 1984). It covers all  53  surfaces o f podocytes f a c i n g the urinary space, i n c l u d i n g the a p i c a l surface o f c e l l bodies and f o o t processes a b o v e the l e v e l o f slit d i a p h r a g m s ; it is not expressed on the basal surface o f podocytes or on the a b l u m i n a l face of endothelial cells ( K e r j a s c h k i et a l . , 1984; S a w a d a et a l . , 1986). T h e s e d e t a i l e d s t u d i e s a l s o p r o v i d e d  some insights  into  p o d o c a l y x i n ' s f u n c t i o n . It had previously been proposed that the purpose o f its p r o m i n e n t n e g a t i v e charge w a s to p r o v i d e a c h a r g e - s e l e c t i v e barrier f o r g l o m e r u l a r f i l t r a t i o n . H o w e v e r , the m a i n barrier p e r f o r m i n g this f u n c t i o n is located at the l e v e l o f the G B M , where p o d o c a l y x i n e x p r e s s i o n is l a c k i n g , so it is instead postulated to be i n v o l v e d i n m a i n t e n a n c e o f structural integrity of foot processes (Farquhar, 1 9 7 5 ; K e r j a s c h k i et a l . , 1984; S a w a d a e t a l . , 1986).  P o d o c a l y x i n e x p r e s s i o n and l o c a l i z a t i o n d u r i n g d e v e l o p m e n t has also been f o l l o w e d c a r e f u l l y in rat ( S c h n a b e l et a l . , 1989). R o d e n t k i d n e y s p r o v i d e an e x c e l l e n t m o d e l f o r s t u d y i n g k i d n e y d e v e l o p m e n t f o r t w o reasons. F i r s t , throughout d e v e l o p m e n t new renal vesicles are c o n t i n u a l l y f o r m e d at the periphery o f the k i d n e y cortex, w i t h more mature nephrons near the c o r t i c a l - m e d u l l a r y j u n c t i o n , so all stages of maturation are v i s i b l e in a s i n g l e k i d n e y . A n d s e c o n d , this process is not c o m p l e t e d until one w e e k after birth in rodents, i n contrast to the situation i n h u m a n s , so g l o m e r u l a r d e v e l o p m e n t can easily be investigated i n n e w b o r n rodents (Reeves et a l . , 1978). T h e earliest detectable p o d o c a l y x i n is e x p r e s s e d on c a p i l l a r y e n d o t h e l i a l c e l l s i n the m e s e n c h y m a l tissue s u r r o u n d i n g the renal v e s i c l e ( S c h n a b e l et a l . , 1989). S u b s e q u e n t l y , the first p o d o c a l y x i n e x p r e s s i o n on c e l l s that e v e n t u a l l y b e c o m e p o d o c y t e s is detected as the p r e - B o w m a n ' s space l u m e n f o r m s . P o d o c a l y x i n is i n i t i a l l y l o c a l i z e d to the a p i c a l s u r f a c e o f these g l o m e r u l a r  54  epithelial c e l l s , but it then m o v e s a l o n g the lateral surface t o w a r d the basal surface as c e l l - c e l l j u n c t i o n s migrate in a s i m i l a r f a s h i o n . P o d o c a l y x i n is a l w a y s expressed on the entire apical surface and a l o n g the lateral m e m b r a n e to the p o s i t i o n i m m e d i a t e l y above tight j u n c t i o n s . Its e x p r e s s i o n extends a l o n g the tops o f f o o t processes, but it is never expressed on the basal surface f a c i n g the G B M . U p o n replacement o f tight j u n c t i o n s w i t h slit d i a p h r a g m s , p o d o c a l y x i n e x p r e s s i o n is m a i n t a i n e d o n the entire p o d o c y t e surface a b o v e this l e v e l . M o r e o v e r , in i m m a t u r e p o d o c y t e s p o d o c a l y x i n is detected i n the e n d o p l a s m i c r e t i c u l u m , G o l g i apparatus, and carrier vesicles o f the biosynthesis pathway demonstrating that it is synthesized at a h i g h rate in these cells.  S i n c e n e u t r a l i z a t i o n o f the e p i t h e l i a l p o l y a n i o n ( p o d o c a l y x i n ) leads to alterations in p o d o c y t e m o r p h o l o g y , d i s e a s e m o d e l s w e r e used to g a i n f u r t h e r i n s i g h t s into the m e c h a n i s m s i n v o l v e d . F o r e x a m p l e , in P A N n e p h r o s i s , actin f i l a m e n t r e o r g a n i z a t i o n occurs ( W h i t e s i d e et a l . , 1993); since p o d o c a l y x i n is l i n k e d to actin through N H E R F 2 and ezrin in podocytes ( L i et a l . , 2 0 0 2 ; O r l a n d o et a l . , 2 0 0 1 ; T a k e d a et a l . , 2 0 0 1 ) , c o i m m u n o p r e c i p i t a t i o n and sequential detergent extraction experiments were p e r f o r m e d in order to investigate potential disruptions o f this c o m p l e x i n disease states ( T a k e d a et a l . , 2001).  In  PAN  nephrosis,  there  is  little,  if  any,  disruption  of  the  p o d o c a l y x i n / N H E R F 2 / e z r i n c o m p l e x , but the o v e r a l l a m o u n t o f p h o s p h o r y l a t e d (active) e z r i n decreases and the entire c o m p l e x d i s s o c i a t e s f r o m the actin c y t o s k e l e t o n . U p o n protamine sulfate p e r f u s i o n , N H E R F 2 and e z r i n r e m a i n b o u n d to a c t i n , but p o d o c a l y x i n dissociates f r o m the c o m p l e x . S i a l i d a s e treatment leads to d i s s o c i a t i o n o f p o d o c a l y x i n f r o m the N H E R F 2 / e z r i n c o m p l e x , w h i c h also dissociates f r o m a c t i n . T h u s , i n a l l three  55  m o d e l s , the association o f p o d o c a l y x i n w i t h the actin cytoskeleton is disrupted, p r o v i d i n g a p o s s i b l e e x p l a n a t i o n f o r the m o r p h o l o g i c a l changes noted in podocyte foot processes. H o w exactly an alteration in the surface charge o f p o d o c a l y x i n affects the c o m p l e x is not k n o w n , but it m a y be that neutralization of the charge induces a c o n f o r m a t i o n a l change in the c y t o p l a s m i c tail o f p o d o c a l y x i n w h i c h prevents b i n d i n g . A l l of the early studies of the e p i t h e l i a l p o l y a n i o n and p o d o c a l y x i n in the k i d n e y suggest a very important role f o r this m o l e c u l e in k i d n e y d e v e l o p m e n t . G e n e r a t i o n o f p o d o c a l y x i n - n u l l m i c e d e m o n s t r a t e d exactly how important this m o l e c u l e is for glomerular development.  1.6.4  Lessons from the Podocalyxin Knockout  P o d o c a l y x i n k n o c k o u t m i c e were generated by h o m o l o g o u s r e c o m b i n a t i o n in w h i c h the m a j o r i t y o f e x o n s f i v e , s i x , s e v e n , and eight were d e l e t e d ; loss o f e x p r e s s i o n w a s c o n f i r m e d by N o r t h e r n blot ( D o y o n n a s et a l . , 2 0 0 1 ) . S i n c e C D 3 4 - n u l l m i c e d o not have any major defects i n tissues that n o r m a l l y c o - e x p r e s s p o d o c a l y x i n and C D 3 4 ( C h e n g et a l . , 1 9 9 6 ; S u z u k i et a l . , 1996), a b n o r m a l i t i e s were not e x p e c t e d i n these tissues i n p o d o c a l y x i n - n u l l m i c e either. Instead, c e l l s that express o n l y p o d o c a l y x i n , p a r t i c u l a r l y podocytes, were expected to have more o b v i o u s defects ( D o y o n n a s et a l . , 2001).  1.6.4.1 Podocalyxin is Essential for Podocyte Morphogenesis T h e expected defects in podocytes l a c k i n g p o d o c a l y x i n made the g l o m e r u l i an e x c e l l e n t i n i t i a l target f o r analysis in p o d o c a l y x i n - n u l l a n i m a l s ( D o y o n n a s et a l . , 2 0 0 1 ) . F o r the m o s t part, h o w e v e r , p o d o c y t e maturation is unaffected i n k n o c k o u t m i c e . N e p h r i n is a  56  c o m p o n e n t o f the slit d i a p h r a g m (discussed in section 1.6.5.1, b e l o w ) , and g l o m e r u l a r epithelial protein ( G L E P P ) - l is a transmembrane tyrosine phosphatase o f podocytes; both are c o n s i d e r e d markers o f podocyte d i f f e r e n t i a t i o n ( D o y o n n a s et a l . , 2 0 0 1 ; K a w a c h i et a l . , 2 0 0 2 ; W a n g et a l . , 2 0 0 0 ) . I m m u n o h i s t o c h e m i c a l s t a i n i n g f o r these t w o proteins is n o r m a l i n m i c e l a c k i n g p o d o c a l y x i n ( D o y o n n a s et a l . , 2 0 0 1 ) . H o w e v e r , ultrastructural a n a l y s i s o f p o d o c y t e s by t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y ( T E M )  reveals d r a m a t i c  m o r p h o l o g i c a l a b n o r m a l i t i e s . T h e r e is a s i g n i f i c a n t r e d u c t i o n i n the n u m b e r o f m a j o r processes and a complete absence o f foot processes and slit d i a p h r a g m s i n p o d o c a l y x i n null m i c e . M o r e o v e r , podocyte c e l l bodies c o m p l e t e l y e n v e l o p the c a p i l l a r y l o o p s , and there is a s t r i k i n g presence o f j u n c t i o n a l c o m p l e x e s between adjacent podocytes. T h e s e defects b l o c k normal filtrate p r o d u c t i o n , as the bladders o f k n o c k o u t m i c e are c o m p l e t e l y empty j u s t before birth, i n contrast to w i l d t y p e littermates. O t h e r observations i n c l u d e v a c u o l e s w i t h i n p o d o c y t e s , t h i c k e n i n g o f the c a p i l l a r y e n d o t h e l i a l c e l l l a y e r , e n d o t h e l i a l fenestrae, a n d an apparent r e d u c t i o n i n the v o l u m e o f u r i n a r y  fewer spaces  ( D o y o n n a s et a l . , 2001). T h e electron m i c r o g r a p h i n F i g u r e 1-9, i n c l u d e d f o r orientation purposes, depicts a normal kidney podocyte extending major processes and processes around a glomerular capillary. S i m i l a r l y , cross-sections taken  foot  through  podocytes and c a p i l l a r i e s o f w i l d t y p e and p o d o c a l y x i n - n u l l m i c e are d e p i c t e d in F i g u r e 1-10, where m a n y o f the differences described above are clearly v i s i b l e .  57  Figure 1-9: Electron Micrograph of a Normal Glomerular Podocyte with Extended Major Processes and Foot Processes Surrounding a Blood Vessel. O r i g i n a l m a g n i f i c a t i o n : 7 3 0 0 X . R e p r o d u c e d a n d adapted w i t h k i n d p e r m i s s i o n o f S p r i n g e r S c i e n c e and B u s i n e s s M e d i a ( M u n d e l and K r i z , 1995).  58  Figure 1-10: T E M ' s of Kidneys from Wildtype and Podocalyxin Knockout Mice. J u n c t i o n a l c o m p l e x e s ( J C ) are v i s i b l e between podocytes (Pod) i n p o d o c a l y x i n - n u l l m i c e . T h e s e m i c e a l s o l a c k m a j o r processes ( M P ) and foot processes ( F P ) and have t h i c k e n e d e n d o t h e l i a l c e l l s ( E C ) . R B C : red b l o o d c e l l . R e p r o d u c e d w i t h k i n d p e r m i s s i o n o f T h e R o c k e f e l l e r U n i v e r s i t y Press ( D o y o n n a s et a l . , 2 0 0 1 ) .  59  T h e p o d o c y t e phenotypes o b s e r v e d in p o d o c a l y x i n k n o c k o u t m i c e are consistent w i t h a role f o r p o d o c a l y x i n in decreasing c e l l adhesion and affecting c e l l m o r p h o l o g y . A l t h o u g h p o d o c y t e m a t u r a t i o n m a r k e r s appear n o r m a l , the c e l l s have an i m m a t u r e m o r p h o l o g y o v e r a l l . A s i n d e v e l o p i n g g l o m e r u l i , the podocytes in p o d o c a l y x i n - n u l l a n i m a l s retain tight j u n c t i o n s instead o f f o r m i n g slit diaphragms ( D o y o n n a s et a l . , 2001). T h u s , it seems l i k e l y that under n o r m a l c o n d i t i o n s , either p o d o c a l y x i n alters distribution of tight j u n c t i o n proteins by means o f its c y t o p l a s m i c interaction partners, or s i m p l y that e x p r e s s i o n o f h i g h l e v e l s o f this b u l k y , n e g a t i v e l y  c h a r g e d m o l e c u l e o n the a p i c a l s u r f a c e  may  p h y s i c a l l y d i s p l a c e j u n c t i o n s to a more basal l o c a t i o n . T h e l a c k o f f o o t processes m a y i m p l y a m o r e s p e c i f i c role f o r p o d o c a l y x i n in a f f e c t i n g c e l l m o r p h o l o g y t h r o u g h its e x t e n s i v e n e g a t i v e l y c h a r g e d d o m a i n i n a d d i t i o n to its c o n s e r v e d c y t o p l a s m i c tail and interactions w i t h the actin c y t o s k e l e t o n . R e g a r d i n g the increased presence o f c y t o p l a s m i c v a c u o l e s , this is also a characteristic o f human and rodent models o f renal disease, and it m a y be an alternative pathway f o r filtrate production, since the presence of tight j u n c t i o n s and the l a c k o f slit pores b l o c k the n o r m a l pathway ( T o t h and T a k e b a y a s h i , 1992). T h e t h i c k e n i n g o f c a p i l l a r y endothelial c e l l s i n p o d o c a l y x i n k n o c k o u t m i c e may be related to the loss o f endothelial p o d o c a l y x i n or it may be due to an increase in c a p i l l a r y pressure as a result o f the b l o c k a g e in filtrate p r o d u c t i o n ( D o y o n n a s et a l . , 2 0 0 1 ) . T h u s , p o d o c a l y x i n n u l l m i c e certainly demonstrate that p o d o c a l y x i n is a vital c o m p o n e n t o f the d e v e l o p i n g kidney.  60  1.6.4.2 The K i d n e y Defect in P o d o c a l y x i n - N u l l M i c e is the Apparent Cause of Perinatal Lethality in these Animals T h e s t r i k i n g l y abnormal podocytes in p o d o c a l y x i n - d e f i c i e n t a n i m a l s clearly represent a serious defect in these a n i m a l s a n d , in f a c t , it is the apparent cause o f their lethality w i t h i n the first day of birth ( D o y o n n a s et a l . , 2 0 0 1 ) . A l l genotypes are present in expected frequencies throughout d e v e l o p m e n t , but this changes shortly after birth, such that there are no l i v e p o d o c a l y x i n - n u l l m i c e one day later ( T a b l e 1-1). U n f o r t u n a t e l y , death at this early time point precludes a more thorough assessment of potentially  widespread  i m p l i c a t i o n s o f the kidney defect.  61  N u m b e r o f animals/genotype +/+  +/-  -/-  Frequency of -/- mice  15 d  19  28  17  26  16 d  5  21  10  27  17 d  8  15  10  30  18 d  35'  71  30  22  19 d  6  10  8  33  52  100  0  0  A g e o f embryos (post c o i t u m )  A g e o f newborns (post partum) 1 d  Table 1-1: Frequency of podxV , podxt'", and podxt ' Animals during Development. l+  1  A l t h o u g h n o r m a l ratios o f p o d o c a l y x i n - n u l l animals are present throughout d e v e l o p m e n t , they a l l d i e w i t h i n 2 4 hours o f birth. A d a p t e d f r o m ( D o y o n n a s et a l . , 2001).  62  O t h e r n e w b o r n m i c e w i t h anuric renal f a i l u r e have also been described to die w i t h i n the f i r s t day o f b i r t h , m a k i n g it l i k e l y that this is the reason f o r the death o f  podocalyxin  k n o c k o u t a n i m a l s ( B u l l o c k et a l . , 1998; D a v i s et a l . , 1995). It is important to r e m e m b e r , h o w e v e r , that other m i c e w i t h anuric renal f a i l u r e generally have other significant defects or l a c k k i d n e y s entirely. T h e p o d o c a l y x i n k n o c k o u t was thus the most s e l e c t i v e , lethal a n u r i c c o n d i t i o n d e s c r i b e d at the t i m e , but c o n f i r m a t i o n that the k i d n e y defect is the d e f i n i t i v e cause of death is not easy to obtain ( D o y o n n a s et a l . , 2001).  1.6.5  Other Molecules Involved in Glomerular Development  S o m e patients w i t h g l o m e r u l a r diseases have m u t a t i o n s in genes e n c o d i n g structural proteins i n v o l v e d in integrity of the podocyte c y t o s k e l e t o n or slit d i a p h r a g m s , such as a a c t i n i n - 4 , nephrin, p o d o c i n , and C D 2 - a s s o c i a t e d protein ( B o u t e et a l . , 2 0 0 0 ; K a p l a n et a l . , 2000;  K e s t i l a et a l . ,  1998; K i m  et  al., 2003).  However,  most  patients  with  g l o m e r u l o s c l e r o s i s d o not have mutations in these genes. Instead, e x p r e s s i o n of these genes may be dysregulated by mutations in other genes, such as W T 1 ( G a o et a l . , 2004). In f a c t , it has been s h o w n that m u t a t i o n s i n W T 1  c a n be a f a c t o r i n  early-onset  g l o m e r u l o s c l e r o s i s . It is also p o s s i b l e that a b n o r m a l p o d o c y t e f u n c t i o n c a u s e d by mutation o f a single gene may initiate a series of responses that includes dysregulation o f other p o d o c y t e genes. A l t h o u g h there are o b v i o u s l y n u m e r o u s proteins r e q u i r e d f o r g l o m e r u l a r d e v e l o p m e n t , I have selected t w o m o l e c u l e s to discuss that are p a r t i c u l a r l y relevant f o r m y studies. N e p h r i n is i n i t i a l l y expressed on the basolateral surface o f d e v e l o p i n g podocytes at the S - s h a p e d body stage o f g l o m e r u l a r d e v e l o p m e n t , not u n l i k e p o d o c a l y x i n ( K a w a c h i et a l . , 2 0 0 2 ; S c h n a b e l et a l . , 1989)). I have taken advantage of this  63  fact w h e n generating transgenic m i c e (described i n chapter f i v e ) . In a d d i t i o n , W T 1 is essential f o r g l o m e r u l a r d e v e l o p m e n t , and it is k n o w n to be a regulator o f p o d o c a l y x i n expression ( G a o et a l . , 2 0 0 4 ; G u o et a l . , 2 0 0 2 ; K r e i d b e r g et a l . , 1 9 9 3 ; P a l m e r et a l . , 2 0 0 1 ; S t a n h o p e - B a k e r et a l . , 2 0 0 4 ) .  1.6.5.1 Nephrin N e p h r i n is a 180 k D a t r a n s m e m b r a n e protein b e l o n g i n g to the i m m u n o g l o b u l i n (Ig) s u p e r f a m i l y ( r e v i e w e d i n (Salant and T o p h a m , 2 0 0 3 ; T r y g g v a s o n , 1999)). It has eight extracellular I g - l i k e m o t i f s , and i n kidneys it is l o c a l i z e d e x c l u s i v e l y to the slit d i a p h r a g m r e g i o n . It was first i d e n t i f i e d i n a g e n o m e - w i d e screen to locate the gene responsible f o r the a u t o s o m a l recessive c o n g e n i t a l nephrotic s y n d r o m e o f the F i n n i s h type ( C N F ) . T h e f i l t r a t i o n b a r r ie r i n C N F patients is d i s r u p t e d , r e s u l t i n g i n s y m p t o m s o f n e p h r o t i c s y n d r o m e . T h e effects are seen early, w i t h severe p r o t e i n u r i a in utero, loss o f f o o t processes, a n d death w i t h i n t w o years i n the absence o f a k i d n e y transplant. C l o s e to 5 0 different mutations i n the gene e n c o d i n g n e p h r i n (NPHS1)  have been detected i n these  patients.  In the d e v e l o p i n g g l o m e r u l u s , n e p h r i n is e x p r e s s e d o n the basolateral s u r f a c e o f podocytes b e l o w j u n c t i o n a l c o m p l e x e s ( K a w a c h i et a l . , 2 0 0 2 ) . It migrates i n c o n j u n c t i o n w i t h j u n c t i o n a l proteins a n d is e v e n t u a l l y restricted to the site o f the slit d i a p h r a g m . N e p h r i n k n o c k o u t m i c e r a p i d l y d e v e l o p severe p r o t e i n u r i a , they e x h i b i t partial f o o t process effacement, loss o f slit d i a p h r a g m s , a n d n a r r o w i n g o f filtration slits, and they d i e w i t h i n 2 4 hours o f birth (Putaala et a l . , 2 0 0 1 ) . A l t h o u g h nephrin expression is decreased  64  i n P A N , other studies a d d r e s s i n g the role o f n e p h r i n i n g l o m e r u l a r diseases are i n c o n c l u s i v e (reviewed in (Salant and T o p h a m , 2003)).  T h e precise role of nephrin in m a i n t a i n i n g slit d i a p h r a g m integrity is u n k n o w n , but some e v i d e n c e suggests that the s l i t d i a p h r a g m is a z i p p e r - l i k e structure ( r e v i e w e d in ( T r y g g v a s o n , 1999)). A m o d e l f o r the i n v o l v e m e n t of n e p h r i n in this type o f structure relies on it interacting h o m o p h i l i c a l l y w i t h other nephrin m o l e c u l e s on n e i g h b o u r i n g foot processes, as expected f o r I g - l i k e a d h e s i o n m o l e c u l e s . T h e r e are t w o I g - l i k e d o m a i n s p r o x i m a l to the p l a s m a m e m b r a n e and six more d i s t a l , separated by a spacer s e c t i o n . B a s e d o n the predicted 4 0 n m slit pore w i d t h , it is p o s s i b l e that the six distal I g - l i k e d o m a i n s w o u l d interact w i t h those on o p p o s i n g n e p h r i n m o l e c u l e s and that u n p a i r e d cysteine residues w o u l d f o r m i n t e r m o l e c u l a r d i s u l p h i d e bridges. Slit pores m a y then f o r m in b e t w e e n the n o n - i n t e r a c t i n g , p r o x i m a l I g - l i k e d o m a i n s o f n e i g h b o u r i n g  nephrin  m o l e c u l e s . T h i s m o d e l has yet to be p r o v e n , but the necessity for nephrin i n g l o m e r u l a r d e v e l o p m e n t and slit d i a p h r a g m structure is clear.  1.6.5.2 WT1 A s d e s c r i b e d i n section 1.3.1, W T 1 is a z i n c f i n g e r t r a n s c r i p t i o n f a c t o r expressed in g l o m e r u l a r p o d o c y t e s ( P a l m e r et a l . , 2 0 0 1 ; R o b e r t s , 2 0 0 5 ) , and there is c o n s i d e r a b l e e v i d e n c e i m p l i c a t i n g W T 1 i n n o r m a l p o d o c y t e f u n c t i o n ( G u o et a l . , 2 0 0 2 ) . It is first detectable at day nine o f m o u s e d e v e l o p m e n t in the l i n i n g o f the c o e l o m i c cavity and i n the u r o g e n i t a l ridge ( A r m s t r o n g et a l . , 1993). E x p r e s s i o n is m a i n t a i n e d i n m e s o t h e l i a l c e l l s l i n i n g m a j o r organs throughout d e v e l o p m e n t , but it is also strongly expressed i n the  65  renal v e s i c l e and S - s h a p e d b o d y stages by c e l l s that later b e c o m e p o d o c y t e s . It is then restricted to p o d o c y t e s i n the adult k i d n e y ( R o b e r t s , 2 0 0 5 ) . D e n y s - D r a s h (DDS)  generally  involves  development  of  glomerular  nephropathy  g l o m e r u l o s c l e r o s i s ; 9 4 % o f D D S patients have m u t a t i o n s in wtl M o r e o v e r , one o f the c o m m o n wtl  syndrome with  ( G u o et a l . , 2 0 0 2 ) .  m u t a t i o n s seen in these patients c a n a l s o cause  g l o m e r u l o s c l e r o s i s in m i c e (Patek et a l . , 1999). In a d d i t i o n , some patients w i t h nephrotic s y n d r o m e and isolated cases of g l o m e r u l o s c l e r o s i s have wtl  mutations (Ito et a l . , 1 9 9 9 ;  Y a n g et a l . , 1999).  Loss of W T 1  has p r o f o u n d effects o n g l o m e r u l a r d e v e l o p m e n t i n m i c e . T a r g e t e d  i n a c t i v a t i o n of W T 1 by deletion o f the first e x o n prevents m e s e n c h y m a l i n d u c t i o n and results in renal agenesis; null m i c e die between e m b r y o n i c day 13 and 14 ( K r e i d b e r g et a l . , 1993). W T 1  a b l a t i o n by s m a l l i n t e r f e r i n g R N A ( s i R N A )  in developing  kidney  explants c o n f i r m s this result ( D a v i e s et a l . , 2 0 0 4 ) . W h e n s i R N A is added to explants c o r r e s p o n d i n g to e m b r y o n i c day 9 ( E 9 ) , ureteric buds f a i l to g r o w . H o w e v e r , i f s i R N A is instead added at E l 1, ureteric buds r e m a i n intact, but nephrogenesis is severely i m p a i r e d . W T 1 is therefore required at several stages o f d e v e l o p m e n t , where it p l a y s n u m e r o u s roles.  When W T l - n u l l  m i c e are rescued u s i n g a h u m a n wtl  transgene, nephrogenesis  is  recovered, but m i c e can still develop crescentic g l o m e r u l o n e p h r i t i s or m e s a n g i a l sclerosis after b i r t h , depending on the level o f W I T  expression ( G u o et a l . , 2 0 0 2 ) . W T l - d e f i c i e n t  m i c e w i t h a single c o p y o f the h u m a n transgene s u r v i v e at least u n t i l birth. H o w e v e r ,  66  o n l y 2 6 % have t w o k i d n e y s ; 14 % have one k i d n e y , and 6 0 % l a c k k i d n e y s c o m p l e t e l y and d i e w i t h i n 4 8 h o u r s o f b i r t h . T h e  surviving  mice all suffer f r o m  delayed  nephrogenesis and congenital nephrotic syndrome w i t h severe a l b u m i n u r i a and die w i t h i n 2 0 days. T r a n s g e n i c m i c e w i t h t w o c o p i e s of human wtl  all have at least one k i d n e y , and  7 6 % have t w o . T h e y also have a less s t r i k i n g delay in n e p h r o g e n e s i s , but they s t i l l d e v e l o p adult-onset nephrotic s y n d r o m e w i t h a l b u m i n u r i a , and 2 6 % die w i t h i n 150 days. S i m i l a r l y , m i c e w i t h one copy o f m u r i n e wtl develop both k i d n e y s , but also e x h i b i t adultonset n e p h r o t i c s y n d r o m e , and 11 % d i e w i t h i n 150 d a y s . W T 1 is therefore c l e a r l y important f o r nephrogenesis and n o r m a l k i d n e y function throughout life.  1.7 Podocalyxin in the Hematopoietic System A l t h o u g h p o d o c a l y x i n is most h i g h l y expressed in k i d n e y , other s i g n i f i c a n t areas o f expression i n c l u d e the hematopoietic and vascular systems.  1.7.1  Overview of Hematopoiesis  D u r i n g d e v e l o p m e n t o f the hematopoietic system, there are thought to be t w o m a i n w a v e s o f h e m a t o p o i e s i s , although there is still some controversy r e g a r d i n g e x a c t l y w h e n and where each c e l l type is first generated. P r i m i t i v e h e m a t o p o i e s i s begins at E 7 . 5 in m i c e and i n v o l v e s p r o d u c t i o n , in the b l o o d islands of the y o l k sac, of the first c i r c u l a t i n g b l o o d c e l l s ( r e v i e w e d in ( D z i e r z a k et a l . , 1 9 9 8 ; K e l l e r et a l . , 1999)). T h e s e c e l l s are l a r g e , nucleated e r y t h r o i d cells and are required f o r s u r v i v a l and rapid g r o w t h o f the e m b r y o . T h i s is f o l l o w e d by p r o d u c t i o n o f y o l k s a c - d e r i v e d h e m a t o p o i e t i c p r o g e n i t o r s , w h i c h  67  enter the v a s c u l a t u r e a n d c i r c u l a t e u n t i l r e p l a c e d by d e f i n i t i v e h e m a t o p o i e t i c c e l l s . D e f i n i t i v e hematopoiesis begins i n the aorta-gonad-mesonephros ( A G M ) region at E l 0 . 5 11.5 and is characterized by p r o d u c t i o n of enucleated erythrocytes, l y m p h o i d c e l l s , and L T R - H S C s . W i t h i n three days, L T R - H S C s migrate to the fetal l i v e r ( F T L ) , the m a i n site o f fetal h e m a t o p o i e s i s . H e m a t o p o i e s i s also occurs transiently in the fetal spleen ( S P L ) before b e c o m i n g f i r m l y established in the bone m a r r o w ( B M ) , where an elaborate set o f e n v i r o n m e n t a l signals regulate p r o l i f e r a t i o n and differentiation ( r e v i e w e d in ( W i l s o n and T r u m p p , 2006)).  1.7.2  Podocalyxin's Role in the Hematopoietic System  P o d o c a l y x i n is expressed in all h e m a t o p o i e t i c a l l y active tissues throughout d e v e l o p m e n t ( D o y o n n a s et a l . , 2 0 0 5 ) . F o r e x a m p l e , most h e m a t o p o i e t i c c e l l s in E 1 0 - 1 2 m u r i n e y o l k sac and peripheral b l o o d express p o d o c a l y x i n ( F i g u r e 1-11). V i r t u a l l y a l l p o d o c a l y x i n p o s i t i v e c e l l s at this stage express m a r k e r s o f the e r y t h r o i d l i n e a g e , but a very s m a l l percentage instead express the stem c e l l factor receptor c - K i t and the p a n - h e m a t o p o i e t i c m a r k e r C D 4 5 . In c o l o n y f o r m i n g assays, these c e l l s g i v e rise to e r y t h r o i d and m y e l o i d c o l o n i e s , suggesting that p o d o c a l y x i n is expressed on p r i m i t i v e erythrocytes as w e l l as p r i m i t i v e m y e l o i d or m u l t i l i n e a g e progenitors. P o d o c a l y x i n e x p r e s s i o n i n y o l k sac and peripheral b l o o d gradually decreases o v e r t i m e , both in terms o f e x p r e s s i o n levels and frequency o f p o d o c a l y x i n - p o s i t i v e cells.  68  100  100  YS PB FL  75  Spleen BM  \  1'1  1'2  1'3  14  l'5  Day PC  1'6  iV  18  19  18 n NBi—r 2  1—I  4  I  I I 1 Wk  2wk  V  50  V  25  4wk  Day PP  F i g u r e 1-11: P o d o c a l y x i n i s E x p r e s s e d i n H e m a t o p o i e t i c T i s s u e s  throughout  Development.  P C : post c o i t u m , P P : post partum. A d a p t e d f r o m ( D o y o n n a s et a l . , 2 0 0 5 ) .  T h e n , as h e m a t o p o i e s i s shifts to F T L  at E l 5 , 7 5 % o f c e l l s in this tissue express  p o d o c a l y x i n ( F i g u r e 1-11) ( D o y o n n a s et a l . , 2 0 0 5 ) . A t this stage, there are t w o distinct populations of podocalyxin  expressing cells. The cells expressing lower levels  p o d o c a l y x i n are either p r i m i t i v e or d e f i n i t i v e e r y t h r o i d c e l l s , w h i l e the  of  population  e x p r e s s i n g the highest l e v e l s c o n t a i n s d e f i n i t i v e h e m a t o p o i e t i c p r o g e n i t o r s .  Again,  p o d o c a l y x i n expression in this tissue declines over the next f e w days. S i m i l a r l y , S P L and B M both c o n t a i n p o d o c a l y x i n - p o s i t i v e p o p u l a t i o n s upon a c q u i s i t i o n o f h e m a t o p o i e t i c a c t i v i t y . In perinatal m i c e , most c e l l s e x p r e s s i n g p o d o c a l y x i n are erythroblasts or early hematopoietic  progenitors.  Podocalyxin  expression  then  decreases  to  virtually  undetectable levels by birth. H o w e v e r , there is a distinct burst o f p o d o c a l y x i n expression i n h e m a t o p o i e t i c tissues i m m e d i a t e l y after birth, but this is again f o l l o w e d by a gradual d e c l i n e . T h u s , the establishment o f each h e m a t o p o i e t i c a l l y active tissue c o i n c i d e s w i t h increased expression o f p o d o c a l y x i n .  P o d o c a l y x i n expression in the d e v e l o p i n g hematopoietic system has also been assessed in chicken (McNagny  et a l . , 1 9 9 7 ; S u o n p a a et a l . , 2 0 0 5 ) . A s in m i c e , there are t w o  p o p u l a t i o n s o f p o d o c a l y x i n e x p r e s s i n g c e l l s in early a v i a n e m b r y o s ( M c N a g n y et a l . , 1997). E r y t h r o i d c e l l s express l o w l e v e l s o f p o d o c a l y x i n , w h i l e p o d o c a l y x i n is more h i g h l y expressed on multipotent h e m a t o p o i e t i c progenitors. S i g n i f i c a n t l y , p o d o c a l y x i n positive c e l l s are f o u n d adhering to the ventral w a l l o f the dorsal aorta before E 4 ; this is the precise l o c a t i o n o f the first d e f i n i t i v e H S C s ( M c N a g n y et a l . , 1 9 9 7 ; S u o n p a a et a l . , 2 0 0 5 ) . P o d o c a l y x i n is also f o u n d on hematopoietic progenitor c e l l s i n bone m a r r o w of 1-  70  week o l d c h i c k s . T h u s , p o d o c a l y x i n ' s e x p r e s s i o n pattern is c o m p a r a b l e i n d e v e l o p i n g m a m m a l s and avians.  In adult, p o d o c a l y x i n e x p r e s s i o n is restricted to c e l l s o f the p l a t e l e t / m e g a k a r y o c y t i c lineage and a rare population of c e l l s w i t h a stem c e l l phenotype ( D o y o n n a s et a l . , 2 0 0 5 ; McNagny  et a l . , 1 9 9 7 ; M i e t t i n e n et a l . , 1999). T h e s e  podocalyxin  +  Lin  Sca-1  +  c-Kit  +  (LSK)  c e l l s give rise to m y e l o i d and l y m p h o i d lineages in serial transplantation  e x p e r i m e n t s , suggesting that p o d o c a l y x i n may be a m a r k e r o f L T R - H S C s ( D o y o n n a s et a l . , 2 0 0 5 ) . A l t h o u g h e r y t h r o i d c e l l s in adult do not n o r m a l l y express p o d o c a l y x i n , it is rapidly upregulated by erythroid progenitors in response to h e m o l y t i c a n e m i a ( D o y o n n a s et a l . , 2 0 0 5 ) . T h i s suggests that p o d o c a l y x i n is expressed by early erythroid  progenitors  o n l y w h e n h i g h rates o f erythropoiesis are required, such as throughout d e v e l o p m e n t and under c o n d i t i o n s o f e r y t h r o p o i e t i c  stress. O n e  podocalyxin  mature  mRNA  in  numerous  study  also reported expression  hematopoietic  lineages,  including  of B  l y m p h o c y t e s , T l y m p h o c y t e s , and m y e l o i d c e l l s , but the c o r r e s p o n d i n g p r o t e i n is not expressed i n these c e l l s ( K e r o s u o et a l . , 2004). N o t a b l y , h o w e v e r , p o d o c a l y x i n  mRNA  levels are m u c h higher in k i d n e y and in endothelial c e l l s than i n any hematopoietic c e l l s . T h u s , p o d o c a l y x i n is expressed by hematopoietic stem cells and platelets, but not i n other mature hematopoietic lineages under steady state conditions in adult.  1.7.3  Hematopoiesis in the Podocalyxin Knockout  S i n c e p o d o c a l y x i n is expressed by a subset o f h e m a t o p o i e t i c c e l l s , i n c l u d i n g early h e m a t o p o i e t i c progenitors, the h e m a t o p o i e t i c s y s t e m o f p o d o c a l y x i n k n o c k o u t a n i m a l s  71  was assessed f o r defects ( D o y o n n a s et a l . , 2 0 0 1 ; D o y o n n a s et a l . , 2 0 0 5 ) . E 1 5 F T L ( F i g u r e 1-12), E 1 8 S P L , and E 1 8 B M were stained and e x a m i n e d by f l o w c y t o m e t r y  with  antibodies against hematopoietic progenitors ( S c a - 1 and c - K i t ) , erythroid c e l l s ( T e r l 19), m e g a k a r y o c y t e s and platelets ( C D 4 1 ) , m y e l o i d c e l l s ( M a c l ) , B c e l l s ( B 2 2 0 ) , T c e l l s ( C D 3 ) , and granulocytes ( G r - 1 ) (Figure 1-12 and ( D o y o n n a s et a l . , 2 0 0 1 ) ) . T h e r e are no detectable differences in the frequencies of any h e m a t o p o i e t i c lineages in p o d o c a l y x i n deficient m i c e throughout development. T h e related protein, C D 3 4 , is also expressed by hematopoietic progenitors, thereby p r o v i d i n g the p o s s i b i l i t y o f f u n c t i o n a l c o m p e n s a t i o n by this f a m i l y m e m b e r ( A n d r e w s et a l . , 1 9 8 9 ; B e r e n s o n et a l . , 1 9 8 8 ; D o y o n n a s et a l . , 2 0 0 1 ) . F o r this r e a s o n , c o m p o u n d k n o c k o u t m i c e l a c k i n g both m o l e c u l e s were a l s o generated and a n a l y z e d ( D o y o n n a s et a l . , 2 0 0 5 ) . S u r p r i s i n g l y , h o w e v e r , hematopoiesis is also normal in m i c e l a c k i n g both m o l e c u l e s ( F i g u r e 1-12).  72  100 90 CU O CU  80 70  > ro 60  O) CD  50  I CT>  40  §  30  °  20  c  10 0  • WT  • Podo KO • CD34 KO DDKO  r  i Sca-1  CD41  Mac-1  B220  CD3  c-kit  Gr-1  Figure 1-12: Steady State Levels of All Hematopoietic Lineages in E15 Fetal Liver in Podocalyxin (Podo)-Knockout (KO), CD34-KO, and Double-KO (DKO) Mice are Similar to those in Wildtype (WT) Mice.  73  A l t h o u g h the ratios o f a l l hematopoietic lineages are n o r m a l in p o d o c a l y x i n - , C D 3 4 - , and p o d o c a l y x i n / C D 3 4 - n u l l m i c e , there is a f u n c t i o n a l defect in these h e m a t o p o i e t i c c e l l s . S h o r t - t e r m h o m i n g assays demonstrate that injected c e l l s l a c k i n g either p o d o c a l y x i n or C D 3 4 migrate w i t h 2 0 - 3 0 % l o w e r e f f i c i e n c y to the bone m a r r o w than their w i l d t y p e counterparts, and that loss o f both m o l e c u l e s has an a d d i t i v e effect ( D o y o n n a s et a l . , 2 0 0 5 ) . H o w e v e r , despite this defect, w h e n c e l l s f r o m any o f these m i c e are injected in s u f f i c i e n t numbers, they can f u l l y reconstitute lethally irradiated recipients. T h i s result, a l o n g w i t h the expression pattern of p o d o c a l y x i n , in particular, i m p l i e s that w h i l e these m o l e c u l e s are not essential, they m a y f a c i l i t a t e the c r o s s i n g o f e n d o t h e l i a l barriers f o r entry into or exit f r o m , h e m a t o p o i e t i c m i c r o e n v i r o n m e n t s ( D o y o n n a s et a l . , 2 0 0 5 ) . H e m a t o p o i e t i c cells of the y o l k sac become less adherent i n order to leave b l o o d islands, precursors i n F T L must cross into the vasculature to migrate to S P L and B M , and severe a n e m i a leads to an e f f l u x o f e r y t h r o i d progenitors f r o m the B M in order to establish a d d i t i o n a l sites o f erythropoiesis. E a c h of these situations corresponds to an increase in p o d o c a l y x i n expression.  1.7.4  Podocalyxin in the Vasculature  In a d d i t i o n to its expression in the hematopoietic s y s t e m , p o d o c a l y x i n is also a universal m a r k e r o f v a s c u l a r e n d o t h e l i a l c e l l s ( D o y o n n a s et a l . , 2 0 0 5 ; H o r v a t et a l . , 1 9 8 6 ; M i e t t i n e n et a l . , 1990). It is expressed in b l o o d vessels o f k i d n e y , l u n g , heart, brain, small intestine, and other tissues ( H o r v a t et a l . , 1986). P o d o c a l y x i n is f o u n d on endothelial cells l i n i n g a w i d e range o f vessels, f r o m the c o r o n a r y artery to the sinusoids o f l i v e r and spleen and the s p e c i a l i z e d postcapillary venules in l y m p h nodes termed H E V ( H o r v a t et  74  a l . , 1 9 8 6 ; M i e t t i n e n et a l . , 1 9 9 0 ; Sassetti et a l . , 1998). B y i m m u n o T E M , it has been s h o w n that p o d o c a l y x i n is l o c a l i z e d in a patchy pattern o n the l u m i n a l face o f vascular endothelial cells ( H o r v a t et a l . , 1986).  D e s p i t e the w i d e s p r e a d e x p r e s s i o n o f p o d o c a l y x i n on b l o o d v e s s e l s , the v a s c u l a t u r e appears n o r m a l i n d e v e l o p i n g p o d o c a l y x i n k n o c k o u t a n i m a l s ( D o y o n n a s et a l . , 2 0 0 1 ) . T h e r e are n o detectable d i f f e r e n c e s in the s t a i n i n g pattern o f the e n d o t h e l i a l m a r k e r , PECAM-1  i n E 1 6 b r a i n , k i d n e y , l u n g , or gut. F u r t h e r m o r e , e l e c t r o n m i c r o s c o p y  of  n e w b o r n lungs shows the f o r m a t i o n of w e l l - d e v e l o p e d p u l m o n a r y c a p i l l a r i e s . T h e related p r o t e i n , C D 3 4 , is also u b i q u i t o u s l y expressed in vasculature, h o w e v e r , so it is p o s s i b l e that it c o u l d be f u n c t i o n a l l y c o m p e n s a t i n g for loss o f p o d o c a l y x i n ( D o y o n n a s et a l . , 2 0 0 1 ; F i n a et a l . , 1990). In fact, u p r e g u l a t i o n o f C D 3 4 in l u n g o f E 1 8 p o d o c a l y x i n - d e f i c i e n t a n i m a l s has been detected by real time R T - P C R and i m m u n o h i s t o c h e m i s t r y ( D o y o n n a s et a l . , 2 0 0 1 ) . A l t h o u g h there are no o b v i o u s defects in the vasculature, a p p r o x i m a t e l y 25 % o f p o d o c a l y x i n - d e f i c i e n t e m b r y o s exhibit m i l d to severe e d e m a ( D o y o n n a s et a l . , 2 0 0 1 ) . T h i s m a y be a result o f l e a k y vessels or, alternatively, the k i d n e y defect w o u l d l i k e l y lead to an increase in b l o o d pressure, w h i c h may be relieved by loss o f f l u i d f r o m the vessels. F u n c t i o n a l l y , o f c o u r s e , it w o u l d be easier to assess defects i n the vascular s y s t e m in adult a n i m a l s , but perinatal lethality precludes this analysis.  1.7.5  Podocalyxin Expression in Hemangioblasts  E x p r e s s i o n o f p o d o c a l y x i n by hematopoietic and vascular endothelial cells i m p l i e s that it c o u l d also be expressed by the c o m m o n precursor o f both c e l l types, the hemangioblast.  75  P o d o c a l y x i n is also expressed by C D 4 5  +  and C D 4 5 c e l l s in the A G M r e g i o n at E l 1.5  ( H a r a et a l . , 1999). P o d o c a l y x i n C D 4 5 c e l l s f r o m the A G M d i f f e r e n t i a t e into both +  a n g i o b l a s t s and h e m a t o p o i e t i c c e l l s in vitro, d e p e n d i n g on the g r o w t h c o n d i t i o n s , and they are also capable o f l o n g - t e r m l y m p h o i d and m y e l o i d reconstitution in transplantation e x p e r i m e n t s , suggesting the presence on L T R - H S C s i n this p o p u l a t i o n , a l t h o u g h this cannot be c o n c l u s i v e l y demonstrated without p e r f o r m i n g single c e l l transplants ( H a r a et a l . , 1999). T h u s , a l t h o u g h not c o n f i r m e d , this d a t a is s u g g e s t i v e o f  podocalyxin  expression on hemangioblasts.  1.8  Podocalyxin  in the  Brain  A n o t h e r interesting site of p o d o c a l y x i n expression is the b r a i n , where it is detected in the m a r g i n a l zone o f the cerebral cortex, the c e r e b e l l u m , and the superior c o l l i c u l u s at E 1 5 16 ( G a r c i a - F r i g o l a et a l . , 2 0 0 4 ) . E x p r e s s i o n in m i g r a t i n g c e l l s i n the d e v e l o p i n g c e r e b e l l u m hints that it m a y be i n v o l v e d in e n a b l i n g neurons to migrate and detach f r o m r a d i a l g l i a . In situ h y b r i d i z a t i o n d e m o n s t r a t e s that p o d o c a l y x i n m R N A i s , i n f a c t , expressed in m a n y regions throughout d e v e l o p m e n t and in adult b r a i n , w i t h the highest expression levels in cerebral cortex and c e r e b e l l u m postnatally ( V i t u r e i r a et a l . , 2 0 0 5 ) . In the f o r e b r a i n , p o d o c a l y x i n  is e x p r e s s e d  i n the o l f a c t o r y  bulb,  neocortex,  and  h i p p o c a m p u s . It is expressed i n d e v e l o p i n g neurons and a l o n g s p e c i f i c axonal pathways. T h e f u n c t i o n a l s i g n i f i c a n c e o f p o d o c a l y x i n e x p r e s s i o n i n the b r a i n and any potential defects i n p o d o c a l y x i n k n o c k o u t a n i m a l s are still under i n v e s t i g a t i o n , but its pattern of e x p r e s s i o n hints at a role i n p r o l i f e r a t i o n , m i g r a t i o n , or neuronal d i f f e r e n t i a t i o n i n the central nervous s y s t e m ( V i t u r e i r a et a l . , 2 0 0 5 ) . A n t i - a d h e s i v e f o r c e s m a y a i d i n the  76  detachment and m i g r a t i o n o f neuronal cells and l e a d i n g processes, or p o d o c a l y x i n may be a c t i v e l y i n v o l v e d in a x o n a l path f i n d i n g t h r o u g h its interactions w i t h P D Z d o m a i n c o n t a i n i n g proteins ( O r l a n d o et a l . , 2 0 0 1 ; V i t u r e i r a et a l . , 2 0 0 5 ) .  1.9  Additional  Phenotypes  in Podocalyxin-Null  Animals  P o d o c a l y x i n is expressed by p o d o c y t e s , h e m a t o p o i e t i c p r o g e n i t o r s , platelets, v a s c u l a r e n d o t h e l i a , a subset o f neurons, and m e s o t h e l i a l c e l l s l i n i n g m a n y o r g a n s ; defects are therefore expected in these tissues in m i c e l a c k i n g p o d o c a l y x i n ( D o y o n n a s et a l . , 2 0 0 1 ; H o r v a t et a l . , 1986; K e r j a s c h k i et a l . , 1984; K e r s h a w et a l . , 1997a; M c N a g n y et a l . , 1997; M i e t t i n e n et a l . , 1 9 9 9 ; Sassetti et a l . , 1998). C D 3 4 m a y f u n c t i o n a l l y c o m p e n s a t e f o r p o d o c a l y x i n loss i n h e m a t o p o i e t i c p r o g e n i t o r s and b l o o d v e s s e l s , potential n e u r o n a l defects are being actively investigated, and podocytes are c l e a r l y a b n o r m a l ( D o y o n n a s et a l . , 2 0 0 1 ) . T h e f i n a l area o f interest is therefore the l i n i n g o f body c a v i t i e s . S t r i k i n g l y , a p p r o x i m a t e l y 3 0 % o f p o d o c a l y x i n - n u l l m i c e are born w i t h a h e r n i a t i o n o f the gut, known  as an o m p h a l o c e l e ( D o y o n n a s  et a l . , 2 0 0 1 ) . T h i s  is a c t u a l l y  a normal  p h y s i o l o g i c a l process that o c c u r s at E 1 2 as the r a p i d l y g r o w i n g organs e x c e e d the l i m i t i n g space o f the peritoneal c a v i t y ; h o w e v e r , i n n o r m a l m i c e it is r e s o l v e d as the peritoneal c a v i t y expands, generally by E 1 6 ( K a u f m a n , 1998). U s i n g t i m e d m a t i n g s , it was d e t e r m i n e d that r e s o l u t i o n o f the o m p h a l o c e l e is d e l a y e d i n a l l p o d o c a l y x i n - n u l l m i c e , but 7 0 % do retract the gut before birth ( D o y o n n a s et a l . , 2 0 0 1 ) . T h e delay may be a result o f increased adhesion upon loss of p o d o c a l y x i n f r o m exposed surfaces. T h u s , i n another tissue where C D 3 4 cannot f u n c t i o n a l l y compensate f o r loss o f p o d o c a l y x i n , there is an apparent increase in cell adhesion.  77  1.10  Podocalyxin  in  Cancer  U p to this point I have concentrated on the n o r m a l f u n c t i o n and e x p r e s s i o n pattern o f podocalyxin,  but  I will  now  f o c u s o n cases w h e r e  podocalyxin  expression  is  d y s r e g u l a t e d . P o d o c a l y x i n has been i m p l i c a t e d i n n u m e r o u s m a l i g n a n t s i t u a t i o n s , i n c l u d i n g breast cancer, testicular cancer, prostate cancer, and l e u k e m i a ( C a s e y et a l . , 2 0 0 6 ; K e l l e y et a l . , 2 0 0 5 ; Schopperle et a l . , 2 0 0 3 ; S o m a s i r i et a l . , 2004).  1.10.1 Podocalyxin in Breast Cancer A c c o r d i n g to a n a l y s i s o f a tissue m i c r o a r r a y o f 2 7 2 i n v a s i v e breast c a r c i n o m a s and c o r r e s p o n d i n g l o n g - t e r m o u t c o m e data, w e have s h o w n that p o d o c a l y x i n upregulation is c o r r e l a t e d w i t h poor o u t c o m e in a d i s t i n c t subset o f t u m o u r s ( S o m a s i r i et a l . , 2 0 0 4 ) . I m m u n o h i s t o c h e m i s t r y was used to c o m p a r e p o d o c a l y x i n expression in tumour samples. A score of " 0 " corresponds to a l a c k of p o d o c a l y x i n staining in t u m o u r c e l l s , and was f o u n d in 6 0 % o f cases. L e s s than 10 % o f tumour cells were stained (group 1) i n 23 % o f s a m p l e s , 12 % of cases e x h i b i t e d intense staining in less than half of the c e l l s or diffuse staining i n more than 10 % (group 2). T h e f i n a l group (group 3) made up 6 % o f the array and had intense p o d o c a l y x i n staining in the majority of tumour cells. S u r v i v a l rates were c o m p a r e d between patients f r o m each group, and a statistically significant difference was noted between group 3 and the rest o f the patients, c o m b i n e d ( F i g u r e 1-13). T h e mean s u r v i v a l time was 9.0±1.8 years f o r patients in group 3 , w h i l e those patients w i t h tumours e x p r e s s i n g l o w or no p o d o c a l y x i n l i v e d on average 15±0.5 years. A l t h o u g h there are no  78  s i g n i f i c a n t d i f f e r e n c e s in h i s t o l o g i c a l subtype, t u m o u r s i z e , or l y m p h node metastasis b e t w e e n g r o u p s , there are p r o p o r t i o n a l l y m o r e h i g h grade, estrogen receptor negative  tumours  in  the  high  podocalyxin  group.  Furthermore,  (ER)-  podocalyxin  o v e r e x p r e s s i o n is a statistically s i g n i f i c a n t independent predictor of poor o u t c o m e w i t h more than an e i g h t - f o l d relative risk c o m p a r e d to p o d o c a l y x i n l o w or negative samples.  79  Group 0 Group 1 Group 2  1  0  5  10  — Group 3  15  Total Follow-Up (years)  Figure 1-13: Survival Rates for Patients Diagnosed with Breast Tumours Shown to be Expressing Varying Levels of Podocalyxin. G r o u p s 0 - 2 : low/no p o d o c a l y x i n e x p r e s s i o n a n d g r o u p 3 : h i g h p o d o c a l y x i n e x p r e s s i o n ( S o m a s i r i et a l . , 2 0 0 4 ) .  80  T h e m e c h a n i s m l e a d i n g to increased p o d o c a l y x i n expression has not yet been determined ( S o m a s i r i et a l . , 2 0 0 4 ) . H o w e v e r , the h u m a n podxl gene is located o n c h r o m o s o m e 7 q 3 2 q 3 3 , w h i c h is i n between t w o regions p r e v i o u s l y i d e n t i f i e d as c h r o m o s o m a l g a i n sites i n ductal breast c a r c i n o m a and breast t u m o u r c e l l lines ( A u b e l e et a l . , 2 0 0 0 ; F o r o z a n et a l . , 2 0 0 0 ; K e r s h a w et a l . , 1997b). M o r e o v e r , the p o d o c a l y x i n b i n d i n g p r o t e i n , N H E R F 1 is often e x p r e s s e d at l o w e r l e v e l s i n E R - n e g a t i v e breast t u m o u r s i n c o m p a r i s o n to E R p o s i t i v e t u m o u r s , and m a n y o f the t u m o u r s e x p r e s s i n g h i g h l e v e l s o f p o d o c a l y x i n were E R - n e g a t i v e , w h i c h m a y be a c o n t r i b u t i n g f a c t o r ( S o m a s i r i et a l . , 2 0 0 4 ; S t e m m e r R a c h a m i m o v et a l . , 2 0 0 1 ) . T h u s , a l t h o u g h the events l e a d i n g u p to it c a n o n l y be h y p o t h e s i z e d at this stage, p o d o c a l y x i n is u p r e g u l a t e d i n a subset o f breast t u m o u r s i n patients w i t h p o o r o u t c o m e s . T h e r e i s c u r r e n t l y a l a r g e r s c r e e n o f breast t u m o u r s underway to c o n f i r m these results.  1.10.2 Podocalyxin in Prostate C a n c e r G e n e t i c a p p r o a c h e s w e r e u s e d to i d e n t i f y p o d o c a l y x i n d y s r e g u l a t i o n as a f a c t o r i n prostate cancer ( C a s e y et a l . , 2 0 0 6 ) . S i n c e prostate c a n c e r a g g r e s s i v e n e s s , w h i c h varies w i d e l y a m o n g patients, is i n f l u e n c e d b y f a m i l y h i s t o r y , a genetic c o m p o n e n t is suspected ( K l e i n et a l . , 1998). A g e n o m e - w i d e s c r e e n o f o v e r 5 0 0 a f f e c t e d s i b l i n g s  strongly  i m p l i c a t e d c h r o m o s o m e 7 q 3 2 - q 3 3 i n t u m o u r aggressiveness ( W i t t e et a l . , 2 0 0 0 ) , and this result has been c o n f i r m e d i n subsequent studies (Paiss et a l . , 2 0 0 3 ; W i t t e et a l . , 2 0 0 3 ) . In prostate t u m o u r s , this region also e x h i b i t s a h i g h f r e q u e n c y o f a l l e l i c i m b a l a n c e , w i t h the podxl gene contained w i t h i n the smallest region o f i m b a l a n c e ( N e v i l l e et a l . , 2 0 0 2 ) .  81  M u t a t i o n a l a n a l y s i s w a s p e r f o r m e d o n g e n o m i c podxl f r o m the probands o f 17 f a m i l i e s i d e n t i f i e d p r e v i o u s l y ( C a s e y et a l . , 2 0 0 6 ) . T h e r e l a t i o n s h i p b e t w e e n prostate c a n c e r , tumour  aggressiveness,  a n d podxl  v a r i a n t s w a s then assessed i n a f a m i l y - b a s e d  a s s o c i a t i o n study. S e v e r a l c o m m o n m u t a t i o n s were i d e n t i f i e d , i n c l u d i n g a v a r i a b l e i n f r a m e d e l e t i o n i n the first e x o n , f o u r m i s s e n s e m u t a t i o n s , and t w o silent variants. T h e presence o f one or t w o c o p i e s o f the i n - f r a m e d e l e t i o n variant, w h i c h results i n loss o f serine and p r o l i n e residues i n the e x t r a c e l l u l a r d o m a i n , increases the relative r i s k , w i t h t w o c o p i e s d o u b l i n g the risk o f d e v e l o p i n g more aggressive prostate cancer. T h e presence o f m i s s e n s e m u t a t i o n s i n e x o n t w o increases the r i s k o f d e v e l o p i n g prostate c a n c e r b y a p p r o x i m a t e l y 5 0 % , but has n o effect o n aggressiveness. T h e other mutations were not r i s k f a c t o r s , a n d a d d i t i o n a l v a r i a n t s o u t s i d e o f the podxl  locus d i d not show any  a s s o c i a t i o n w i t h prostate cancer either, i m p l y i n g that the mutations d e s c r i b e d a b o v e are g e n u i n e r i s k f a c t o r s . W h i l e the f u n c t i o n a l i m p l i c a t i o n s o f these m u t a t i o n s are not y e t k n o w n , it m a y be that they result i n decreased negative charge o n p o d o c a l y x i n , w h i c h m a y d i s r u p t the a s s o c i a t i o n o f p o d o c a l y x i n w i t h the actin c y t o s k e l e t o n , o r c e l l m o t i l i t y and i n v a s i v e n e s s m a y be increased as a result o f perturbation o f d o w n s t r e a m targets.  1.10.3 Podocalyxin in Testicular Cancer P o d o c a l y x i n e x p r e s s i o n i n testicular c a n c e r , the most c o m m o n type o f s o l i d t u m o u r i n y o u n g adult m a l e s , w a s the first report o f p o d o c a l y x i n in m a l i g n a n t c e l l s ( S c h o p p e r l e et a l . , 2 0 0 3 ) . It i s i m p o r t a n t to f i n d m a r k e r s to d i s t i n g u i s h b e t w e e n the t w o types o f testicular g e r m c e l l t u m o u r s , s e m i n o m a t o u s a n d n o n s e m i n o m a t o u s ( N S G C T ) , because they require different treatments. P o d o c a l y x i n is preferentially expressed b y N S G C T , and  82  not i n protein lysates f r o m n o r m a l tissue. P o d o c a l y x i n is also f o u n d in the supernatants o f cultured e m b r y o n a l c a r c i n o m a c e l l l i n e s , and it m a y therefore be a useful s e r u m m a r k e r f o r d e t e c t i o n o f N S G C T ( S c h o p p e r l e et a l . , 1992). I n t e r e s t i n g l y , the N S G C T f o r m o f p o d o c a l y x i n is c o n s i d e r a b l y larger than the f o r m f o u n d in k i d n e y , suggesting that it m a y undergo a d d i t i o n a l p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s i n these t u m o u r s ( S c h o p p e r l e et a l . , 2003).  1.10.4 Podocalyxin in Leukemia P o t e n t i a l e x p r e s s i o n o f p o d o c a l y x i n i n l e u k e m i a w a s assessed f o r several reasons: the related p r o t e i n , C D 3 4 is expressed by m a n y , but not a l l , l e u k e m i c blasts, p o d o c a l y x i n is e x p r e s s e d by n o r m a l h e m a t o p o i e t i c p r o g e n i t o r s , and the p o d o c a l y x i n  transcriptional  regulator, W T 1 is expressed by the majority o f blasts i n acute m y e l o i d l e u k e m i a ( A M L ) a n d acute l y m p h o b l a s t i c l e u k e m i a ( A L L )  ( D o y o n n a s et a l . , 2 0 0 5 ; K e l l e y et a l . , 2 0 0 5 ;  K e r o s u o et a l . , 2 0 0 4 ; M e n s s e n et a l . , 1 9 9 5 ; P a l m e r et a l . , 2 0 0 1 ) . A M L and A L L tissue m i c r o a r r a y s and b i o p s y s p e c i m e n s were assayed f o r p o d o c a l y x i n e x p r e s s i o n ( K e l l e y et a l . , 2 0 0 5 ) . P o d o c a l y x i n w a s detected i n blasts i n 7 7 % o f 3 9 A M L cases, w i t h strong expression in 4 1 % o f samples. It was also detected in 81 % o f 27 A L L cases, w i t h strong e x p r e s s i o n in 2 2 % . A l t h o u g h C D 3 4 is also expressed i n m a n y l e u k e m i c blasts, there is no s i g n i f i c a n t c o r r e l a t i o n b e t w e e n C D 3 4 and p o d o c a l y x i n e x p r e s s i o n .  Podocalyxin  e x p r e s s i o n i n m y e l o i d sarcomas w a s also assessed in order to address a proposed role in f a c i l i t a t i n g tissue i n f i l t r a t i o n ; it w a s expressed i n 87 % o f 15 cases w i t h strong e x p r e s s i o n i n 5 3 % . S i n c e p o d o c a l y x i n e x p r e s s i o n is regulated by W T 1 i n p o d o c y t e s , e x p r e s s i o n o f W T 1 w a s assessed i n l e u k e m i c blasts: 4 4 % o f A M L cases a n d 7 8 % o f A L L cases  83  e x h i b i t e d nuc le a r W T 1 , but there w a s no c o r r e l a t i o n w i t h p o d o c a l y x i n e x p r e s s i o n . T h i s suggests that p o d o c a l y x i n e x p r e s s i o n is s o m e h o w d y s r e g u l a t e d i n l e u k e m i a or, s i n c e p o d o c a l y x i n is n o r m a l l y expressed by h e m a t o p o i e t i c progenitors, perhaps e x p r e s s i o n by l e u k e m i c blasts j u s t f o l l o w s the n o r m a l expression pattern, w h i c h may be independent o f W T 1 i n h e m a t o p o i e s i s . A g a i n , the f u n c t i o n a l relevance o f p o d o c a l y x i n e x p r e s s i o n i n this type o f c a n c e r is u n k n o w n , but its presence c o u l d be used as a m a r k e r to increase the sensitivity o f assays designed to detect l e u k e m i a .  1.10.5 Podocalyxin in Hepatocellular Carcinoma Hepatocellular carcinoma ( H C C )  is one o f the f i v e l e a d i n g causes o f c a n c e r death  w o r l d w i d e ( P i s a n i et a l . , 1999). U n f o r t u n a t e l y , this type o f cancer is often detected too late, thereby l i m i t i n g treatment o p t i o n s , and m a k i n g the f i v e - y e a r s u r v i v a l rate o n l y f i v e percent ( E l - S e r a g et a l . , 2 0 0 1 ) . F i n d i n g a d d i t i o n a l markers o f H C C m a y facilitate earlier detection and better o u t c o m e ( C h e n et a l . , 2 0 0 4 ) . O n e m a j o r difference between H C C and n o r m a l l i v e r tissue can be in the type o f b l o o d vessels they c o n t a i n . N o r m a l vessels l i n i n g the hepatic s i n u s o i d a l l o w free d i f f u s i o n o f m a c r o m o l e c u l e s , but not larger particles, v i a s m a l l fenestrations ( C h e n et a l . , 2 0 0 4 ) . In contrast, vasculature w i t h i n t u m o u r s is often a b n o r m a l l y p e r m e a b l e , a l l o w i n g passage o f larger m o l e c u l e s , and e v e n metastasis o f c a n c e r o u s c e l l s , t h r o u g h w i d e n e d c e l l - c e l l j u n c t i o n s , larger fenestrations, t r a n s c e l l u l a r holes, and an irregular basement m e m b r a n e ( H a s h i z u m e et a l . , 2 0 0 0 ) . S t r i k i n g l y , C D 3 4 is highly upregulated in endothelial cells of H C C  in c o m p a r i s o n to n o r m a l l i v e r tissue  ( R u c k et a l . , 1995). N o t a b l y , C D 3 4 is g e n e r a l l y not detectable i n most s o l i d t u m o u r s , such as breast cancer, l y m p h o m a , m y e l o m a , or n e u r o b l a s t o m a ( r e v i e w e d i n ( S i l v e s t r i et  84  a l . , 1992)), w h i l e p o d o c a l y x i n has been f o u n d i n s e v e r a l , such as those already d i s c u s s e d , W i l m s ' t u m o u r s ( d e s c r i b e d b e l o w ) , and h i g h grade o v a r i a n t u m o u r s  (unpublished  o b s e r v a t i o n s , M L M c C o y , C B G i l k s , C D R o s k e l l e y , as c i t e d i n ( S o m a s i r i et a l . , 2 0 0 4 ) ) . In a search f o r other markers o f H C C u s i n g c D N A m i c r o a r r a y a n a l y s i s o f n o r m a l tissue and tumours f r o m 58 patients, p o d o c a l y x i n and C D 3 4 were both f o u n d to be upregulated i n H C C ( C h e n et a l . , 2 0 0 4 ) . T h i s result was c o n f i r m e d by i m m u n o h i s t o c h e m i s t r y : 11 o f 12 H C C tissue sections were positive f o r p o d o c a l y x i n , w h i l e a l l 5 n o r m a l tissue sections lacked p o d o c a l y x i n . There was also a highly  statistically significant increase  in  p o d o c a l y x i n and C D 3 4 expression in H C C on a tissue m i c r o a r r a y c o n t a i n i n g 3 5 0 samples ( C h e n et a l . , 2 0 0 4 ) . It is thought that perhaps this d r a m a t i c u p r e g u l a t i o n contributes to the leakiness o f vasculature in H C C . Regardless o f the f u n c t i o n a l i m p l i c a t i o n s o f C D 3 4 and p o d o c a l y x i n e x p r e s s i o n , they m a y be useful markers f o r earlier detection o f this type o f cancer.  1.10.6 Podocalyxin in Wilms' Tumours W i l m s ' t u m o u r is the most f r e q u e n t l y o c c u r r i n g p e d i a t r i c k i d n e y c a n c e r and the f o u r t h most  common  childhood  malignancy  (Miller  et  a l . , 1995).  The  podocalyxin  t r a n s c r i p t i o n a l regulator, W T 1 is mutated i n 1 0 - 1 5 % o f W i l m s ' t u m o u r s , a n d there is also e v i d e n c e to i m p l i c a t e p53 in some cases; h o w e v e r , the m a j o r i t y o f W i l m s ' t u m o u r s h a v e n o k n o w n cause. In contrast to the other c a n c e r s d e s c r i b e d a b o v e , p o d o c a l y x i n e x p r e s s i o n is s i g n i f i c a n t l y reduced in W i l m ' s t u m o u r s r e l a t i v e to n o r m a l fetal k i d n e y , a c c o r d i n g to c D N A m i c r o a r r a y a n a l y s i s o f 6 4 t u m o u r s a m p l e s ( S t a n h o p e - B a k e r et a l . , 2004). Surprisingly,  however,  p o d o c a l y x i n and W T 1  expression levels were  not  85  correlated, at least at the level of m R N A . It must be kept in mind, though, that protein levels, and the potential presence of WT1 mutations in these samples, have not yet been assessed. More in keeping with the other published literature, however, there was a significant increase in podocalyxin expression  in the more aggressive,  anaplastic  tumours. Since p53 is generally mutated in anaplastic Wilms' tumours, and in that it has been shown to negatively regulate podocalyxin expression, this may explain why podocalyxin is expressed more highly in these cases. Functionally, podocalyxin may contribute to the increased metastasis of this subset of tumours.  Podocalyxin has now been associated with a wide variety of cancers, and, strikingly, it is often associated with more aggressive cases. The most likely functional implication of podocalyxin overexpression is increased metastasis, although this has yet to be proven.  1.11 Thesis Objectives 1) The dramatic upregulation of podocalyxin in numerous cancers, and particularly in subsets with poor outcome, is an important area for further investigation. Understanding the functional role of podocalyxin in these situations may facilitate the design of new therapies. I have approached this goal by overexpressing podocalyxin in cell lines in order to determine its function. I have also generated a panel of podocalyxin mutants and overexpressed  these in vitro  as well. T h i s has helped to unravel  podocalyxin's  mechanism of action.  86  2) Although considerable information has been gained by studying normal podocalyxin expression patterns and podocalyxin-null mice, an in vivo gain-of-function model is lacking. I have therefore attempted to generate  transgenic mice  overexpressing  podocalyxin. I am using the versatile Cre-loxP system in order to create mice expressing podocalyxin in select tissues. For example, overexpression in mammary tissue will provide a model for deciphering podocalyxin's role in breast cancer. Alternatively, overexpression in vascular tissue may create leaky vessels. Thus, a single transgenic strain could be crossed to multiple Cre mice in order to produce mice overexpressing podocalyxin in numerous tissues.  3) The absolute requirement for podocalyxin expression in podocytes is important and interesting in itself, but it prevents assessment of podocalyxin-deficient tissues, such as the hematopoietic system and vasculature as well as the brain, in adult mice. I have therefore repaired the kidney defect in podocalyxin-null mice by specifically expressing podocalyxin in podocytes using a tissue-specific  promoter, with the intention of  investigating the effect of podocalyxin loss in other tissues.  87  CHAPTER 2 : MATERIALS AND METHODS  2.1 Cloning and Mutagenesis of Podocalyxin Murine podxl cDNA was a generous gift from Dr. David Kershaw; chicken podocalyxin cDNA was cloned from HD100 hematopoietic progenitor cells (McNagny et al., 1997).  2.1.1  Conditional Podocalyxin Overexpression Transgenic  Construct  Murine podxl cDNA was excised from pBluescript using BamHI and Xhol restriction enzymes, and the transgenic p C C A L L - 2 vector (generously provided by Dr. Corrinne Lobe) was digested with Bglll and Xhol. After ligation of murine podxl into this vector and in vitro assessment of podocalyxin expression in NSO cells, the strategy was modified slightly to include expression of the marker gene, GFP. Thus, the IRES-GFP sequence between Xhol and SacII was excised from the p C C A L L - 2 derivative Z/EG and ligated into the pCCALL-2-/?o<ix/ construct, such that podocalyxin and G F P were expressed concurrently.  2.1.2  Podocyte-Specific Podocalyxin Transgenic  Construct  The podocyte-specific transgenic construct was generated using the murine promoter  provided  by  Dr.  Sue  Quaggin.  Two  oligonucleotides  NPHSI (5'-  T C G A G C G G C C T T A A T T A A G - 3 ' and 5 ' - A A T T C T T A A T T A A G G C C G C - 3 ' ) were first annealed to generate a linker sequence containing a P a d restriction site. This was ligated  88  into the p I R E S 2 - E G F P vector ( B D B i o s c i e n c e s , M i s s i s s a u g a O N ) u s i n g the X h o l a n d E c o R l sites o f the m u l t i p l e c l o n i n g site. M u r i n e podxl  w a s t h e n e x c i s e d f r o m the  p B l u e s c r i p t S K c l o n i n g v e c t o r v i a the S a c I I a n d B g l l l sites a n d l i g a t e d i n t o p I R E S 2 E G F P between S a c I I and B a m H I . F i n a l l y , the m u r i n e NPHS1  p r o m o t e r w a s c l o n e d into  this p l a s m i d b e t w e e n X h o l and the n e w P a d site. A f t e r testing C M V p r o m o t e r - d r i v e n p o d o c a l y x i n e x p r e s s i o n f r o m this construct in vitro, X h o l and S f i l r e s t r i c t i o n e n z y m e s were used to isolate the NPHSl  promoter and m u r i n e podxl f o r generation o f transgenic  mice.  2.1.3  Murine Podocalyxin Expression Vector  T h e m u r i n e podxl e x p r e s s i o n vector used f o r in vitro studies w a s created as d e s c r i b e d i n section 2 . 1 . 2 , but the ubiquitous C M V p r o m o t e r w a s retained and the p o d o c y t e - s p e c i f i c NPHSI promoter w a s not i n c l u d e d .  2.1.4  Chicken Podocalyxin Expression Vectors  A construct had previously  been generated f o r e x p r e s s i o n o f a f u s i o n p r o t e i n that  i n c l u d e d the e x t r a c e l l u l a r ( N - t e r m i n a l ) p o r t i o n o f c h i c k e n podxl expression T C T C T C  vector  (pcDNA3. l-chMEP21Fc).  A C T T T C C  A G T C  PCR,  A T C G T C C - 3  '  i n the p c D N A 3 . 1  using  primers  a n d  5'5'-  G C T C T A G A G T T T C A G G G G G T T G T T T T T T G C - 3 ' , w a s u s e d to a m p l i f y the C - t e r m i n a l p o r t i o n o f c h i c k e n podxl  f r o m the o r i g i n a l p B l u e s c r i p t c l o n i n g c o n s t r u c t ( c l o n e 4 D 1  ( M c N a g n y et a l . , 1997)) a n d to a d d a n X b a l c l o n i n g site at its C - t e r m i n u s . T h i s P C R  89  product w a s ligated into the p C R 2 . 1 - T O P O c l o n i n g vector (Invitrogen) f r o m w h i c h it w a s t h e n e x c i s e d w i t h B a m H I a n d X b a l . T h i s f r a g m e n t w a s l i g a t e d into the same sites o f the p c D N A 3 . 1 - N - t e r m i n a l podxl construct, i n order to generate a f u l l - l e n g t h c h i c k e n podxl e x p r e s s i o n construct.  P o d o c a l y x i n e x p r e s s i o n l e v e l s w e r e l o w i n c e l l s transfected w i t h the pcDNA3.1-/?0<sW construct, so n e w constructs w e r e generated u s i n g the p I R E S 2 - E G F P e x p r e s s i o n vector. C h i c k e n podxl c D N A w a s a m p l i f i e d f r o m the p c D N A 3 . 1 - / ? o f l W construct u s i n g p r i m e r s 5 ' - C C A C T G C T T A C T G G C T T A T C G - 3 ' a n d 5 ' - A C A A C A G A T G G C T G G C A A C - 3'. T h e P C R product w a s l i g a t e d into the p C R 2 . 1 - T O P O c l o n i n g vector, e x c i s e d w i t h E c o R l , a n d l i g a t e d i n t o p I R E S 2 - E G F P . T h i s w a s then s e q u e n c e d after f i r s t c h e c k i n g the i n s e r t ' s orientation.  2.1.5 Chicken Podocalyxin Mutants Ala,  A s p , and A D T H L  c h i c k e n podxl  mutants were generated b y site-directed  mutagenesis u s i n g the T r a n s f o r m e r S i t e - D i r e c t e d M u t a g e n e s i s K i t ( C l o n t e c h , M o u n t a i n V i e w C A ) , a c c o r d i n g to m a n u f a c t u r e r ' s instructions. D r . R e g i s D o y o n n a s generated the a l a n i n e m u t a n t s b y s i t e - d i r e c t e d m u t a g e n e s i s o f the 4 D 1 p B l u e s c r i p t - c h i c k e n podxl c o n s t r u c t . I generated the other mutants as d e s c r i b e d b e l o w . T h e p c D N A 3 . 1 - c h i c k e n podxl v e c t o r w a s i n i t i a l l y used as a template f o r c r e a t i o n o f the aspartic a c i d mutants using primers 5 ' - C A C C A A C G C T T C G A C C A A A A G A A G - 3 ' AGGTGATGGAAGACGGCTCTGAAAT-3' CCTGAAACACTAGTGGGCCCGT-3'  (putative P K C site) a n d 5 ' -  ( p u t a t i v e C K I I s i t e ) , as w e l l as 5 ' -  f o r m u t a t i o n o f t h e X b a l site t o S p e l f o r  90  s e l e c t i o n purposes. R e d lettering denotes c o d o n s o f interest. H o w e v e r , c o n v e r s i o n o f the t h r e o n i n e r e s i d u e i n t h e p u t a t i v e C K I I site t o a n a s p a r t i c a c i d r e s i d u e  involved  m u t a g e n e s i s o f a l l three r e s i d u e s i n the c o d o n , w h i c h p r o v e d d i f f i c u l t . Instead, t h e p r e v i o u s l y mutated p c D N A 3 . 1 - c h i c k e n podxl C K I I - a l a n i n e mutant w a s e v e n t u a l l y used as a t e m p l a t e b e c a u s e c o n v e r s i o n o f the a l a n i n e r e s i d u e t o a s p a r t i c a c i d r e q u i r e d mutagenesis o f j u s t t w o residues.  Similarly, 5 ' - G A G G A C C T A G A G G A A T A G G A T A C G C A T T - 3 '  w a s u s e d to insert a  p r e m a t u r e stop c o d o n i m m e d i a t e l y p r e c e d i n g t h e C - t e r m i n a l D T H L  sequence b y  m u t a t i n g the last g l u t a m i c a c i d residue t o a stop c o d o n , thereby c r e a t i n g the A D T H L mutant.  T h e A t a i l mutant w a s created b y u s i n g the 4 D 1 p B l u e s c r i p t - c h i c k e n podxl construct as a P C R template and the primers 5 ' - T C T C T C A C T T T C C A G T C A T C G T C C - 3 ' and 5 ' G C T C T A G A G C C T A G A A G C G T T G G T G A C A G C A G C - 3 ' t o insert a p r e m a t u r e stop c o d o n after the j u x t a m e m b r a n e C C H Q R F sequence. T h e P C R p r o d u c t w a s then l i g a t e d i n t o p C R 2 . 1 - T O P O , e x c i s e d w i t h B a m H I and X b a l , and l i g a t e d i n t o the p c D N A 3 . 1 - N -  t e r m i n a l podxl construct.  T h e a l t e r n a t i v e l y s p l i c e d f o r m o f c h i c k e n podxl w a s generated b y a m p l i f i c a t i o n o f the C'terminal portion o f  podxl f r o m a p B l u e s c r i p t c l o n i n g v e c t o r c o n t a i n i n g podxl's s p l i c e  v a r i a n t ( 4 B 3 ( M c N a g n y et a l . , 1997)). T h e P C R p r o d u c t created w i t h the p r i m e r s 5 ' T C T C T C  A C T T T C C  A G T C  A T C G T C C - 3  '  a n d  5 ' -  91  G C T C T A G A C G G G A A A T A G G T T C T C C T T C T G C - 3 ' was ligated into p C R 2 . 1 - T O P O , excised with B a m H I and X b a l , and inserted into the p c D N A 3 . 1 - N - t e r m i n a l podxl construct.  In addition, the above variants were all later cloned into the p I R E S 2 - E G F P expression vector.  The  primers  5'-CCACTGCTTACTGGCTTATCG-3'  and  5'-  A C A A C A G A T G G C T G G C A A C - 3 ' were used to amplify all forms of chicken podxl from their respective p c D N A 3 . 1 constructs. The P C R products were inserted into p C R 2 . 1 T O P O , excised with E c o R l , and ligated into the multiple cloning site of p I R E S 2 - E G F P .  The last mutant, the flag-tagged podxl construct lacking the majority of the extracellular domain, was generated in several steps. The first step involved isolation of podxl's cytoplasmic tail, transmembrane region, and a small portion of its extracellular domain from the p I R E S 2 - E G F P - w t podxl construct using the H i n d l l l restriction sites, and insertion into the empty p I R E S 2 - E G F P vector. Secondly, the signal peptide and flag-tag were amplified, using the primers 5 ' - T A G C T A G C G A G A T G G C C T T G C A C C T T C T - 3 ' and 5 ' - T A C T C G A G G A T G C C G C C C T T A T C G T C - 3 ' ,  from a p M X - p i e expression  construct containing flag-tagged cd43, generously provided by Dr. Hermann Ziltener and Wooseok Seo. This P C R step also involved addition of N h e l and X h o l restriction sites to the ends of the P C R product. This product was then ligated into p C R 2 . 1 - T O P O and sequenced. The final step required isolation of the signal peptide and flag-tag from p C R 2 . 1 - T O P O using the N h e l and X h o l sites, and ligation into the same sites in the  92  p I R E S 2 - E G F P construct c o n t a i n i n g podxl's t r a n s m e m b r a n e region and c y t o p l a s m i c t a i l . A l l constructs were sequenced after c o m p l e t i o n .  2.2 Cell Culture 2.2.1  Culture Conditions  M C F - 7 and M D C K c e l l s were routinely m a i n t a i n e d in A d v a n c e d D - M E M / F 1 2 m e d i u m ( I n v i t r o g e n # 1 2 6 3 4 - 0 1 0 , B u r l i n g t o n O N ) s u p p l e m e n t e d w i t h 5 % fetal b o v i n e s e r u m ( F B S ) , H E P E S , g l u t a m i n e , p e n i c i l l i n , and s t r e p t o m y c i n .  N S O m o u s e m y e l o m a c e l l s ( g e n e r o u s l y p r o v i d e d by D r . G a r y M c L e a n and D r . J o h n Schrader) were r o u t i n e l y m a i n t a i n e d in D - M E M s u p p l e m e n t e d w i t h 10 % F B S , s o d i u m p y r u v a t e , n o n - e s s e n t i a l a m i n o a c i d s , g l u t a m i n e , p e n i c i l l i n , and s t r e p t o m y c i n . C H O c e l l s were c u l t u r e d i n D - M E M s u p p l e m e n t e d w i t h 10 % F B S , g l u t a m i n e , (3-mercaptoethanol, p e n i c i l l i n , and s t r e p t o m y c i n . F D C P - 1 cells were m a i n t a i n e d i n R P M I s u p p l e m e n t e d w i t h 10 % F B S , 2 % W E H I - 3 B  c o n d i t i o n e d m e d i u m as a source o f I L - 3 , g l u t a m i n e , (3-  mercaptoethanol, p e n i c i l l i n , and s t r e p t o m y c i n .  R l e m b r y o n i c stem c e l l s ( E S C s ) were r o u t i n e l y m a i n t a i n e d in D - M E M  supplemented  w i t h 15 % F B S , 10 ng/ml L I F , s o d i u m pyruvate, non-essential a m i n o a c i d s , g l u t a m i n e , (3m e r c a p t o e t h a n o l , p e n i c i l l i n , and s t r e p t o m y c i n . E S C s were cultured o n m o u s e e m b r y o n i c f i b r o b l a s t feeder layers or gelatin coated plates.  93  2.2.2  Transfection Techniques  M C F - 7 and M D C K  cells were transfected with 3 0 p g D N A  u s i n g the  DMRIE-C  t r a n s f e c t i o n reagent ( I n v i t r o g e n ) a c c o r d i n g to m a n u f a c t u r e r ' s i n s t r u c t i o n s . D N A  was  d i l u t e d in 3 0 0 p i s e r u m - f r e e D M E M / F 1 2 and s l o w l y a d d e d to 15 u l D M R I E - C ,  also  p r e v i o u s l y d i l u t e d i n 3 0 0 p i D M E M / F 1 2 . T h i s w a s m i x e d g e n t l y , i n c u b a t e d f o r 15 minutes, and diluted w i t h 2.4 m l D M E M / F 1 2 . T h e transfection m i x t u r e w a s transferred to 10 c m dishes c o n t a i n i n g 3 0 - 5 0 % c o n f l u e n t c e l l s and incubated f o r 6 - 8 hours. M e d i a was r e p l a c e d w i t h D M E M / F 1 2 c o n t a i n i n g 5 % F B S and g l u t a m i n e , and stable c l o n e s w e r e selected based o n resistance to 4 0 0 pg/ml G 4 1 8 .  15 x 10 N S 0 c e l l s were electroporated i n 5 0 0 u l c o l d P B S w i t h 2 0 p g D N A i n 0 . 4 c m 6  cuvettes ( 2 0 0 V , 9 5 0 p F , t i m e constant ~ 3 0 m s e c ) . C e l l s were then w a s h e d w i t h culture m e d i a , and replated as b u l k cultures and i n 9 6 w e l l plates f o r c l o n a l s e l e c t i o n . C u l t u r e i n 1.5 mg/ml G 4 1 8 was used to generate stable transfectants.  CHO  c e l l s w e r e t r a n s f e c t e d i n 6 0 m m d i s h e s w i t h the L i p o f e c t a m i n e P l u s reagent  (Invitrogen) a c c o r d i n g to m a n u f a c t u r e r ' s instructions. P l u s reagent (8 p i ) w a s added to 2 p g D N A , p r e v i o u s l y d i l u t e d i n 2 5 0 p i m e d i a l a c k i n g s e r u m and a n t i b i o t i c s . T h i s m i x t u r e was incubated f o r 15 minutes at r o o m temperature w h i l e 12 p i o f L i p o f e c t a m i n e reagent was d i l u t e d i n 2 5 0 p i serum-free m e d i a . T h e s e c o m p o n e n t s were c o m b i n e d and incubated f o r 15 m i n u t e s before they were transferred to plates o f c e l l s c o n t a i n i n g 2 m l o f f r e s h s e r u m - f r e e m e d i a . A f t e r 3 - h o u r i n c u b a t i o n at 3 7 ° C , 3 m l o f m e d i a c o n t a i n i n g s e r u m was added, and expression was assessed 2 days later.  94  5.6 x IO  6  E S C s w e r e electroporated i n 0.8 m l P B S c o n t a i n i n g 2 0 p g D N A ( 2 4 0 V , 5 0 0  p F , t i m e constant ~ 7 m s e c ) . C e l l s were then a l l o w e d to recover o n ice f o r 2 0 m i n u t e s , w a s h e d w i t h c u l t u r e m e d i a , a n d t r a n s f e r r e d to 2 1 0 - c m plates. S t a b l e c l o n e s w e r e obtained by c u l t u r i n g i n 150 pg/ml G 4 1 8 .  2.3 Expression Analysis 2.3.1  Antibodies  T h e antibodies used i n this thesis are s h o w n i n ( T a b l e 2 - 1 and T a b l e 2 - 2 ) .  95  Antibody a-ezrin  Type ms IgG!  Concentration 25 pg/ml  Procedure Confocal  Source 3C12: Abeam (Cambridge M A )  a-ezrin (biotin)  ms IgG,  25 pg/ml  Confocal  3C12: NeoMarkers (Fremont C A )  a-flag  ms IgG[  1 pg/ml  Western  M 2 : Sigma-Aldrich (Oakville O N )  a-flag (biotin)  ms IgG!  1 pg/ml  Confocal,  M 2 : Sigma-Aldrich  FC a-GFP  rb IgG  10 pg/ml  IF  Invitrogen  (pAb) a-NHERFl  rb (pAb)  3 u,g/ml  Confocal  Abeam  a-PECAM-1  rat I g G  5 pg/ml  IHC  B D Biosciences  a-ch podocalyxin  ms IgG[  neat  Confocal,  (McNagny et al.,  FC  1992)  Western  (McNagny et al.,  a-ch podocalyxin  2a  ms IgGi  1:5  1992) a-ms podocalyxin  rat IgG!  5 pg/ml  Confocal,  M B L (Woburn M A )  FC a-ms podocalyxin  rat IgG,  5 pg/ml  IHC  (Hara et al., 1999)  ms IgG;  Isotype  25 pg/ml  Confocal  D A K O (Mississauga  control  ON)  96  Antibody ms I g G , (biotin)  Type Isotype  Concentration 25 u.g/ml  Confocal  Isotype  5 p,g/ml  FC, IHC  Isotype  Cedarlane (Hornby ON)  control rb I g G  R & D Systems (Minneapolis M N )  control rat I g G ,  Source  Procedure  3 pg/ml  Confocal  Jackson ImmunoResearch  control  Laboratories (Westgrove P A ) phalloidin  Alexa  25 units/ml  Confocal  Invitrogen  Fluor-568  Table 2 - 1 : Primary Antibodies used in this Thesis.  p A b : p o l y c l o n a l antibody c h : c h i c k e n , rb: rabbit, m s : mouse F C : flow cytometry, IF: immunofluorescence, I H C : immunohistochemistry.  97  Antibody  Label  Concentration  Source  Procedure  a - m s Ig  APC  2 pg/ml  FC  B D Biosciences  a - m s Ig  HRP  0.1 pg/ml  Western  DAKO  a - m s IgG,  A l e x a Fluor 488  8 - 2 0 pg/ml  Confocal  Invitrogen  a - m s IgG[  A l e x a Fluor 568  2 0 pg/ml  Confocal  Invitrogen  a-rat IgG  biotin  1 u,g/ml  FC  Southern B i o t e c h (Birmingham A L )  a-rat IgG  biotin  5 pg/ml  IHC  Vector Laboratories (Burlington O N )  a-rb IgG  A l e x a Fluor 488  2 0 u.g/ml  Confocal  Invitrogen  a - r b IgG  A l e x a Fluor 488  4 pg/ml  IF  Invitrogen  streptavidin  A l e x a Fluor 568  5 pg/ml  Confocal  Invitrogen  streptavidin  APC  0.5 pg/ml  FC  B D Biosciences  streptavidin  PE  1.25 p,g/ml  FC  B D Biosciences  Table 2-2: Secondary Antibodies used in this Thesis. A P C : a l l o p h y c o c y a n i n , H R P : horse radish peroxidase, P E : p h y c o e r y t h r i n rb: rabbit, m s : mouse F C : f l o w c y t o m e t r y , IF: i m m u n o f l u o r e s c e n c e , I H C : i m m u n o h i s t o c h e m i s t r y .  98  2.3.2  Flow Cytometry and Cell Sorting  C e l l s were w a s h e d w i t h F A C S b u f f e r ( 1 0 % F B S a n d 0 . 0 5 % s o d i u m a z i d e i n P B S ) , labelled with primary antibodies (Table 2-1) f o r 2 0 - 3 0 minutes o n i c e , washed with F A C S buffer, i n c u b a t e d w i t h s e c o n d a r y a n t i b o d i e s ( T a b l e 2 - 2 ) f o r 2 0 m i n u t e s o n i c e , w a s h e d a g a i n , and stained w i t h the v i a b i l i t y m a r k e r 7 - a m i n o - a c t i n o m y c i n D ( 7 A A D ) ( B D B i o s c i e n c e s , M i s s i s s a u g a O N ) . In m o s t e x p e r i m e n t s , 10 0 0 0 v i a b l e c e l l s were c o l l e c t e d with a F A C S Calibur flow cytometer (Becton D i c k i n s o n ) and analyzed with  FlowJo  software (Tree Star Inc, A s h l a n d O R ) . C e l l sorting w a s p e r f o r m e d b y A n d y J o h n s o n w i t h a B D F A C S V a n t a g e c e l l sorter i n the B i o m e d i c a l R e s e a r c h C e n t r e ' s F A C S f a c i l i t y .  MCF-7  c e l l s t r a n s f e c t e d w i t h m u r i n e podxl  w e r e a s s e s s e d u s i n g rat a n t i - m o u s e  p o d o c a l y x i n , biotinylated anti-rat I g G , , and phycoerythrin ( P E ) - or a l l o p h y c o c y a n i n ( A P C ) - c o n j u g a t e d streptavidin ( S A ) . M C F - 7 cells transfected w i t h c h i c k e n podxl mutants were assessed u s i n g m o u s e a n t i - c h i c k e n p o d o c a l y x i n o r b i o t i n y l a t e d m o u s e a n t i - f l a g and A P C - c o n j u g a t e d a n t i - m o u s e Ig or A P C - c o n j u g a t e d S A . C e l l s transfected w i t h the empty vector were used as negative controls f o r p o d o c a l y x i n s t a i n i n g .  T r a n s f e c t e d E S C s were assessed f o r m u r i n e p o d o c a l y x i n e x p r e s s i o n u s i n g rat a n t i - m o u s e p o d o c a l y x i n , b i o t i n y l a t e d anti-rat I g G [ , a n d p h y c o e r y t h r i n ( P E ) - c o n j u g a t e d  streptavidin  ( S A ) . R a t IgG[ was used as a negative c o n t r o l f o r p o d o c a l y x i n s t a i n i n g .  99  2.3.3  Western blotting  Cells  were  lysed  with  RIPA  buffer  plus  protease  inhibitors,  including  p h e n y l m e t h a n e s u l f o n y l f l u o r i d e ( P M S F ) and a protease i n h i b i t o r c o c k t a i l ( S i g m a - A l d r i c h # P 8 3 4 0 ) . R I P A buffer contained 10 m M phosphate buffer, 150 m M s o d i u m c h l o r i d e , 1 % NP-40,  1 %  sodium deoxycholate,  0.1 %  sodium dodecyl  sulfate ( S D S ) , 2  mM  e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A ) , and 5 0 m M s o d i u m f l u o r i d e . P r o t e i n s w e r e s e p a r a t e d by  SDS-polyacrylamide  gel electrophoresis  ( P A G E ) and transferred  to  n i t r o c e l l u l o s e m e m b r a n e s . M e m b r a n e s were b l o c k e d f o r 3 0 m i n u t e s w i t h 5 % s k i m m i l k in T r i s buffered saline ( T B S )  p l u s 0 . 0 5 % T w e e n 2 0 ( T B S - T ) , f o l l o w e d by a 2 - h o u r  i n c u b a t i o n i n p r i m a r y a n t i b o d y ( m o u s e a n t i - f l a g or m o u s e a n t i - c h i c k e n  podocalyxin)  d i l u t e d i n T B S - T and 3 5 - m i n u t e washes w i t h T B S - T ( T a b l e 2 - 1 ) . M e m b r a n e s were then p r o b e d w i t h s e c o n d a r y a n t i b o d y (horse r a d i s h p e r o x i d a s e - c o u p l e d a n t i - m o u s e  IgG)  d i l u t e d i n 2 - 3 % B S A in T B S - T f o r 4 5 minutes and w a s h e d 3 x 5 m i n u t e s w i t h T B S - T ( T a b l e 2 - 2 ) . W e s t e r n blots were d e v e l o p e d after e x p o s i n g f i l m ( K o d a k ) to m e m b r a n e s that were p r e v i o u s l y incubated w i t h enhanced c h e m i l u m i n e s c e n c e ( E C L ) reagent ( P e r k i n E l m e r , W o o d b r i d g e O N ) f o r 1 minute.  2.3.4  RT-PCR  m R N A w a s isolated u s i n g p M A C S m R N A i s o l a t i o n kits ( M i l t e n y i B i o t e c , A u b u r n  CA)  a c c o r d i n g to manufacturers i n s t r u c t i o n s . c D N A was generated u s i n g T h e r m o s c r i p t  RT-  P C R kits (Invitrogen) a c c o r d i n g to m a n u f a c t u r e r ' s instructions. M u r i n e podxl c D N A was detected by P C R w i t h p r i m e r s 5 ' - G A G G A T T T G T G C A C T C T A C A T G T G - 3 ' a n d 5 ' -  100  TACTCGAGTGGGTTGTCATGGTAACC-3'. AAGTTCATCTGCACCACCG-3'  and  G F P w a s detected w i t h p r i m e r s 5 ' -  5'-TCCTTGAAGAAGATGGTGCG-3'.  H y p o x a n t h i n e p h o s p h o r i b o s y l transferase ( H P R T ) was used as a positive c o n t r o l , w i t h 5 ' CTCGAAGTGTTGGATACAGG-3'and  5 ' - T G G C C T A T A G G C T C A T A G T G 3 ' primers.  2.4 Additional Characterization of ESC Clones for Conditional Podocalyxin Overexpressing Transgenic 2.4.1  p-galactosidase Assay  In order to assess E S C clones f o r (3-glactosidase e x p r e s s i o n , they were w a s h e d w i t h P B S , and f i x e d f o r 5 m i n u t e s at r o o m temperature i n 0 . 2 % g l u t a r a l d e h y d e , 0.01 % s o d i u m deoxycholate, 0.02 %  NP-40,  100 m m m a g n e s i u m c h l o r i d e , a n d 5 m m e t h y l e n e -  bis(oxyethylenenitrilo)tetraacetic a c i d ( E G T A ) i n P B S . C e l l s were then w a s h e d 3 t i m e s and stained overnight  at 3 7 ° C u s i n g 1 m g / m l X - g a l , 5 m M K F e ( C N ) , 5 3  6  mM  K F e ( C N ) , and 2 m M m a g n e s i u m c h l o r i d e i n P B S . 4  2.4.2  6  PCR to Detect Transgene  Genomic G A G G  transgenic  m u r i n e podxl  was  A T T T G T G C A C T C T A C  detected  A T G T G - 3  TACTCGAGTGGGTTGTCATGGTAACC-3'. AAGTTCATCTGCACCACCG-3'  and  by  PCR '  with  primers  and  5'5 ' -  G F P was detected w i t h primers 5 ' -  5'-TCCTTGAAGAAGATGGTGCG-3'.  101  2.5 Adhesion Assays M C F - 7 c e l l s were plated i n triplicate in 6 w e l l plates (5 x 1 0 c e l l s per w e l l ) and cultured 5  f o r 2 4 hours. S u s p e n s i o n c e l l s i n c l u d e d those in culture m e d i a as w e l l as those obtained after w a s h i n g w i t h P B S f o r 1 m i n u t e . A d h e r e n t c e l l s were r e m o v e d w i t h t r y p s i n , d i l u t e d w i t h m e d i a , and counted as w e l l .  2.6 Confocal Microscopy C e l l s were cultured on glass c o v e r slips and f i x e d f o r 2 0 minutes w i t h 37 °C preheated 4 % P F A . A l l subsequent steps w e r e p e r f o r m e d at r o o m temperature. C e l l s w e r e then rehydrated w i t h P B S , p e r m e a b i l i z e d w i t h 0.1 % T r i t o n X - 1 0 0 f o r 1 0 - 1 5 m i n u t e s (or 0.5 % T r i t o n X - 1 0 0 f o r 10 minutes f o r actin l a b e l l i n g ) , and w a s h e d 2 x 1 0 minutes w i t h P B S before i m m u n o l a b e l l i n g . C o v e r slips were b l o c k e d f o r 4 0 minutes w i t h 10 % goat s e r u m and 1 % B S A i n P B S , and then r i n s e d b r i e f l y w i t h P B S . S a m p l e s were i n c u b a t e d w i t h p r i m a r y antibodies ( T a b l e 2 - 1 ) i n 1 % B S A f o r 1 hour, f o l l o w e d by 4 1 0 - m i n u t e washes w i t h P B S or 1 % B S A i n P B S . S e c o n d a r y antibodies ( T a b l e 2 - 2 ) were also d i l u t e d i n 1 % B S A in P B S and incubated w i t h samples f o r 1 hour, f o l l o w e d by a single 1 0 - m i n u t e w a s h w i t h P B S or 1 % B S A i n P B S . F o r actin l a b e l l i n g , A l e x a F l u o r 5 6 8 - c o n j u g a t e d p h a l l o i d i n (in 1 % B S A ) was added f o r 15 minutes at this stage, f o l l o w e d by a 1 0 - m i n u t e w a s h w i t h P B S . N u c l e i were l a b e l l e d w i t h 0.5 pg/ml 4 ' , 6 ' - d i a m i d i n o - 2 - p h e n y l i n d o l e ( D A P I ) f o r 2 m i n u t e s , and c e l l s were w a s h e d w i t h P B S or 1 % B S A i n P B S 4 x 10 m i n u t e s . C o v e r s l i p s were m o u n t e d w i t h f l u o r o m o u n t - G ( S o u t h e r n B i o t e c h ) . C e l l s transfected w i t h the  102  empty vector w e r e used as negative c o n t r o l s f o r p o d o c a l y x i n s t a i n i n g . R a b b i t I g G w a s used as a negative control f o r N H E R F - 1 s t a i n i n g .  B i o t i n y l a t e d e z r i n and p o d o c a l y x i n d u a l l a b e l l i n g r e q u i r e d a d d i t i o n a l steps to prevent c r o s s - r e a c t i v i t y and b a c k g r o u n d f r o m e n d o g e n o u s b i o t i n . S t r e p t a v i d i n w a s added d u r i n g the b l o c k i n g step at 1 pg/ml to b l o c k c e l l u l a r b i o t i n , a n d this w a s f o l l o w e d by 3 1 0 minute washes w i t h P B S to r e m o v e excess u n b o u n d streptavidin. In contrast to other dual l a b e l l i n g e x p e r i m e n t s , in this case antigens had to be l a b e l l e d s e q u e n t i a l l y . P o d o c a l y x i n w a s l a b e l l e d first, w i t h the p r i m a r y a n t i b o d y against c h i c k e n p o d o c a l y x i n f o l l o w e d by A l e x a F l u o r - 4 8 8 - c o n j u g a t e d a n t i - m l g G , . C o v e r slips were then b l o c k e d w i t h 10 % m o u s e s e r u m and 1 % B S A i n P B S f o r 4 0 m i n u t e s f o l l o w e d by t w o a d d i t i o n a l P B S w a s h e s . E z r i n w a s then l a b e l l e d w i t h b i o t i n y l a t e d a n t i - e z r i n f o l l o w e d by A l e x a F l u o r - 5 6 8 conjugated streptavidin before D A P I l a b e l l i n g , as d e s c r i b e d above. C e l l s transfected w i t h the empty vector were used as negative c o n t r o l s f o r p o d o c a l y x i n s t a i n i n g . M o u s e  IgG[  was used as a negative control f o r e z r i n s t a i n i n g .  S l i d e s were e x a m i n e d u s i n g an O l y m p u s F l u o v i e w F V 1 0 0 0 c o n f o c a l m i c r o s c o p e ( 1 0 0 X o i l i m m e r s i o n o b j e c t i v e , N A : 1.35, z o o m : 1.4 or 6 0 X o i l i m m e r s i o n o b j e c t i v e , N A : 1.40). T r i p l e - l a b e l l e d i m a g e s were c o l l e c t e d s e q u e n t i a l l y , and c o n f o c a l sections were a c q u i r e d i n 0 . 1 8 0 [xm o r 0 . 2 3 0 p m steps. M i c r o g r a p h s w e r e generated w i t h several m e r g e d c o n f o c a l planes or vertical sections o f c o n f o c a l stacks u s i n g O l y m p u s F l u o v i e w F Y 1000 s o f t w a r e ( v e r s i o n 1.3b). P h o t o s w e r e a r r a n g e d w i t h A d o b e P h o t o s h o p and  Adobe  Illustrator software.  103  2.7 Scanning Electron Microscopy (SEM) C e l l s were g r o w n o n glass c o v e r s l i p s and f i x e d u s i n g 2.5 % g l u t a r a l d e h y d e w i t h 1 % tannic a c i d i n 0.1 M c a c o d y l a t e buffer. S E M p r o c e s s i n g was p e r f o r m e d by D e r r i c k H o m e i n the B i o l m a g i n g F a c i l i t y at the U n i v e r s i t y o f B r i t i s h C o l u m b i a : samples were p o s t - f i x e d w i t h b u f f e r e d 1 % o s m i u m t e t r o x i d e , d e h y d r a t e d i n a graded series o f e t h a n o l s , a n d c r i t i c a l p o i n t d r i e d . I m a g e s w e r e then c o l l e c t e d u s i n g a H i t a c h i S 4 7 0 0 F E S E M , a n d photos were arranged u s i n g A d o b e P h o t o s h o p and A d o b e Illustrator software.  2.8 Transmission Electron Microscopy (TEM) 2.8.1  TEM of Cell Lines  C e l l s were g r o w n o n filters (1 urn pore size: B e c t o n D i c k i n s o n ) and then f i x e d f o r 1 hour i n 1.5 % g l u t a r a l d e h y d e and 1.5 % p a r a f o r m a l d e h y d e in 0.1 M s o d i u m c a c o d y l a t e buffer ( p H 7 . 3 ) . T h e f i l t e r s w e r e w a s h e d w i t h 0.1 M s o d i u m c a c o d y l a t e b u f f e r , and then postf i x e d f o r 3 0 m i n u t e s on ice i n b u f f e r e d 1 % o s m i u m tetroxide. T h e y were w a s h e d w i t h d i s t i l l e d water and stained en bloc f o r 3 0 m i n u t e s w i t h 1 % u r a n y l acetate. S a m p l e s were then dehydrated through a graded series o f ethanols and infiltrated w i t h p r o p y l e n e o x i d e and P o l y b e d . A f t e r e m b e d d i n g in P o l y b e d , the samples were p o l y m e r i z e d f o r 2 4 hours at 6 0 ° C . T h i n s e c t i o n s w e r e p r e p a r e d , stained w i t h u r a n y l acetate and l e a d citrate, and v i e w e d and p h o t o g r a p h e d o n a P h i l i p s 3 0 0 electron m i c r o s c o p e operated at 6 0 k V by A . W a y n e V o g l at the U n i v e r s i t y o f B r i t i s h C o l u m b i a . N e g a t i v e s were scanned into d i g i t a l  104  f o r m a t and contrast adjusted u s i n g the Image A d j u s t m e n t s t o o l . F i g u r e s were arranged using A d o b e P h o t o s h o p and A d o b e Illustrator software.  2.8.2 T E M of Kidneys K i d n e y s w e r e i s o l a t e d f r o m E 1 8 m i c e , cut i n t o 4 - 6 p i e c e s , a n d f i x e d i n 2 . 5  %  glutaraldehyde i n 0.1 M cacodylate buffer o v e r n i g h t at 4 ° C . D e r r i c k H o m e c o m p l e t e d the p r o c e s s i n g and i m a g i n g at the U n i v e r s i t y o f B r i t i s h C o l u m b i a ' s B i o l m a g i n g F a c i l i t y . Samples were washed with cacodylate buffer, post-fixed with 1 % o s m i u m tetroxide, w a s h e d w i t h d i s t i l l e d water, stained en bloc w i t h 2 % u r a n y l acetate, and rinsed w i t h d i s t i l l e d water. S a m p l e s were then d e h y d r a t e d u s i n g a graded series o f e t h a n o l s and e m b e d d e d i n resin. T h i n sections were i m a g e d u s i n g a H i t a c h i H 7 6 0 0 T E M , and f i g u r e s were arranged u s i n g A d o b e P h o t o s h o p and A d o b e Illustrator software.  2.9 Immunofluorescence of Tissue Sections 2.9.1  Sample Preparation  M i c e were anesthetized w i t h 2 5 mg/ml avertin i n t r a p e r i t o n e a l l y and p e r f u s e d w i t h 4 % p a r a f o r m a l d e h y d e ( P F A ) . T i s s u e s were r e m o v e d and f i x e d i n 2 % P F A f o r 2 hours at 4 ° C , f o l l o w e d by i n c u b a t i o n i n 2 0 % s u c r o s e o v e r n i g h t at 4 ° C . E x c e s s l i q u i d w a s r e m o v e d , s a m p l e s were p l a c e d in T i s s u e T e k e m b e d d i n g m e d i u m ( S a k u r a F i n e t e k U S A I n c , T o r r a n c e C A ) , f r o z e n i n l i q u i d n i t r o g e n , a n d stored at - 8 0 ° C . T i s s u e s  were  105  sectioned u s i n g a cryostat set at 6 - 1 2 p m , d r i e d o v e r n i g h t , and rehydrated i n P B S f o r 1 hour at 4 °C.  2.9.2  Tissue Staining  T i s s u e sections were b l o c k e d o v e r n i g h t w i t h 2 5 % goat s e r u m and 10 % B S A i n 0.3 % T r i t o n X - 1 0 0 i n P B S . S e c t i o n s were then incubated w i t h a n t i - G F P ( T a b l e 2 - 1 ) i n 10 % goat s e r u m , 10 % B S A , and 0.3 % T r i t o n X - 1 0 0 i n P B S f o r 1.5-2 hours at 4 °C. S a m p l e s were w a s h e d 5 x 12 m i n u t e s w i t h 0.3 % T r i t o n X - 1 0 0 i n P B S at 4 ° C . S e c t i o n s w e r e i n c u b a t e d w i t h s e c o n d a r y a n t i b o d y ( A l e x a F l u o r - 4 8 8 - c o n j u g a t e d goat a n t i - r a b b i t I g G ) ( T a b l e 2 - 2 ) i n 10 % goat s e r u m , 10 % B S A , and 0.3 % T r i t o n X - 1 0 0 i n P B S f o r 1 . 5 - 2 hours at 4 ° C a n d w a s h e d as a b o v e . C o v e r s l i p s w e r e m o u n t e d w i t h f l u o r o m o u n t - G . T i s s u e s e c t i o n s f r o m C 5 7 B L / 6 ( B 6 ) m i c e w e r e used as n e g a t i v e c o n t r o l s f o r G F P staining. S l i d e s were e x a m i n e d u s i n g a Z e i s s m i c r o s c o p e w i t h a 4 0 X objective o r a 6 3 X o i l i m m e r s i o n objective.  2.10  Immunohistochemistry  T i s s u e s were e m b e d d e d i n T i s s u e T e k , f r o z e n i n l i q u i d n i t r o g e n , and stored at - 8 0 ° C . F r o z e n s a m p l e s were s e c t i o n e d , f i x e d i n acetone, and rehydrated i n P B S . S a m p l e s were stained u s i n g the V e c t a s t a i n A B C K i t , a c c o r d i n g to m a n u f a c t u r e r ' s instructions ( V e c t o r Laboratories). Slides were blocked with  10 % F B S i n P B S , l a b e l l e d w i t h  primary  antibodies ( a n t i - m o u s e p o d o c a l y x i n o r P E C A M - 1 ) f o r 3 0 - 4 5 minutes ( T a b l e 2 - 1 ) , w a s h e d w i t h P B S , labelled w i t h secondary antibody (biotinylated anti-rat I g G ) ( T a b l e 2 - 2 ) f o r 3 0 -  106  4 5 m i n u t e s , and w a s h e d a g a i n . R a t I g G , w a s used as a negative control f o r p o d o c a l y x i n and P E C A M - 1  s t a i n i n g . S l i d e s w e r e then i n c u b a t e d i n 0 . 3 % h y d r o g e n p e r o x i d e i n  methanol f o r 1 5 - 3 0 minutes to q u e n c h endogenous peroxidase. T h i s w a s f o l l o w e d by 2 3 minute P B S w a s h e s , i n c u b a t i o n i n the A B C reagent f o r 3 0 - 4 5 m i n u t e s , and another P B S w a s h . S l i d e s were d e v e l o p e d w i t h the d i a m i n o b e n z i d i n e ( D A B ) substrate, and w a s h e d i n P B S . N u c l e i were l a b e l l e d w i t h 1 % m e t h y l green f o r 2 - 3 minutes and slides were w a s h e d w i t h P B S and dilute a m m o n i a . S a m p l e s were then dehydrated through a graded series o f ethanols, f o l l o w e d b y methanol and x y l e n e , and c o v e r s l i p s were sealed w i t h P e r m o u n t . Slides were examined using a Zeiss microscope with a 2 0 X objective or a 6 3 X o i l i m m e r s i o n objective.  2.11 Mice A l l m i c e w e r e m a i n t a i n e d o n a B 6 b a c k g r o u n d i n the B i o m e d i c a l R e s e a r c h C e n t r e ' s mouse f a c i l i t y . M i c e were genotyped f o r the w i l d t y p e o r k n o c k o u t allele o f podxl b y P C R using  primers  5 ' - G A G G A T T T G T G C A C T C T A C A T G T G - 3 ' ,  T A T C G C C T T C T T G A C G A G T T C T T - 3 ' , and  5 ' -  5'-AGTGAGAGACACATTGGGTAACT-  3 ' . T h e first p r i m e r is a c o m m o n p r i m e r , w i t h a sequence f o u n d in the f i f t h e x o n o f podxl, the s e c o n d p r i m e r ' s sequence is f o u n d i n the neomycin resistance cassette o f k n o c k o u t m i c e , and the sequence o f the third p r i m e r is f o u n d i n the f i f t h intron o f w i l d t y p e a n i m a l s . T h e e x p e c t e d P C R p r o d u c t s are 5 5 0 b p ( k n o c k o u t ) a n d 7 6 0 b p ( w i l d t y p e ) i n l e n g t h . P o d o c y t e - s p e c i f i c p o d o c a l y x i n t r a n s g e n i c m i c e w e r e g e n o t y p e d f o r p r e s e n c e o f the transgene u s i n g the G F P - s p e c i f i c p r i m e r s 5 ' - A A G T T C A T C T G C A C C A C C G - 3 '  and 5 ' -  T C C T T G A A G A A G A T G G T G C G - 3 ' , w h i c h produced a 3 6 0 b p P C R product.  107  CHAPTER 3 : OVEREXPRESSION OF WILDTYPE AND MUTANT PODOCALYXIN IN EPITHELIAL C E L L S  3.1  Rationale  A t the outset o f this study, there w e r e o n l y t w o p r e v i o u s p u b l i c a t i o n s based o n in vitro a n a l y s i s o f p o d o c a l y x i n ' s f u n c t i o n . T h e f i r s t d e s c r i b e d p o d o c a l y x i n as a l i g a n d f o r L s e l e c t i n i n H E V (Sassetti et a l . , 1 9 9 8 ) , w h i l e the s e c o n d suggested that p o d o c a l y x i n i n h i b i t e d c e l l - c e l l a d h e s i o n and disrupted c e l l j u n c t i o n s ( T a k e d a et a l . , 2 0 0 0 ) . It was clear that further a n a l y s i s w a s r e q u i r e d i n order to d e c i p h e r p o d o c a l y x i n ' s f u n c t i o n a n d to understand its r o l e s m e c h a n i s t i c a l l y . L a t e r papers p r o v i d e d b i o c h e m i c a l e v i d e n c e to suggest that p o d o c a l y x i n c o u l d interact w i t h the a c t i n c y t o s k e l e t o n t h r o u g h e z r i n a n d m e m b e r s o f the N H E R F f a m i l y o f adapter proteins ( L i et a l . , 2 0 0 2 ; O r l a n d o et a l . , 2 0 0 1 ; S c h m i e d e r et a l . , 2 0 0 4 ; T a k e d a et a l . , 2 0 0 1 ) , but the f u n c t i o n a l i m p l i c a t i o n s o f these interactions were unclear. P o d o c a l y x i n w a s therefore expressed i n numerous c e l l lines i n order to c l a r i f y its f u n c t i o n and m e c h a n i s m o f action.*  •  S o m e data presented i n this chapter can be f o u n d i n the f o l l o w i n g articles:  1) N i e l s e n , J . S . , M c C o y , M L . , C h e l l i a h , S . , V o g l , A . W . , R o s k e l l e y , C D . , a n d M c N a g n y , K . M . T h e C D 3 4 - r e l a t e d m o l e c u l e , P o d o c a l y x i n , is a potent i n d u c e r o f m i c r o v i l l u s f o r m a t i o n . Proc Natl Acad Sci USA,  submitted.  2) S o m a s i r i , A . , N i e l s e n , J . S . , M a k r e t s o v , N . , M c C o y , M . L . , P r e n t i c e , L . , G i l k s , C . B . , C h i a , S . K . , G e l m o n , K . A . , Kershaw, D . B . , Huntsman, D . G . , M c N a g n y , K . M . and Roskelley, C D . (2004) Overexpression  o f the a n t i - a d h e s i n p o d o c a l y x i n  is a n  independent predictor o f breast cancer progression. Cancer Res, 64, 5 0 6 8 - 5 0 7 3 .  108  In a d d i t i o n , a p a r t i c u l a r l y interesting feature o f p o d o c a l y x i n ' s n o r m a l e x p r e s s i o n pattern is its t e n d e n c y  to be e x p r e s s e d i n c e l l s w i t h  unique and intricate cell  surface  m o r p h o l o g i e s . F o r e x a m p l e , p o d o c a l y x i n is expressed by m e g a k a r y o c y t e s , w h i c h extend l o n g processes w h e n generating platelets ( M c N a g n y et a l . , 1 9 9 7 ; M i e t t i n e n et a l . , 1999). A s d i s c u s s e d i n s e c t i o n 1.6, it is a l s o e s s e n t i a l f o r f o r m a t i o n o f the elaborate f o o t p r o c e s s e s a s s o c i a t e d w i t h m a t u r e k i d n e y p o d o c y t e s . A s s e s s i n g the p o s s i b i l i t y p o d o c a l y x i n h a v i n g a m o r e g l o b a l role i n r e g u l a t i n g c e l l s u r f a c e m o r p h o l o g y  of was  therefore another m a j o r goal o f this study.  K i d n e y and breast e p i t h e l i a l c e l l lines were the m a i n m o d e l systems used f o r our in vitro studies since i n a d d i t i o n to p o d o c a l y x i n ' s essential role in n o r m a l k i d n e y d e v e l o p m e n t ( D o y o n n a s et a l . , 2 0 0 1 ) , o u r early studies a l s o d e s c r i b e d p o d o c a l y x i n as a p r e d i c t i v e m a r k e r o f poor o u t c o m e in h u m a n breast cancer ( r e v i e w e d i n section 1.10.1) ( S o m a s i r i et a l . , 2 0 0 4 ) . S p e c i f i c a l l y , t w o c e l l lines were used f o r the m a j o r i t y o f e x p e r i m e n t s : M D C K and M C F - 7 c e l l s . M D C K c e l l s were chosen because they are a w e l l - c h a r a c t e r i z e d k i d n e y c e l l l i n e that had been used i n other p o d o c a l y x i n - r e l a t e d p u b l i c a t i o n s ( L i et a l . , 2 0 0 2 ; O r l a n d o et a l . , 2 0 0 1 ) . M C F - 7 breast c a r c i n o m a c e l l s were used since they are a w e a k l y i n v a s i v e e p i t h e l i a l c e l l l i n e that expresses l o w l e v e l s o f e n d o g e n o u s p o d o c a l y x i n , i n c o m p a r i s o n to the more i n v a s i v e , h i g h l y p o d o c a l y x i n - p o s i t i v e M D A - 2 3 1 c e l l l i n e ( F i g u r e 3 - 1 ) ( S o m a s i r i et a l . , 2 0 0 4 ) . O f note, p o d o c a l y x i n is absent f r o m the n o n - i n v a s i v e T 4 7 D breast c e l l l i n e . T h u s , M D C K and M C F - 7 c e l l lines were appropriate f o r o v e r e x p r e s s i o n studies to address the f u n c t i o n o f p o d o c a l y x i n .  109  F i g u r e 3-1:  UokD—|  ••—Podocalyxin  42kDH  $=ERK 1/2  Endogenous  Human  Podocalyxin Expression i n Breast C a r c i n o m a  Lines. Western blot analysis demonstrates that metastatic M D A - 2 3 1 cells express considerably higher levels of podocalyxin than the less invasive T 4 7 D and M C F - 7 cell lines (Somasiri et al., 2004).  110  A f t e r f i n d i n g a s u i t a b l e assay s y s t e m f o r a s s e s s i n g p o d o c a l y x i n ' s f u n c t i o n , v a r i o u s p o d o c a l y x i n mutants were generated a n d tested u s i n g this system. T h e s e studies p r o v i d e d important m e c h a n i s t i c insights into p o d o c a l y x i n ' s f u n c t i o n s .  3.2  Expression  of Full-Length  Podocalyxin  in MCF-7 and  MDCK  Cells  3.2.1 Expression Analysis M u r i n e p o d o c a l y x i n w a s c l o n e d i n t o the p I R E S 2 - E G F P e x p r e s s i o n v e c t o r to e n a b l e e c t o p i c e x p r e s s i o n in vitro. T h i s v e c t o r f a c i l i t a t e s e x p r e s s i o n o f a n y gene o f interest, a l o n g w i t h G F P v i a an internal r i b o s o m e entry site ( I R E S ) s e q u e n c e . In a d d i t i o n , a n e o m y c i n resistant cassette a l l o w s f o r selection o f stably expressing transfectants.  M C F - 7 a n d M D C K c e l l s were transfected w i t h the p o d o c a l y x i n - c o n t a i n i n g p l a s m i d , o r the empty vector as a c o n t r o l . Stably transfected clones were isolated based o n e x p r e s s i o n o f p o d o c a l y x i n or G F P i n c o m b i n a t i o n w i t h the n e o m y c i n - r e s i s t a n c e gene ( F i g u r e 3 - 2 ) .  Ill  1  uu Empty vector Podocalyxin  Podocalyxin Figure 3-2: Ectopic Murine Podocalyxin Expression in Transfected MCF-7 Breast Epithelial Cells.  112  3.2.2  Podocalyxin Decreases Cell Adhesion in Epithelial Cells  U p o n i n i t i a l e x a m i n a t i o n o f transfected c e l l s , the most o b v i o u s d i f f e r e n c e between those t r a n s f e c t e d w i t h p o d o c a l y x i n or e m p t y v e c t o r w a s that c e l l s e c t o p i c a l l y e x p r e s s i n g p o d o c a l y x i n f o r m e d large clusters o f s u s p e n s i o n c e l l s rather than c o n f l u e n t m o n o l a y e r s o f adherent c e l l s , as s h o w n in F i g u r e 3 - 3 . Q u a n t i t a t i v e e x p e r i m e n t s revealed that there w e r e c l o s e to f o u r t i m e s as m a n y c e l l s i n s u s p e n s i o n i n the p o d o c a l y x i n - t r a n s f e c t e d p o p u l a t i o n ( F i g u r e 3 - 4 ) . T h e s e c e l l s m a i n t a i n e d v i a b i l i t y , as assessed by f l o w c y t o m e t r y and r e p l a t i n g o f d i s a g g r e g a t e d c e l l s . T h e s e e x p e r i m e n t s d i d not e x p l a i n , h o w e v e r , the m e c h a n i s m l e a d i n g to decreased cell-substrate a d h e s i o n . It is s o m e w h a t c o u n t e r i n t u i t i v e that a protein expressed on the a p i c a l surface o f e p i t h e l i a l c e l l s c o u l d l e a d to decreased adhesion at the basolateral surface. Further experiments (described b e l o w ) p r o v i d e d some insights into this p h e n o m e n o n .  M o r e o v e r , i n support o f p r e v i o u s l y p u b l i s h e d w o r k ( T a k e d a et a l . , 2 0 0 0 ) , there were also apparent alterations i n c e l l - c e l l j u n c t i o n s i n M C F - 7 c e l l s o v e r e x p r e s s i n g  podocalyxin  ( F i g u r e 3 - 5 ) ( S o m a s i r i et a l . , 2 0 0 4 ) : instead o f a w e l l a l i g n e d a p i c a l bar o f s t a i n i n g , as i n the vector c o n t r o l , p o d o c a l y x i n - p o s i t i v e p o p u l a t i o n s d i s p l a y e d d i s o r g a n i z e d l o c a l i z a t i o n o f the tight j u n c t i o n p r o t e i n , Z O - l . T h u s , the data c o n f i r m e d that p o d o c a l y x i n f u n c t i o n s as an a n t i - a d h e s i o n m o l e c u l e , a f f e c t i n g both c e l l - c e l l j u n c t i o n s , a n d c e l l - s u b s t r a t e interactions.  113  Empty vector  Podocalyxin  Figure 3-3: Transfected MCF-7 Cells Ectopically Expressing Murine Podocalyxin Exhibited Decreased Cell-Substratum Interactions. S c a l e bar: 5 0 u m .  114  c 100  g  '55  c 80 0) a in  3 </>  Isi  C  60 40  a> o o  20  T  1  0 E m p t y vector  Podocalyxin  Figure 3-4: Podocalyxin Decreases Cell Substrate Adhesion. A f t e r M C F - 7 c e l l s transfected w i t h empty vector or vector e n c o d i n g m u r i n e p o d o c a l y x i n were sorted based o n p o d o c a l y x i n e x p r e s s i o n , 5 x 1 0 c e l l s were plated per w e l l i n 6 - w e l l 5  plates. O n e d a y later, s u s p e n s i o n a n d adherent c e l l s were c o u n t e d i n t r i p l i c a t e w e l l s . Representative o f three independent experiments.  115  Podocalyxin ZO-1  I  •  1  .  S ]  Podocalyxin  Figure 3-5: Podocalyxin-Induced Alteration of Cell-Cell Junctions. M C F - 7 c e l l s transfected w i t h e m p t y vector o r vector e n c o d i n g m u r i n e p o d o c a l y x i n a n d l a b e l l e d w i t h antibodies against Z O - 1 (green) a n d p o d o c a l y x i n (red). C e l l j u n c t i o n s were a b n o r m a l a n d m o n o l a y e r s were disrupted u p o n e c t o p i c e x p r e s s i o n o f p o d o c a l y x i n . S c a l e bar: 15 UMTI ( S o m a s i r i et a l . , 2 0 0 4 ) .  116  3.2.3  Podocalyxin Recruits NHERF1 to the Apical Surface of Cells  T h e p r e v i o u s e x p e r i m e n t s c l e a r l y supported the role o f p o d o c a l y x i n i n d i s r u p t i n g c e l l a d h e s i o n , but h o w it a c c o m p l i s h e d this w a s not i m m e d i a t e l y o b v i o u s . S i n c e the a c t i n c y t o s k e l e t o n p l a y s an important role in c e l l a d h e s i o n , m o l e c u l e s that have been suggested to l i n k p o d o c a l y x i n to actin were e x a m i n e d . A l t h o u g h b i o c h e m i c a l e v i d e n c e suggested that p o d o c a l y x i n c o u l d interact w i t h both N H E R F 1 and N H E R F 2 , p o d o c a l y x i n had o n l y been s h o w n to c o l o c a l i z e w i t h N H E R F 2 ( L i et a l . , 2 0 0 2 ; T a k e d a et a l . , 2 0 0 1 ) . T a k i n g advantage o f the fact that M C F - 7 c e l l s express N H E R F 1 , its l o c a l i z a t i o n w i t h respect to p o d o c a l y x i n was assessed by c o n f o c a l m i c r o s c o p y .  In c e l l s e x p r e s s i n g both m o l e c u l e s , p o d o c a l y x i n a n d N H E R F 1 were c l e a r l y a p i c a l l y c o l o c a l i z e d ( F i g u r e 3 - 6 ) . In c e l l s transfected w i t h empty vector, h o w e v e r , N H E R F 1 w a s consistently expressed throughout  the c y t o p l a s m ( F i g u r e 3 - 6 C , l e f t p a n e l ) .  Thus,  p o d o c a l y x i n e x p r e s s i o n led to a d r a m a t i c increase i n a p i c a l l y l o c a l i z e d N H E R F 1 w i t h a c o n c o m i t a n t decrease i n c y t o p l a s m i c s t a i n i n g ( F i g u r e 3 - 6 C , right panel). S i n c e N H E R F f a m i l y p r o t e i n s f u n c t i o n as s c a f f o l d i n g p r o t e i n s and interact w i t h a c t i n , it had been suggested that they c o u l d p l a y a role i n proper l o c a l i z a t i o n o f p o d o c a l y x i n ( C h e n g et a l . , 2 0 0 5 ; L i et a l . , 2 0 0 2 ; M e d e r et a l . , 2 0 0 5 ; S c h m i e d e r et a l . , 2 0 0 4 ) . It w a s therefore very i n t r i g u i n g that p o d o c a l y x i n , not N H E R F 1 , w a s responsible f o r a p i c a l l o c a l i z a t i o n o f both m o l e c u l e s ( F i g u r e 3 - 6 ) . R e g a r d l e s s , the interaction o f p o d o c a l y x i n w i t h this s c a f f o l d i n g protein i m p l i e d a l i n k to the c y t o s k e l e t o n and a potential c l u e to p o d o c a l y x i n ' s role in blocking cell adhesion.  117  Empty vector  Podocalyxin  Podocalyxin  B  NHERF1  Podocalyxin NHERF1 DAPI  Figure 3-6: Confocal Images Demonstrated Apical Recruitment of NHERF1 by Podocalyxin. M C F - 7 c e l l s transfected w i t h e m p t y vector or vector e n c o d i n g m u r i n e p o d o c a l y x i n were labelled with D A P I  ( b l u e ) a n d a n t i b o d i e s against p o d o c a l y x i n (red)  and N H E R F 1  (green). Y e l l o w represents c o l o c a l i z a t i o n o f p o d o c a l y x i n a n d N H E R F 1 . I m a g i n g  by  M a r c i a L. M c C o y , U n i v e r s i t y o f B r i t i s h C o l u m b i a .  118  3.2.4 Morphological Changes: Podocalyxin Induces Microvillus Formation in Epithelial Cells A f t e r g a i n i n g s o m e insights into p o d o c a l y x i n ' s f u n c t i o n as an a n t i - a d h e s i o n m o l e c u l e , it was p o s s i b l e to concentrate o n its potential role i n i n f l u e n c i n g c e l l m o r p h o l o g y . E l e c t r o n m i c r o s c o p y w a s used to assess the c e l l surface o f e p i t h e l i a l c e l l s transfected  with  p o d o c a l y x i n or e m p t y vector. In p a r t i c u l a r , the f o r m a t i o n o f m i c r o v i l l i was assessed by e l e c t r o n m i c r o s c o p y because there is s o m e e v i d e n c e to suggest that the p o d o c a l y x i n b i n d i n g p r o t e i n , N H E R F 1 m a y be i n v o l v e d i n the generation o f such structures. F o r e x a m p l e , the intestinal brush border m i c r o v i l l i o f one strain o f N H E R F l - n u l l m i c e are s t r i k i n g l y d i s o r g a n i z e d in c o m p a r i s o n to those o f w i l d t y p e littermates ( M o r a l e s et a l . , 2 0 0 4 ) . F u r t h e r m o r e , w h e n M C F - 7 c e l l s are treated w i t h e s t r a d i o l , they u p r e g u l a t e N H E R F 1 m R N A , and the n u m b e r and length o f m i c r o v i l l i on the c e l l surface increases ( E d i g e r et a l . , 1 9 9 9 ; V i c et a l . , 1982). W h i l e this certainly does not p r o v e that N H E R F is responsible f o r m i c r o v i l l u s f o r m a t i o n , i n c o m b i n a t i o n w i t h the above data it is suggestive e n o u g h to warrant f u r t h e r i n v e s t i g a t i o n . T h u s , m i c r o v i l l u s f o r m a t i o n was assessed i n podocalyxin-transfected M C F - 7 cells.  T h e d i f f e r e n c e between the t w o c e l l p o p u l a t i o n s was s t r i k i n g : a l t h o u g h c o n t r o l c e l l s had s o m e m i c r o v i l l i , p o d o c a l y x i n - t r a n s f e c t e d c e l l s were c o v e r e d w i t h these c e l l surface p r o t r u s i o n s , as s h o w n by s c a n n i n g e l e c t r o n m i c r o s c o p y ( S E M ) ( F i g u r e 3 - 7 ) . A  very  s i m i l a r result w a s o b s e r v e d i n transfected M D C K c e l l s ( F i g u r e 3 - 8 ) , d e m o n s t r a t i n g that this d r a m a t i c phenotype was not an artifact of one particular c e l l l i n e . B o t h c e l l lines were  119  a l s o e x a m i n e d by t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y ( T E M ) , w i t h c o n s i s t e n t results (Figure 3-9).  120  Empty vector  Podocalyxin  Figure 3-7: Podocalyxin Induced Microvillus F o r m a t i o n in M C F - 7 Cells.  S E M ' s o f M C F - 7 cells transfected w i t h empty vector or vector e n c o d i n g  murine  p o d o c a l y x i n . S c a l e bar: 2 p m . Representative o f t w o independent experiments.  121  Empty vector  Podocalyxin  Figure 3-8: Podocalyxin Also Induced Microvillus Formation in M D C K Epithelial Cells. S E M ' s of M D C K  cells transfected with empty vector or vector encoding  murine  p o d o c a l y x i n . S c a l e bar: 2 u m .  122  Empty vector  In  Podocalyxin  1  MCF-7  fa • Q  M mn wm  MDCK  Figure 3 - 9 : TEM's of Epithelial Cells Transfected with Empty Vector or Vector Encoding Murine Podocalyxin. S c a l e bar: 1 p m . Representative o f t w o independent e x p e r i m e n t s . I m a g i n g b y A . W a y n e V o g l , University of British Columbia.  123  3.3  Generation  and Analysis  of Podocalyxin  Mutants  In order to g a i n further i n s i g h t s into the m e c h a n i s m s i n v o l v e d i n p o d o c a l y x i n - i n d u c e d N H E R F - r e c r u i t m e n t and m i c r o v i l l u s f o r m a t i o n , a panel o f c h i c k e n p o d o c a l y x i n mutants was generated, as depicted i n F i g u r e 3-10. M o s t mutations were i n the c y t o p l a s m i c tail as the h i g h l e v e l o f c o n s e r v a t i o n i n this r e g i o n suggested f u n c t i o n a l i m p o r t a n c e , a n d m u t a t i o n s w e r e e x p e c t e d to b l o c k o r p r o m o t e i n t e r a c t i o n w i t h k n o w n a n d u n k n o w n b i n d i n g partners. T h e mutants ranged f r o m those l a c k i n g entire d o m a i n s to those w i t h s i n g l e p o i n t m u t a t i o n s , as f o l l o w s : 1) serine a n d t h r e o n i n e residues o f t w o potential p h o s p h o r y l a t i o n sites were mutated, alone or i n c o m b i n a t i o n , to alanine residues to b l o c k p h o s p h o r y l a t i o n i n the A l a mutants; 2) the same residues were converted to aspartic a c i d residues to m i m i c c o n s t i t u t i v e p h o s p h o r y l a t i o n i n the A s p c o n s t r u c t s ; 3) the A D T H L mutant l a c k e d the C - t e r m i n a l D T H L sequence essential f o r i n t e r a c t i o n w i t h proteins; 4) A t a i l  l a c k e d the entire c y t o p l a s m i c tail w i t h the exception  j u x t a m e m b r a n e sequence C C H Q R F ,  NHERF o f the  w h i c h w a s retained as a m e m b r a n e a n c h o r (this  mutant l a c k e d a l l potential p h o s p h o r y l a t i o n sites, the C - t e r m i n a l N H E R F - b i n d i n g site, and a putative e z r i n - b i n d i n g site); 5) A E C l a c k e d the majority o f the extracellular d o m a i n ( i n c l u d i n g the m u c i n d o m a i n and the c y s t e i n e - b o n d e d g l o b u l a r d o m a i n ) a n d i n s t e a d e n c o d e d an e x t r a c e l l u l a r f l a g - t a g , as w e l l as p o d o c a l y x i n ' s transmembrane r e g i o n and its full-length  cytoplasmic  tail;  a n d 6 ) the f i n a l  construct,  A l t . splice,  contained  p o d o c a l y x i n ' s n a t u r a l l y o c c u r r i n g s p i c e variant, w h i c h l a c k s m u c h o f the c y t o p l a s m i c tail.  124  Wildtype  Ala  Asp  ADTHL  Atail  Alt. splice  AEC  Figure 3-10: Schematic of Podocalyxin Mutants. L i g h t b l u e : e x t r a c e l l u l a r d o m a i n , green: f l a g - t a g , r e d : t r a n s m e m b r a n e d o m a i n , p u r p l e : c y t o p l a s m i c t a i l ( i n c l u d i n g C - t e r m i n a l D T H L i n w i l d t y p e , A l a , A s p , and A E C ) ,  yellow:  c y t o p l a s m i c t a i l o f s p l i c e variant, h o r i z o n t a l l i n e s : g l y c o s y l a t i o n s , A : p o i n t m u t a t i o n to a l a n i n e , and D : point mutation to aspartic a c i d .  125  A v i a n p o d o c a l y x i n w a s c h o s e n as the basis f o r these experiments f o r three reasons: 1) it is 8 5 % i d e n t i c a l to m a m m a l i a n p o d o c a l y x i n i n its i n t r a c e l l u l a r d o m a i n , 2) as w i t h the m u r i n e protein used i n section 3 . 2 , w i l d t y p e a v i a n p o d o c a l y x i n also i n d u c e d m i c r o v i l l u s f o r m a t i o n i n M C F - 7 c e l l s , and 3) I c o u l d s e l e c t i v e l y detect its ectopic e x p r e s s i o n u s i n g a s p e c i e s - s p e c i f i c m o n o c l o n a l antibody that reacts w i t h native, f i x e d , and denatured f o r m s o f the m o l e c u l e ( M c N a g n y et a l . , 1997).  3.3.1  Podocalyxin Mutant Expression Analysis  3.3.1.1 Podocalyxin-Positive Cells were Continuously Lost from Bulk Populations T h e mutants d e s c r i b e d a b o v e w e r e c l o n e d i n t o the p c D N A 3 . 1 e x p r e s s i o n v e c t o r a n d transfected i n t o M C F - 7 c e l l s . U n f o r t u n a t e l y , there w a s a c o n s i s t e n t p r o b l e m w i t h a l l p o d o c a l y x i n overexpression studies: i n b u l k populations of transfected cells, cells e x p r e s s i n g p o d o c a l y x i n were c o n t i n u o u s l y lost f r o m cultures. A l t h o u g h this w a s partly due to l o s s o f s u s p e n s i o n c e l l s w h e n c h a n g i n g m e d i a , m o d i f i c a t i o n o f the c u l t u r e techniques to retain suspension c e l l s d i d not c o m p l e t e l y s o l v e the p r o b l e m . I n i t i a l l y , this led to r e c l o n i n g o f constructs into an alternative e x p r e s s i o n vector ( p I R E S 2 - E G F P ) . A s this d i d not s o l v e the p r o b l e m e i t h e r , F A C S w a s used to s e r i a l l y sort c u l t u r e s f o r p o d o c a l y x i n e x p r e s s i o n . A l t h o u g h this e n a b l e d g e n e r a t i o n o f p o p u l a t i o n s c o n t a i n i n g h i g h e r p r o p o r t i o n s o f p o d o c a l y x i n - p o s i t i v e c e l l s , e x p r e s s i o n w a s s t i l l not stable. F i g u r e 3-11 s h o w s F A C S p r o f i l e s o f transfected c e l l s before s o r t i n g , and after o n e , t w o , or three rounds o f s o r t i n g . A l t h o u g h the percentage o f p o d o c a l y x i n - p o s i t i v e c e l l s d i d increase  126  after each r o u n d , the c e l l s that were cultured after each sort are s h o w n i n the white b o x e s , and this l e v e l o f p o d o c a l y x i n expression was clearly not m a i n t a i n e d . A l t h o u g h not perfect either, the m o s t s u c c e s s f u l strategy f o r g e n e r a t i n g stably e x p r e s s i n g c u l t u r e s w a s to isolate and e x p a n d s i n g l e p o d o c a l y x i n - p o s i t i v e c l o n e s . U s i n g this strategy, at least three c l o n e s w e r e i s o l a t e d f o r each mutant. M o s t e x p e r i m e n t s w e r e p e r f o r m e d w i t h these c l o n e s , and k e y results were c o n f i r m e d w i t h b u l k populations.  127  > GFP Figure 3 - 1 1 : Podocalyxin Expression After Multiple Rounds of Cell Sorting. T r a n s f e c t e d M C F - 7 c e l l s were sorted based o n p o d o c a l y x i n and G F P expression. C e l l s i n w h i t e b o x e s w e r e e x p a n d e d a n d s e r i a l l y sorted. T h u s , c e l l s i n the first w h i t e b o x were sorted and e x p a n d e d . These c e l l s gave rise to the c e l l s depicted i n the second F A C S plot. S i m i l a r l y , o n l y the c e l l s i n the s e c o n d w h i t e b o x were r e t a i n e d ; these gave rise to a l l o f the c e l l s depicted i n the third F A C S blot, and so o n .  128  3.3.1.2 Podocalyxin Mutants were All Expressed and were of Expected Molecular Weights W e s t e r n b l o t t i n g was used to assess the expression o f each mutant in c l o n a l p o p u l a t i o n s ( F i g u r e 3 - 1 2 ) . T h i s also e n a b l e d c o n f i r m a t i o n that each f o r m o f p o d o c a l y x i n w a s o f the appropriate m o l e c u l a r weight. T h e l o w e r band w a s n o n - s p e c i f i c , as it w a s also o b s e r v e d i n e m p t y vector transfected c e l l s ( V e c t o r 6). F u l l - l e n g t h p o d o c a l y x i n w a s a p p r o x i m a t e l y 160 k D a , as e x p e c t e d ( W i l d t y p e 7 and W i l d t y p e  1 3 ) , as w e r e m u t a n t s c o n t a i n i n g  S e r / T h r ^ A l a or S e r / T h r ^ A s p point mutations ( A s p 7 , A l a 3 , and A l a 6 ) . C y t o p l a s m i c tail deletions (Atail 2 and A t a i l 8) and the alternatively s p l i c e d f o r m o f p o d o c a l y x i n ( A l t . splice 5 ) , w h i c h a l l l a c k most o f p o d o c a l y x i n ' s c y t o p l a s m i c t a i l , were n o t i c e a b l y shorter, as up to 71 a m i n o a c i d s had been deleted. E x p e c t e d l y , t r u n c a t i o n o f the f o u r C - t e r m i n a l a m i n o a c i d s made little d i f f e r e n c e to the size o f the protein ( A D T H L 2 and A D T H L 15). T h e mutant l a c k i n g m o s t o f the e x t r a c e l l u l a r d o m a i n w a s detected u s i n g an a n t i - f l a g a n t i b o d y , w h i c h r e c o g n i z e d the f l a g - t a g inserted i n p l a c e o f the d e l e t e d p o r t i o n p o d o c a l y x i n . A s predicted, this mutant was c o n s i d e r a b l y  of  shorter than f u l l - l e n g t h  p o d o c a l y x i n due to the absence o f the h e a v i l y g l y c o s y l a t e d m u c i n d o m a i n ( A E C 8).  129  Figure 3-12: Western Blots Demonstrated Expression of Podocalyxin Mutants of the Expected Molecular Weights in Clonal Populations. N o t e the s l i g h t l y s m a l l e r size o f p o d o c a l y x i n mutants w i t h shorter c y t o p l a s m i c tails. C e l l s transfected w i t h the e m p t y v e c t o r were u s e d as n e g a t i v e c o n t r o l s . S i z e m a r k e r s w e r e measured i n k D a . Representative o f three independent experiments.  130  3.3.2  Mutation of  Phosphorylation  Sites  and  Analysis  of  Podocalyxin's Splice Variant did not Provide any Novel Insights into Podocalyxin's Function A l t h o u g h m a n y e x p e r i m e n t s were p e r f o r m e d w i t h a l l mutants, there w a s n o detectable d i f f e r e n c e between w i l d t y p e p o d o c a l y x i n and the p h o s p h o r y l a t i o n mutants ( A s p and A l a ) or between p o d o c a l y x i n ' s s p l i c e variant ( A l t . splice) and the f u l l c y t o p l a s m i c tail deletion ( A t a i l ) . F o r this reason, repetitive i n f o r m a t i o n has been o m i t t e d by i n c l u d i n g a n a l y s i s o f j u s t f i v e constructs i n each f i g u r e : empty vector, w i l d t y p e p o d o c a l y x i n , A D T H L ,  Atail,  and A E C . W e s t e r n blotting was used to detect protein expression o f the mutants described i n the f o l l o w i n g e x p e r i m e n t s ( F i g u r e 3 - 1 3 ) , and surface e x p r e s s i o n w a s c o n f i r m e d by f l o w c y t o m e t r y , as s h o w n i n F i g u r e 3 - 1 4 . E x p r e s s i o n l e v e l s were c o m p a r a b l e , w i t h the e x c e p t i o n o f the extracellular d e l e t i o n , A E C , w h i c h c o u l d not be accurately c o m p a r e d due to the use o f an independent antibody f o r its detection.  131  chPodocalyxin  Figure 3-13: Western Blots Demonstrated Expression of Podocalyxin in Clonal Populations Used for Subsequent Experiments. N o t e the slight decrease i n size w h e n the c y t o p l a s m i c tail w a s deleted. S i z e markers were measured i n k D a .  132  Wildtype  ADTHL  Atail  AEC  >• Podocalyxin Figure 3-14: Podocalyxin was Expressed at Comparable Levels at the Cell Surface in Clonal Populations. T r a n s f e c t e d c e l l s w e r e c l o n a l l y s o r t e d , e x p a n d e d , a n d l a b e l l e d w i t h a n t i b o d i e s against c h i c k e n p o d o c a l y x i n or the f l a g - t a g ( A E C mutant). D o t t e d l i n e s represent b a c k g r o u n d staining m e a s u r e d o n e m p t y vector-transfected c e l l s . Representative o f three independent experiments.  133  3.3.3  Interaction of Podocalyxin with NHERF1  3.3.3.1 Podocalyxin's C-terminal DTHL Sequence is Required for Interaction with NHERF 1 P r e v i o u s e x p e r i m e n t s demonstrated that p o d o c a l y x i n and N H E R F 1 were c o l o c a l i z e d in M C F - 7 c e l l s ( F i g u r e 3 - 6 ) , and it is k n o w n that p o d o c a l y x i n ' s f o u r C - t e r m i n a l a m i n o acids are the P D Z r e c o g n i t i o n site f o r N H E R F proteins ( L i et a l . , 2 0 0 2 ; T a k e d a et a l . , 2 0 0 1 ; T a n et a l . , 2 0 0 6 ) . If p o d o c a l y x i n and N H E R F 1 were t r u l y i n t e r a c t i n g i n M C F - 7 c e l l s , then c o l o c a l i z a t i o n w o u l d be lost in c e l l s e x p r e s s i n g p o d o c a l y x i n truncations l a c k i n g the P D Z b i n d i n g site. C o n f o c a l m i c r o s c o p y w a s therefore used to assess this i n t e r a c t i o n . A s s h o w n i n m e r g e d i m a g e s t a k e n at the a p i c a l s u r f a c e o f t r a n s f e c t e d c e l l s , podocalyxin  showed  strong c o l o c a l i z a t i o n w i t h N H E R F 1 , w h i l e both  wildtype  C-terminal  truncation mutants (Atail and A D T H L ) d i d not ( F i g u r e 3 - 1 5 ) . In contrast, the A E C mutant l a c k i n g the extracellular d o m a i n but r e t a i n i n g the entire c y t o p l a s m i c tail o f p o d o c a l y x i n was f o u n d m a i n l y apically l o c a l i z e d and c o l o c a l i z e d with N H E R F 1 . T h i s  further  demonstrates that p o d o c a l y x i n recruits N H E R F 1 to the a p i c a l d o m a i n t h r o u g h its C terminal D T H L motif.  134  NHERF1 Podocalyxin  Vector  Atail  F i g u r e 3-15:  H Wildtype  ADTHL  AEC  Isotype  Confocal Analysis of N H E R F 1 a n d Podocalyxin at the A p i c a l Surface  of Transfected M C F - 7 Cells.  C e l l s w e r e l a b e l l e d w i t h D A P I ( b l u e ) a n d a n t i b o d i e s against N H E R F 1 (green) a n d p o d o c a l y x i n (red). Y e l l o w represents c o l o c a l i z a t i o n o f N H E R F 1 and p o d o c a l y x i n . T h e i s o t y p e c o n t r o l s a m p l e w a s l a b e l l e d w i t h D A P I (blue), a n t i - p o d o c a l y x i n (red), a n d an isotype c o n t r o l f o r N H E R F 1 (green) to demonstrate the s p e c i f i c i t y o f N H E R F 1 l a b e l l i n g . C e l l s transfected w i t h the e m p t y vector were u s e d as negative c o n t r o l s f o r p o d o c a l y x i n staining. S c a l e bar: 5 u m . Representative o f t w o independent experiments.  135  A p i c a l recruitment o f N H E R F 1 c o u l d be c l e a r l y v i s u a l i z e d i n vertical slices o f c o n f o c a l stacks ( F i g u r e 3 - 1 6 ) . C y t o p l a s m i c N H E R F 1 s t a i n i n g was v i s i b l e i n c e l l s transfected w i t h C - t e r m i n a l deletions o f p o d o c a l y x i n , w h i l e an increase i n a p i c a l N H E R F 1 s t a i n i n g w a s a c c o m p a n i e d b y a decrease i n c y t o p l a s m i c s t a i n i n g i n the w i l d t y p e and A E C s a m p l e s . T h e s e e x p e r i m e n t s c o n f i r m e d that p o d o c a l y x i n and N H E R F 1 are bona fide i n t e r a c t i o n partners in vivo. Importantly, the p o d o c a l y x i n - d e p e n d e n t recruitment o f N H E R F 1 to the a p i c a l c e l l surface m a y have i m p l i c a t i o n s f o r f u n c t i o n a l r e g u l a t i o n o f N H E R F f a m i l y members.  136  NHERF1  Podocalyxin Vector  Atail  Wildtype  AEC  ADTHL  Isotype  Figure 3-16: Analysis of NHERF1 and Podocalyxin in Vertical Slices of Confocal Stacks. Transfected M C F - 7 cells were labelled with D A P I  (blue) and antibodies against  N H E R F 1 (green) and p o d o c a l y x i n (red). Y e l l o w represents c o l o c a l i z a t i o n o f N H E R F 1 and p o d o c a l y x i n . The isotype control sample was labelled with D A P I (blue), antip o d o c a l y x i n ( r e d ) , a n d a n i s o t y p e c o n t r o l f o r N H E R F 1 (green) to d e m o n s t r a t e the s p e c i f i c i t y o f N H E R F 1 l a b e l l i n g . C e l l s transfected w i t h the e m p t y v e c t o r were used as n e g a t i v e c o n t r o l s f o r p o d o c a l y x i n s t a i n i n g . S c a l e bar: 5 u.m. R e p r e s e n t a t i v e o f t w o independent experiments.  137  3.3.4  Analysis of Essential Sequences Required for Morphological Changes  T h e effect o f loss o f the p o d o c a l y x i n / N H E R F l interaction o n m i c r o v i l l u s f o r m a t i o n w a s then assessed by e l e c t r o n m i c r o s c o p y u s i n g c l o n a l l y i s o l a t e d M C F - 7 c e l l s transfected w i t h p o d o c a l y x i n mutant constructs ( F i g u r e 3 - 1 7 A and F i g u r e 3 - 1 8 A ) .  3.3.4.1 Interaction with NHERF1 is Not Required for Formation of Microvilli TEM  d e m o n s t r a t e d that, as e x p e c t e d , c o n t r o l c e l l s h a d f e w m i c r o v i l l i w h i l e c e l l s  transfected w i t h f u l l - l e n g t h p o d o c a l y x i n d i s p l a y e d increased m i c r o v i l l i ( F i g u r e 3 - 1 7 B ) . S t r i k i n g l y , c e l l s e x p r e s s i n g A D T H L or A t a i l mutants generated m i c r o v i l l i i n s i m i l a r n u m b e r s to f u l l - l e n g t h p o d o c a l y x i n  transfectants. S E M  analysis confirmed  these  o b s e r v a t i o n s ( F i g u r e 3 - 1 7 C ) . T h i s d e m o n s t r a t e d that d i r e c t i n t e r a c t i o n o f p o d o c a l y x i n w i t h N H E R F proteins was surprisingly not required f o r f o r m a t i o n o f m i c r o v i l l i .  138  Vector  Wildtype  ADTHL  Atail  Figure 3-17: Podocalyxin Induced Microvillus Formation in a NHERF-Independent Manner. ( A ) S c h e m a t i c o f p o d o c a l y x i n a n d mutants, as d e s c r i b e d i n F i g u r e 3 - 1 0 . ( B ) T E M ' s o f transfected M C F - 7 c e l l s . S c a l e bar: 1 p m . ( C ) S E M ' s o f transfected M C F - 7 c e l l s . S c a l e bar: 2 p m . Representative o f t w o independent e x p e r i m e n t s . T E M i m a g i n g b y A . W a y n e V o g l , University of British Columbia.  139  3.3.4.2 Podocalyxin's Extracellular Domain is Required for Formation of Microvilli C o n v e r s e l y , T E M revealed that the extracellular d o m a i n o f p o d o c a l y x i n w a s essential f o r m i c r o v i l l u s f o r m a t i o n : there w a s no increase i n m i c r o v i l l u s n u m b e r i n c e l l s e x p r e s s i n g the A E C mutant b e a r i n g o n l y the f l a g - t a g g e d transmembrane a n d c y t o p l a s m i c d o m a i n o f p o d o c a l y x i n ( F i g u r e 3 - 1 8 B ) , e v e n t h o u g h this m u t a n t w a s a b l e to a p i c a l l y  recruit  N H E R F 1 (see F i g u r e 3 - 1 5 , above). T h i s result w a s c o n f i r m e d v i a S E M a n a l y s i s ( F i g u r e 3-18C).  140  Vector  Wildtype  AEC  F i g u r e 3-18:  Deletion of the M a j o r i t y of P o d o c a l y x i n ' s E x t r a c e l l u l a r  Domain  Abolished Microvillus Formation.  ( A ) S c h e m a t i c o f w i l d t y p e p o d o c a l y x i n a n d A E C mutant. ( B ) T E M ' s  of transfected  M C F - 7 c e l l s . S c a l e bar: 1 u m . ( C ) S E M ' s o f transfected M C F - 7 c e l l s . S c a l e bar: 2 p m . Representative of t w o independent experiments. T E M i m a g i n g by A . W a y n e  Vogl,  University of British Columbia.  141  In order to quantitate this effect, a l l m i c r o v i l l i o b s e r v e d i n six r a n d o m 15 0 0 0 X  SEM  f i e l d s w e r e enumerated f o r each mutant ( F i g u r e 3-19). F u l l - l e n g t h , A D T H L , a n d A t a i l transfectants a l l h a d at least t w i c e as m a n y m i c r o v i l l i as v e c t o r - c o n t r o l t r a n s f e c t a n t s . It is t h e r e f o r e  p o s s i b l e to c o n c l u d e that the e x t r a c e l l u l a r  and  AEC  domain,  t r a n s m e m b r a n e r e g i o n , and s i x a m i n o a c i d s o f p o d o c a l y x i n ' s c y t o p l a s m i c t a i l w e r e sufficient to induce N H E R F 1 - i n d e p e n d e n t f o r m a t i o n o f m i c r o v i l l i .  142  Microvilli / 50 pm 2  Vector  Wildtype  ADTHL  Atail  AEC  Figure 3-19: Microvillus Counts for Transfected Cells. M i c r o v i l l i i n s i x r a n d o m 5 0 u m f i e l d s were enumerated f o r M C F - 7 c e l l s transfected w i t h 2  each p o d o c a l y x i n construct. E r r o r bars represent standard d e v i a t i o n . R e p r e s e n t a t i v e o f t w o independent experiments.  143  It has been s h o w n that m a i n t e n a n c e o f p o d o c y t e f o o t process i n t e g r i t y is c r i t i c a l l y dependent  on  the  negatively-charged  glycosylations  decorating  podocalyxin's  e x t r a c e l l u l a r d o m a i n ( A n d r e w s , 1 9 7 9 ; K e r j a s c h k i et a l . , 1 9 8 4 ; S e i l e r et a l . , 1975); it w a s t h e r e f o r e not p a r t i c u l a r l y s u r p r i s i n g that d e l e t i o n o f the entire m u c i n d o m a i n a l s o p r e v e n t e d m i c r o v i l l u s f o r m a t i o n i n M C F - 7 c e l l s . H o w e v e r , the f a c t that the conserved, N H E R F - b i n d i n g ,  c y t o p l a s m i c tail of p o d o c a l y x i n  highly  was dispensable  for  m i c r o v i l l u s f o r m a t i o n w a s quite u n e x p e c t e d . It is k n o w n that the integrity o f the actin c y t o s k e l e t o n is essential f o r m a i n t a i n i n g c e l l shape, and that l i n k a g e o f actin to integral m e m b r a n e proteins is i m p o r t a n t f o r generating and s u p p o r t i n g c e l l surface p r o t r u s i o n s , i n c l u d i n g m i c r o v i l l i ( R e v e n u et a l . , 2 0 0 4 ) . S i n c e N H E R F 1 and N H E R F 2 c a n c o n n e c t p o d o c a l y x i n to the actin c y t o s k e l e t o n through ezrin ( M o r a l e s et a l . , 2 0 0 4 ; T a k e d a et a l . , 2 0 0 1 ; T a n et a l . , 2 0 0 6 ) , it w a s i n i t i a l l y a s s u m e d that p o d o c a l y x i n ' s i n v o l v e m e n t i n the f o r m a t i o n o f m i c r o v i l l i w o u l d require i n t e r a c t i o n w i t h N H E R F p r o t e i n s .  Moreover,  N H E R F 2 interactions w i t h p o d o c a l y x i n have recently been s h o w n to c o i n c i d e c l o s e l y w i t h the f o r m a t i o n o f a " p r e - a p i c a l d o m a i n " in M D C K c e l l s (as d i s c u s s e d i n s e c t i o n 1.5.4), and it w a s postulated that the a s s o c i a t i o n w a s a prerequisite f o r f o r m a t i o n o f this d o m a i n ( M e d e r et a l . , 2 0 0 5 ) . H o w e v e r , the current data suggested that, a l t h o u g h the C t e r m i n a l tail o f p o d o c a l y x i n w a s s u f f i c i e n t to a c t i v e l y recruit N H E R F 1 to a p i c a l p l a s m a m e m b r a n e d o m a i n s , the f o r m a t i o n o f m i c r o v i l l i a n d , i n d e e d , the a p i c a l t a r g e t i n g  of  p o d o c a l y x i n to the p l a s m a m e m b r a n e o c c u r r e d i n the absence o f any direct i n teracti on with N H E R F .  144  3.3.5 Interaction with NHERF is Unnecessary for Colocalization of Podocalyxin with Ezrin Since microvilli formation coincides with  dramatic reorganization  o f the a p i c a l  m e m b r a n e d o m a i n , p o l y m e r i z a t i o n o f f - a c t i n at the core o f m i c r o v i l l i , and recruitment o f m e m b e r s o f the E R M f a m i l y of. proteins to the a p i c a l d o m a i n , p r e s u m a b l y to act as l i n k e r s b e t w e e n the c y t o s k e l e t o n and t r a n s m e m b r a n e proteins ( r e v i e w e d i n ( L o u v e t V a l l e e , 2 0 0 0 ) ) , the l o c a l i z a t i o n o f e z r i n and f - a c t i n w e r e assessed i n transfected c e l l s . Interestingly, cells transfected with all f o r m s o f extracellular  domain-containing  p o d o c a l y x i n , regardless o f whether o r not they c o n t a i n e d a N H E R F - b i n d i n g s e q u e n c e , d i s p l a y e d increased a p i c a l recruitment o f e z r i n a n d strong c o l o c a l i z a t i o n o f p o d o c a l y x i n w i t h this p r o t e i n ( F i g u r e 3 - 2 0 ) . Increased a p i c a l r e c r u i t m e n t o f e z r i n w a s e s p e c i a l l y evident i n vertical sections o f c o n f o c a l stacks, where strong e z r i n s t a i n i n g c o r r e s p o n d e d to c e l l s e x p r e s s i n g e c t o p i c p o d o c a l y x i n ( F i g u r e 3 - 2 1 ) . T h u s , recruitment o f e z r i n to the a p i c a l m e m b r a n e o c c u r r e d i n the absence o f a n y direct i n teracti on o f p o d o c a l y x i n w i t h N H E R F . In contrast, recruitment o f e z r i n appeared to be s o m e w h a t less p r o n o u n c e d i n the A E C mutant, a g a i n d e m o n s t r a t i n g the i m p o r t a n c e o f the e x t r a c e l l u l a r d o m a i n i n podocalyxin function.  145  Ezrin Podocalyxin Vector  Wildtype  ADTHL  Atail  AEC  Isotype  Figure 3-20: Confocal Analysis of Podocalyxin and Ezrin at the Apical Surface of Transfected M C F - 7 Cells. Transfected cells were labelled with D A P I (blue) and antibodies against ezrin (red) and podocalyxin (green). Yellow represents colocalization of ezrin and podocalyxin. The isotype control sample was labelled with D A P I (blue), anti-podocalyxin (green), and an isotype control for ezrin (red) to demonstrate the specificity of ezrin labelling. Cells transfected with the empty vector were used as negative controls for podocalyxin staining. Scale bar: 5 pm. Representative of two independent experiments.  146  Empty vector  Podocalyxin  Ezrin  Podocalyxin  Merge  Figure 3-21: Vertical Sections of Confocal Stacks Demonstrated Increased Apical Recruitment of Ezrin in Cells Transfected with Podocalyxin. Cells transfected with podocalyxin or empty vector were assessed for ezrin (red) and podocalyxin (green) in vertical sections of confocal stacks. Increased apical recruitment of ezrin was especially evident when staining was compared between neighbouring cells with and without ezrin. Cells transfected with the empty vector were used as negative controls for podocalyxin staining. Scale bar: 5 pm. Representative of two independent experiments.  147  3.3.6  Recruitment of f-actin O c c u r s in the A b s e n c e of  NHERF  Binding F u r t h e r m o r e , a p i c a l recruitment o f f - a c t i n w a s also independent o f N H E R F - b i n d i n g . A l l forms of podocalyxin  d e m o n s t r a t e d a p i c a l c o l o c a l i z a t i o n w i t h f - a c t i n , but a p i c a l  r e c r u i t m e n t w a s n o t a b l y less robust i n the absence o f the e x t r a c e l l u l a r d o m a i n ( F i g u r e 3 - 2 2 a n d F i g u r e 3 - 2 3 ) . C o n s i s t e n t w i t h e l e c t r o n m i c r o s c o p y results, c e l l s e x p r e s s i n g e x t r a c e l l u l a r d o m a i n - c o n t a i n i n g p o d o c a l y x i n mutants e x h i b i t e d a clear punctate s t a i n i n g pattern o n the a p i c a l surface o f c e l l s i n p r o t r u d i n g structures i n d i c a t i v e o f m i c r o v i l l i . T o c o n f i r m that these structures bear a l l the structural h a l l m a r k s t y p i c a l o f m i c r o v i l l i , they w e r e e x a m i n e d at h i g h m a g n i f i c a t i o n by T E M . A s w i t h f u l l - l e n g t h p o d o c a l y x i n , c e l l s e x p r e s s i n g A D T H L and A t a i l mutants each c l e a r l y demonstrated the presence o f a c t i n f i l a m e n t s i n the m i c r o v i l l a r c o r e , as s h o w n both i n l o n g i t u d i n a l and c r o s s - s e c t i o n s o f i n d i v i d u a l m i c r o v i l l i ( F i g u r e 3 - 2 4 ) . In s u m m a r y , p o d o c a l y x i n was able to recruit actin to m i c r o v i l l i i n the absence o f the b u l k o f its c y t o p l a s m i c d o m a i n , but the e x t r a c e l l u l a r d o m a i n was essential f o r m i c r o v i l l u s f o r m a t i o n .  148  f-actin Podocalyxin  Vector  Wildtype  ADTHL  Atail  AEC  _ •  Figure 3-22: Confocal Images of the Apical Surface of Cells Show that f-Actin was Recruited in Podocalyxin-Transfected Cells. M C F - 7 cells transfected with wildtype and mutant podocalyxin were assessed for f-actin (red), podocalyxin (green), and nuclear (blue) labelling at the apical surface. Cells transfected with the empty vector were used as negative controls for podocalyxin staining. Scale bar: 5 u.m. Representative of two independent experiments.  149  f-actin Podocalyxin Vector  Wildtype  ADTHL  Atail  AEC  F i g u r e 3-23: V e r t i c a l Sections of C o n f o c a l Stacks Demonstrated A p i c a l R e c r u i t m e n t of f - A c t i n i n P o d o c a l y x i n - T r a n s f e c t e d C e l l s .  M C F - 7 c e l l s transfected w i t h w i l d t y p e and mutant p o d o c a l y x i n were assessed f o r f - a c t i n (red), p o d o c a l y x i n (green), and n u c l e a r (blue) l a b e l l i n g i n v e r t i c a l sections o f c o n f o c a l s t a c k s . C e l l s t r a n s f e c t e d w i t h the e m p t y v e c t o r w e r e u s e d as n e g a t i v e c o n t r o l s  for  p o d o c a l y x i n staining. S c a l e bar: 5 u m . Representative o f t w o independent experiments.  150  Figure 3-24:  A c t i n Filaments were Visible in Individual M i c r o v i l l i .  H i g h m a g n i f i c a t i o n T E M ' s demonstrated the presence o f actin f i l a m e n t s i n m i c r o v i l l i o f c e l l s transfected w i t h w i l d t y p e p o d o c a l y x i n as w e l l as A D T H L and A t a i l mutants. S c a l e bars: 0.1 p m . ( A ) L o n g i t u d i n a l sections. ( B ) C r o s s sections. I m a g i n g by A . W a y n e V o g l , University of British C o l u m b i a .  151  3.4 Discussion 3.4.1  Summary  P o d o c a l y x i n was overexpressed i n t w o epithelial c e l l lines i n order to assess its effects o n c e l l a d h e s i o n and m o r p h o l o g y . In a d d i t i o n to d e c r e a s i n g c e l l - s u b s t r a t e a d h e s i o n and c a u s i n g alterations i n c e l l - c e l l j u n c t i o n s , p o d o c a l y x i n was f o u n d to recruit N H E R F 1 and i n d u c e m i c r o v i l l u s f o r m a t i o n . E x p r e s s i o n o f p o d o c a l y x i n mutants i n e p i t h e l i a l c e l l s provided some insights into p o d o c a l y x i n ' s  mechanism of action. The  podocalyxin-  dependent a p i c a l recruitment o f N H E R F 1 was unexpected, but the requirement f o r the C t e r m i n a l D T H L sequence w a s not s u r p r i s i n g . In contrast, the w e l l - c o n s e r v e d c y t o p l a s m i c tail was f o u n d to be dispensable f o r m i c r o v i l l u s f o r m a t i o n , w h i l e the extracellular d o m a i n was essential.  3.4.2  Podocalyxin's Role in Determining Cell Morphology  E x p e r i m e n t a l e v i d e n c e f r o m the 1 9 7 0 ' s suggested that h i g h l y - g l y c o s y l a t e d and h e a v i l y s i a l y l a t e d g l y c o p r o t e i n s p l a y a k e y role in m a i n t a i n i n g the integrity o f the f o o t processes o f k i d n e y p o d o c y t e s , as d i s c u s s e d i n section 1.6.3 ( A n d r e w s , 1 9 7 9 ; S e i l e r et a l . , 1 9 7 7 ; S e i l e r et a l . , 1975). W i t h the s u b s e q u e n t i d e n t i f i c a t i o n o f p o d o c a l y x i n as the m a j o r c o m p o n e n t o f the p o d o c y t e g l y c o c a l y x and the tight c o r r e l a t i o n between its e x p r e s s i o n and p o d o c y t e f o o t p r o c e s s m o r p h o g e n e s i s in vivo, this m o l e c u l e b e c a m e the p r i m e candidate as a regulator o f f o o t process f o r m a t i o n ( D e k a n et a l . , 1 9 9 1 ; K e r j a s c h k i et a l . , 1 9 8 4 ; S a w a d a et a l . , 1 9 8 6 ; S c h n a b e l et a l . , 1989). G e n e targeting studies a l l o w e d us to  152  c o n f i r m that this m o l e c u l e is i n d e e d r e q u i r e d f o r the g e n e r a t i o n o f f o o t p r o c e s s e s : although  d e f i c i e n t a n i m a l s generate p o d o c y t e  p r e c u r s o r s , these f a i l to u n d e r g o  m o r p h o g e n e s i s (described i n section 1.6.4) ( D o y o n n a s et a l . , 2 0 0 1 ) . M o r e o v e r , this defect is rescued b y k i d n e y - s p e c i f i c ectopic expression o f p o d o c a l y x i n (see chapter 5). W i t h the current e x p e r i m e n t s , these observations can be g e n e r a l i z e d : rather than b e i n g a p o d o c y t e specific phenomenon, expression o f podocalyxin was also sufficient f o r induction o f m o r p h o g e n e s i s , as m e a s u r e d t h r o u g h m i c r o v i l l u s f o r m a t i o n , i n t w o d i f f e r e n t e p i t h e l i a l c e l l l i n e s , suggesting that it is a master-regulator o f this process. Importantly, endogenous p o d o c a l y x i n is e x p r e s s e d and a p i c a l l y targeted i n n o r m a l m a m m a r y e p i t h e l i u m in vivo ( S o m a s i r i et a l . , 2 0 0 4 ) and m a y be required f o r the e x t e n s i v e r e m o d e l i n g that o c c u r s i n this tissue.  A l t h o u g h the exact m e c h a n i s m by w h i c h p o d o c a l y x i n i n d u c e s m i c r o v i l l u s f o r m a t i o n is unclear, m y experiments suggest that an essential c o m p o n e n t o f p o d o c a l y x i n ' s a c t i v i t y as a c e l l m o r p h o g e n i s its e x t r a c e l l u l a r d o m a i n : mutants l a c k i n g this d o m a i n , t h o u g h apically targeted, were unable to induce m i c r o v i l l u s f o r m a t i o n . Several  previous  e x p e r i m e n t s in vivo, though indirect, support this n o t i o n : treatment o f k i d n e y p o d o c y t e s to neutralize the negatively charged s i a l i c a c i d residues o n the podocyte surface leads to a d r a m a t i c loss o f p o d o c y t e i n t e r d i g i t a t i n g f o o t processes ( A n d r e w s , 1 9 7 9 ; G e l b e r g et a l . , 1 9 9 6 ; S e i l e r et a l . , 1 9 7 7 ; S e i l e r et a l . , 1975). T h i s is b e l i e v e d to be due to a direct effect on podocalyxin  f o r several  reasons: p o d o c a l y x i n  is the most h i g h l y  expressed  s i a l o g l y c o p r o t e i n o n these c e l l s , its expression d u r i n g e m b r y o g e n e s i s perfectly c o i n c i d e s w i t h f o r m a t i o n o f these structures, and d i s r u p t i o n o f the podxl gene leads to f a i l u r e to  153  produce f o o t processes d u r i n g e m b r y o g e n e s i s ( D o y o n n a s et a l . , 2 0 0 1 ; K e r j a s c h k i et a l . , 1 9 8 4 ; S c h n a b e l et a l . , 1989). It w a s therefore not s u r p r i s i n g that deletion o f the entire m u c i n d o m a i n also prevented m i c r o v i l l u s f o r m a t i o n . A l t h o u g h the precise m e c h a n i s m by w h i c h this effect occurs is e l u s i v e , w e propose t w o m o d e l s . T h e first m o d e l p r o v i d e s a b i o p h y s i c a l explanation f o r the i n d u c t i o n o f m i c r o v i l l u s f o r m a t i o n by p o d o c a l y x i n . In this m o d e l , generation o f a d d i t i o n a l p l a s m a m e m b r a n e m a y s i m p l y serve to e v e n l y disperse the b u l k y , n e g a t i v e l y c h a r g e d m u c i n d o m a i n s ( F i g u r e 3 - 2 5 ) . T h i s m o d e l is consistent w i t h the p r e v i o u s reports o f a d o s e - d e p e n d e n t w e a k e n i n g o f c e l l j u n c t i o n s i n d u c e d by e c t o p i c e x p r e s s i o n o f p o d o c a l y x i n a n d the t i g h t c o r r e l a t i o n b e t w e e n  podocalyxin  o v e r e x p r e s s i o n and breast c a n c e r metastatic i n d e x ( S o m a s i r i et a l . , 2 0 0 4 ; T a k e d a et a l . , 2 0 0 0 ) . It is also consistent w i t h recent reports o f p o d o c a l y x i n as a " p r e - a p i c a l " d o m a i n f o r m i n g protein ( M e d e r et a l . , 2 0 0 5 ) .  154  Low / no podocalyxin  f-actin  F i g u r e 3-25:  cell junctions  l l  III  integrins  microvilli  | podocalyxin  T w o Models of Podocalyxin-Induced Microvillus  protein X  Formation.  Ectopic expression of podocalyxin recruited f-actin to the apical surface and led to formation of microvilli. This may lead to a decrease in basolateral actin available for stabilizing integrin-mediated cell-substratum interactions. Two models are proposed to explain podocalyxin's mechanism of action.  155  O n the other h a n d , a s u r p r i s i n g result o f m y studies is the apparent d i s p e n s a b i l i t y o f p o d o c a l y x i n ' s c y t o p l a s m i c d o m a i n , i n c l u d i n g a l l p o t e n t i a l p h o s p h o r y l a t i o n sites and m o t i f s w i t h the capacity f o r d i r e c t l y b i n d i n g e z r i n or N H E R F f a m i l y proteins ( L i et a l . , 2 0 0 2 ; M e d e r et a l . , 2 0 0 5 ; S c h m i e d e r et a l . , 2 0 0 4 ; S e r r a d o r et a l . , 2 0 0 2 ; T a k e d a et a l . , 2 0 0 1 ) . T h e f o r m a t i o n o f m i c r o v i l l i is tightly l i n k e d to the recruitment of f - a c t i n and E R M proteins to the a p i c a l m e m b r a n e d o m a i n . W i t h the d i s c o v e r y that p o d o c a l y x i n b i n d s the N H E R F f a m i l y o f e z r i n - b i n d i n g proteins, several groups have suggested that this f a m i l y o f adaptors is r e q u i r e d f o r p o d o c a l y x i n f u n c t i o n ( L i et a l . , 2 0 0 2 ; M e d e r et a l . , 2 0 0 5 ; S c h m i e d e r et a l . , 2 0 0 4 ; T a k e d a et a l . , 2 0 0 1 ; W e i n m a n , 2 0 0 1 ) . U n e x p e c t e d l y ,  however,  m y results demonstrated that f o r m a t i o n o f m i c r o v i l l i does not require direct interaction o f p o d o c a l y x i n w i t h N H E R F proteins. In support o f this f i n d i n g , a l t h o u g h one strain o f N H E R F l - n u l l m i c e i m p l i c a t e d this adapter protein i n intestinal m i c r o v i l l u s f o r m a t i o n or o r g a n i z a t i o n ( M o r a l e s et a l . , 2 0 0 4 ) , a s e c o n d strain f a i l e d to support the o b s e r v a t i o n ( S h e n o l i k a r et a l . , 2 0 0 2 ) . N o t a b l y , p o d o c a l y x i n is not e x p r e s s e d i n the intestinal b r u s h border, so i f N H E R F 1 is indeed i n v o l v e d in m i c r o v i l l u s f o r m a t i o n i n this tissue, it w o u l d have to be i n d e p e n d e n t o f p o d o c a l y x i n ( M c N a g n y et a l . , 1 9 9 7 ) . R e g a r d l e s s , m y data suggest that p o d o c a l y x i n m a y be able to r e c r u i t e z r i n a n d f - a c t i n by i n t e r a c t i n g w i t h another m o l e c u l e v i a its e x t r a c e l l u l a r d o m a i n . T h i s i n t e r a c t i o n m a y then transduce the s i g n a l to i n i t i a t e m i c r o v i l l u s f o r m a t i o n ( F i g u r e 3 - 2 5 ) . T h i s p r o v i d e s an a l t e r n a t i v e hypothesis to the b i o p h y s i c a l m o d e l p r o p o s e d above and depends u p o n loss o f a s p e c i f i c b i n d i n g site in p o d o c a l y x i n ' s e x t r a c e l l u l a r d o m a i n .  156  3.4.3  Significance of Recruitment of NHERF by Podocalyxin  Although  my  results preclude a direct f u n c t i o n a l role for  NHERF/podocalyxin  i n t e r a c t i o n s i n f o r m a t i o n o f m i c r o v i l l i and e s t a b l i s h i n g the a p i c a l c e l l d o m a i n , a p i c a l recruitment o f N H E R F 1 by p o d o c a l y x i n is l i k e l y to be very important f o r other aspects o f p o d o c a l y x i n and N H E R F f u n c t i o n . N H E R F proteins have been s h o w n to be i n v o l v e d i n a v a r i e t y o f processes i n c l u d i n g i o n transport, s i g n a l t r a n s d u c t i o n , g r o w t h c o n t r o l , a n d receptor i n t e r n a l i z a t i o n , and they have a l s o been s h o w n to b i n d a w i d e variety o f l i g a n d s and undergo o l i g o m e r i z a t i o n ( r e v i e w e d i n section 1.4.2) ( S h e n o l i k a r et a l . , 2 0 0 4 ; T h e l i n et a l . , 2 0 0 5 ; V o l t z et a l . , 2 0 0 1 ; W e i n m a n , 2 0 0 1 ; W e i n m a n et a l . , 2 0 0 5 ) . T h e fact that p o d o c a l y x i n is a potent i n d u c e r o f N H E R F recruitment to m i c r o v i l l i m a y suggest a new l i n k b e t w e e n the f o r m a t i o n o f these s p e c i a l i z e d structures and n u m e r o u s b i o l o g i c a l processes ( F i g u r e 3 - 2 6 ) . R e g a r d l e s s o f m i c r o v i l l u s f o r m a t i o n , the p o d o c a l y x i n - d e p e n d e n t l o c a l i z a t i o n o f N H E R F proteins c o u l d be quite important i n their regulation.  157  NHERF + no Podocalyxin  NHERF + high Podocalyxin  signal transduction  receptor internalization I I  KEY:  f-actin  cell junctions  integrins  III microvilli  NHERF-1  Figure 3-26: Model of Podocalyxin's Role in the Function of N H E R F Family Members. Although speculative, podocalyxin may regulate NHERF-dependent activities by altering its subcellular localization.  158  3.4.4  Podocalyxin and Microvilli in Adhesion / Anti-Adhesion  P o d o c a l y x i n has been s h o w n to act as a p r o - or a n t i - a d h e s i n d e p e n d i n g u p o n its c e l l u l a r context, as d i s c u s s e d i n sections 1.5.2 and 1.5.3. F o r e x a m p l e , w h e n expressed in H E V p o d o c a l y x i n acts as a l i g a n d f o r L - s e l e c t i n on l y m p h o c y t e s , but it is l i k e l y that this p r o adhesive f u n c t i o n is a s p e c i a l e x c e p t i o n rather than the general rule. T o act as a selectin l i g a n d p o d o c a l y x i n must be decorated w i t h an H E V - s p e c i f i c carbohydrate m o d i f i c a t i o n that is not f o u n d on m o s t other cells ( M i c h i e et a l . , 1 9 9 3 ; Sassetti et a l . , 1 9 9 8 ; S e g a w a et a l . , 1997). In contrast, w h e n o v e r e x p r e s s e d i n M D C K c e l l s , w h i c h l a c k the appropriate e n z y m e s f o r m o d i f y i n g p o d o c a l y x i n f o r s e l e c t i n - b i n d i n g , the m o l e c u l e w a s f o u n d to b l o c k c e l l - c e l l aggregation and m o d i f y c e l l j u n c t i o n s ( T a k e d a et a l . , 2 0 0 0 ) . L i k e w i s e , we have p r e v i o u s l y s h o w n that p o d o c a l y x i n - d e f i c i e n t m i c e have v a r i o u s d e v e l o p m e n t a l defects ( o m p h a l o c e l e and anuria) that are consistent w i t h e x c e s s i v e c e l l a d h e s i o n in its absence ( r e v i e w e d i n sections 1.6.4.1 and 1.9) ( D o y o n n a s et a l . , 2 0 0 1 ) . F i n a l l y , w e have f o u n d that p o d o c a l y x i n e x p r e s s i o n is u p r e g u l a t e d i n metastatic breast c a n c e r c e l l s and decreases cell-substrate a d h e s i o n w h e n overexpressed in vitro ( ( S o m a s i r i et a l . , 2 0 0 4 ) and F i g u r e 3 - 3 ) . O u r current results suggest that the a b i l i t y to act as either a p r o - or a n t i adhesin may  be c l o s e l y  l i n k e d to p o d o c a l y x i n ' s  a b i l i t y to generate m i c r o v i l l i .  P o d o c a l y x i n e x p r e s s i o n o n m i c r o v i l l i o f H E V , w h i c h are k n o w n to express a d h e s i o n m o l e c u l e s at t h e i r t i p s , c o u l d f a c i l i t a t e the reported L - s e l e c t i n - d e p e n d e n t l e u k o c y t e r o l l i n g and t r a n s e n d o t h e l i a l m i g r a t i o n ( G i r a r d et a l . , 1 9 9 9 ; P i c k e r et a l . , 1 9 9 1 ; v o n A n d r i a n et a l . , 1 9 9 5 ) . C o n v e r s e l y , in m o s t other c e l l types the p o d o c a l y x i n - c o a t e d , m i c r o v i l l i - r i c h , a p i c a l d o m a i n m a y protect c e l l s f r o m n o n - s p e c i f i c a d h e s i o n . T h u s , p o d o c a l y x i n o v e r e x p r e s s i o n i n breast m a y p r o m o t e t u m o u r c e l l d i s s e m i n a t i o n by  159  i n i t i a t i n g a general d i s r u p t i o n o f c e l l adhesion, p a r t i c u l a r l y under c o n d i t i o n s where a p i c a l m e m b r a n e d o m a i n s are e x p a n d e d due to b r e a k d o w n i n p o l a r i t y . T h i s w o u l d h e l p initiate metastatic spread by a different, although not necessarily m u t u a l l y e x c l u s i v e , m e c h a n i s m from  the  well  described  epithelial-mesenchymal transition  which  specifically  downregulates c e l l - c e l l j u n c t i o n s ( K a n g and M a s s a g u e , 2 0 0 4 ) .  It is also t e m p t i n g to speculate that the recruitment o f f - a c t i n to the a p i c a l m e m b r a n e o f c e l l s to generate m i c r o v i l l i m i g h t deplete a c t i n f r o m the basal s u r f a c e o f these c e l l s ( S c h m i e d e r et a l . , 2 0 0 4 ) , thereby p r e v e n t i n g stable i n t e r a c t i o n s o f i n t e g r i n s w i t h the extracellular matrix. T h i s hypothesis is supported by the o b s e r v a t i o n that cells transfected w i t h f u l l - l e n g t h p o d o c a l y x i n or mutants l a c k i n g its c y t o p l a s m i c tail generate abundant m i c r o v i l l i and e x h i b i t decreased c e l l - s u b s t r a t u m a d h e s i o n , w h i l e c e l l s transfected w i t h p o d o c a l y x i n l a c k i n g the e x t r a c e l l u l a r d o m a i n d o not generate m i c r o v i l l i a n d r e m a i n adherent ( ( S o m a s i r i et a l . , 2 0 0 4 ) , F i g u r e 3 - 2 5 , and u n p u b l i s h e d o b s e r v a t i o n s ) . F u r t h e r studies d e l i n e a t i n g the m e c h a n i s m s by w h i c h a p i c a l p o d o c a l y x i n and basolateral integrins compete f o r actin m a y c l a r i f y this process.  160  CHAPTER 4 : GENERATION OF TRANSGENIC MICE CONDITIONALLY OVEREXPRESSING PODOCALYXIN  4.1 Rationale T h e r e w e r e f o u r m a j o r reasons f o r a t t e m p t i n g t o generate a m o u s e  overexpressing  podocalyxin: a)  Loss-of-function  and gain-of-function  experiments  are t w o v e r y  powerful  approaches f o r e l u c i d a t i n g the n o r m a l f u n c t i o n o f a p o o r l y u n d e r s t o o d p r o t e i n . T h e p o d o c a l y x i n k n o c k o u t mouse has a d r a m a t i c phenotype i n that it dies w i t h i n 2 4 hours o f b i r t h , but this prevents us f r o m f u l l y u n d e r s t a n d i n g  podocalyxin's  f u n c t i o n s , e s p e c i a l l y i n adult a n i m a l s ( r e v i e w e d i n section 1.6.4) ( D o y o n n a s et a l . , 2001).  Much  overexpression  information  has been obtained f r o m  experiments  in vitro  podocalyxin  (see c h a p t e r 3 ) , b u t there are m a n y  questions  r e m a i n i n g , w h i c h m a y be answered u s i n g in vivo overexpression studies. b)  In vitro e x p e r i m e n t s  suggest  that p o d o c a l y x i n  mainly functions  to block  i n a p p r o p r i a t e c e l l a d h e s i o n . B y o v e r e x p r e s s i n g p o d o c a l y x i n i n tissues w h e r e a d h e s i o n is i m p o r t a n t , n u m e r o u s b i o l o g i c a l p r o c e s s e s c o u l d be altered i n an attempt t o c l a r i f y their i m p o r t a n c e and g a i n m e c h a n i s t i c insights. F o r e x a m p l e , b l o c k i n g a d h e s i o n o f l e u k o c y t e s m a y m i m i c diseases such as l e u k o c y t e a d h e s i o n d e f i c i e n c y s y n d r o m e s , by p r e v e n t i n g l e u k o c y t e r o l l i n g and f i r m arrest ( B u n t i n g et al., 2002). Overexpression  o f podocalyxin i n vascular endothelia may  mimic  161  s o m e c h a r a c t e r i s t i c s o f other d i s o r d e r s , l i k e a r t h r i t i s , by i n c r e a s i n g v a s c u l a r p e r m e a b i l i t y ( D o r i a et a l . , 2 0 0 6 ) . c)  P o d o c a l y x i n k n o c k o u t m i c e die w i t h i n one day o f birth, p r e s u m a b l y as a result o f a m a j o r k i d n e y defect. T h i s precludes a n a l y s i s o f defects i n other tissues in adult m i c e . T h e r e f o r e , by c r o s s i n g an i n d u c i b l e p o d o c a l y x i n overexpressor mouse to a p o d o c y t e - s p e c i f i c C r e m o u s e , it w o u l d be p o s s i b l e to generate m i c e s p e c i f i c a l l y e x p r e s s i n g p o d o c a l y x i n i n the area where it is thought to be absolutely essential. T h i s m a y e n a b le the m i c e to o v e r c o m e the lethal p h e n o t y p e , thereby a l l o w i n g a n a l y s i s o f other p o d o c a l y x i n - d e f i c i e n t tissues in adult.  d)  W e h a v e r e c e n t l y s h o w n that p o d o c a l y x i n is an e x c e l l e n t p r e d i c t o r o f  poor  o u t c o m e i n h u m a n breast c a n c e r ( r e v i e w e d i n s e c t i o n 1.10.1) ( S o m a s i r i et a l . , 2004).  It is p o s s i b l e that the a n t i - a d h e s i v e f u n c t i o n o f p o d o c a l y x i n a l l o w s f o r  m o r e r a p i d d i s s e m i n a t i o n o f t u m o u r c e l l s . T h i s p o s s i b i l i t y , as w e l l as other potential roles f o r p o d o c a l y x i n in breast cancer progression c o u l d be addressed by s p e c i f i c a l l y o v e r e x p r e s s i n g p o d o c a l y x i n i n m a m m a r y tissue. A f t e r i n d u c i n g the formation of tumours  u s i n g the  mouse  mammary tumour  virus,  disease  progression c o u l d be f o l l o w e d in these m i c e and c o m p a r e d to non-transgenics. T h e r e are therefore a n u m b e r o f i n t e r e s t i n g q u e s t i o n s that c o u l d be a d d r e s s e d u s i n g conditional podocalyxin overexpressing mice.  4.2 The Cre-loxP System T h e C r e - l o x P s y s t e m is a v e r s a t i l e tool a l l o w i n g i n d u c i b l e e x p r e s s i o n , or d e l e t i o n , o f genes o f i n t e r e s t ( r e v i e w e d i n ( L o b e a n d N a g y ,  1998)). C r e  is a  bacteriophage  162  r e c o m b i n a s e that s e l e c t i v e l y i n d u c e s s i t e - s p e c i f i c r e c o m b i n a t i o n b e t w e e n 3 4 bp l o x P consensus sequences ( F i g u r e 4 - 1 ) . T h u s , after i n s e r t i n g a gene i n b e t w e e n t w o  loxP  sequences, C r e can be used to delete the i n t e r v e n i n g sequences, l e a v i n g b e h i n d a si n g le l o x P site. It is p o s s i b l e to m a k e n u m e r o u s t i s s u e - s p e c i f i c k n o c k o u t s by generating just a single mouse l i n e w i t h a transgene c o n s i s t i n g o f the gene o f interest f l a n k e d by l o x P sites and then c r o s s i n g it w i t h any o f the m a n y m i c e a v a i l a b l e that express C r e i n a t i s s u e s p e c i f i c or t e m p o r a l l y regulated m a n n e r . L i k e w i s e , this s y s t e m c a n a l s o be used to c o n d i t i o n a l l y overexpress a gene. In this case, the l o x P sites f l a n k a spacer that separates the p r o m o t e r f r o m the gene o f interest and i n c l u d e s a stop c o d o n and p o l y - a d e n y l a t i o n ( p o l y A ) sequences. T h i s element can subsequently be deleted, thereby f a c i l i t a t i n g gene expression f r o m a c o m m o n promoter. T h u s , the C r e - l o x P system was deemed suitable f o r i n d u c i b l e expression o f p o d o c a l y x i n in m i c e .  163  1> D>—  + Cre recombinase  V  Figure 4-1: Schematic of Cre-Mediated Recombination. C r e r e c o m b i n a s e ( h e x a g o n ) m e d i a t e s h o m o l o g o u s r e c o m b i n a t i o n b e t w e e n l o x P sites (triangles), thereby e x c i s i n g the i n t e r v e n i n g ( b l a c k ) sequence a n d c o n n e c t i n g the f l a n k i n g (purple) sequences.  164  4.3 Transgenic Construct T h e r e are several basic l o x P - c o n t a i n i n g constructs a v a i l a b l e f o r g e n e r a t i n g transgenic m i c e ; the one that I u s e d to generate the c o n d i t i o n a l p o d o c a l y x i n  overexpressing  transgenic is c a l l e d Z / E G , and it encodes several useful features ( F i g u r e 4 - 2 and ( L o b e et a l . , 1 9 9 9 ; N o v a k et a l . , 2000)). I n i t i a l l y , p o d o c a l y x i n and G F P e x p r e s s i o n were b l o c k e d by a /acZ/neomycin resistance  (Pgeo) f u s i o n  spacer cassette, w h i c h i n c l u d e d a stop c o d o n  and three p o l y A sites. T h i s was f l a n k e d by l o x P sites, a n d w a s therefore e x c i s a b l e by C r e - m e d i a t e d s i t e - s p e c i f i c r e c o m b i n a t i o n . T h e C M V enhancer/(3-actin promoter was used to d r i v e e x p r e s s i o n o f the gene o f interest (podxl, upstream o f an I R E S sequence and GFP,  i n this c a s e ) , w h i c h w a s inserted  e n a b l i n g e x p r e s s i o n o f the reporter in concert  w i t h p o d o c a l y x i n . T h u s , the p r o m o t e r i n i t i a l l y d r o v e  u b i q u i t o u s e x p r e s s i o n o f (3-  galactosidase ((3-gal), but C r e - e x c i s i o n l e d to e x p r e s s i o n , i n s t e a d , o f p o d o c a l y x i n and G F P (Figure 4-2).  165  CMV-f3 actin promoter | pgeo 3pA  loxP  )\  Podocalyxin | IRES-GFP  loxP  i  + Cre  CMV-p actin promoter Podocalyxin  IRES-GFP  loxP Figure 4-2: Schematic of Transgenic Construct Before and After Cre-Mediated Recombination. T h e o r i g i n a l construct e n c o d e d the  Pgeo f u s i o n gene f o l l o w e d by p o l y A ( p A ) signals and  f l a n k e d by l o x P sites. P o d o c a l y x i n a n d G F P w e r e e n c o d e d d o w n s t r e a m a n d w e r e therefore not e x p r e s s e d . C r e - m e d i a t e d r e c o m b i n a t i o n d e l e t e d the spacer e l e m e n t a n d i n d u c e d e x p r e s s i o n o f p o d o c a l y x i n and G F P .  166  4.4 In Vitro Validation of Transgenic Construct P r i o r to u s i n g the construct to generate t r a n s g e n i c m i c e , a s i m i l a r p r e c u r s o r p l a s m i d l a c k i n g G F P was first tested in vitro by transfecting the m u r i n e m y e l o m a c e l l l i n e , N S O , w h i c h w a s c h o s e n f o r ease o f t r a n s f e c t i o n (see s e c t i o n 2 . 1 . 1 ) . A f t e r p r o d u c i n g stable n e o m y c i n resistant lines c o n t a i n i n g the construct, the c e l l s were transiently transfected w i t h the p C I - C r e e x p r e s s i o n p l a s m i d ( g e n e r o u s l y p r o v i d e d by D r . F a b i o R o s s i ) . F l o w c y t o m e t r y w a s then used to assess p o d o c a l y x i n e x p r e s s i o n ( F i g u r e 4-3). A s e x p e c t e d , due to the transient nature o f the C r e t r a n s f e c t i o n , s o m e c e l l s expressed p o d o c a l y x i n , w h i l e m a n y d i d not; the e f f i c i e n c y w o u l d be expected to increase w i t h stable C r e transfection. T h u s , this construct was f o u n d to f u n c t i o n appropriately and the Z/EG-podxl  plasmid was  used to generate stable e m b r y o n i c stem c e l l ( E S C ) lines.  167  Empty vector  Podocalyxin  X  _>,  o o  CSI  o feC#  T3 O CL  I'' ><• I" " 111 • • I 200 400 600 800 1000 1 1 1 1 1  0  0  • ii I iiii I iiii |iiii |iiii | 200 <400 600 800 1000  Forward scatter (size) Figure 4-3: Cre-Mediated Recombination Induced Podocalyxin Expression in NS0 Cells Transfected with the Construct.  168  4.5 Selection of Transgene Positive ES Cells T h e l i n e a r i z e d construct was i n t r o d u c e d into m u r i n e R l E S C s by e l e c t r o p o r a t i o n . C e l l s w e r e then a l l o w e d to r e c o v e r , after w h i c h t i m e t h e y w e r e s e l e c t e d f o r  neomycin  resistance. A p p r o x i m a t e l y 4 0 0 drug-resistant E S C c o l o n i e s were p i c k e d and transferred to 9 6 w e l l plates. T h o s e c o l o n i e s that c o n t i n u e d to g r o w were r e p l i c a plated onto feeder layers f o r c o n t i n u e d e x p a n s i o n and preparation o f s t o c k s and onto g e l a t i n - c o a t e d plates f o r a n a l y s i s ( F i g u r e 4 - 4 ) . A s s e s s m e n t o f c l o n e s i n c l u d e d c o n f i r m a t i o n o f transgene presence by P C R ( F i g u r e 4 - 5 ) and screening c e l l s , before and after d i f f e r e n t i a t i o n , f o r Pgalactosidase e x p r e s s i o n . T h i s ensured that the transgene had integrated i n t o a site that w o u l d f a c i l i t a t e e x p r e s s i o n in m a n y c e l l types. F i v e c l o n e s met these c r i t e r i a and were m a i n t a i n e d f o r further screening: 2 A 7 , 2 B 6 , 2 G 1 2 , 3 E 3 , and 3 H 7 .  169  DNA /  1 G418 selection  Pick colonies & ^ screen for p-gal /  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  1  /  /  / Freeze stocks  \ Confirm transgene presence by PCR  \ Assess Cre-mediated transgene expression F i g u r e 4-4: O v e r v i e w o f S e l e c t i o n P r o c e s s .  R I E S C s were electroporated, and c e l l s that had incorporated the construct (green circles) w e r e s e l e c t e d b a s e d o n r e s i s t a n c e to n e o m y c i n ( G 4 1 8 ) . T h e s e c o l o n i e s w e r e  then  screened f o r f3-galactosidase e x p r e s s i o n , and p o s i t i v e clones (blue) were further assessed after generating f r o z e n stocks.  170  o  Podocalyxin PCR product  F i g u r e 4-5: P C R - B a s e d D e t e c t i o n o f T r a n s g e n e i n G e n o m i c D N A o f E S C s . Podocalyxin c D N A , encoded by the transgenic construct, was detected in P-glactosidasepositive clones, but not in parental R l E S C s .  171  4.6 Transgene Expression Analysis After Cre-Mediated Recombination in ESCs T h e next step i n the s e l e c t i o n process r e q u i r e d testing the f i v e c l o n e s f o r proper C r e mediated e x c i s i o n o f the spacer and subsequent expression o f p o d o c a l y x i n . R T - P C R w a s used to c o n f i r m the presence o f p o d o c a l y x i n m R N A after C r e - t r a n s f e c t i o n ( F i g u r e 4 - 6 ) . It was also noted that parental R l E S C s expressed a l o w level o f endogenous p o d o c a l y x i n . T h i s w a s not u n e x p e c t e d because it has been s h o w n that h u m a n E S C s a l s o express p o d o c a l y x i n ( W e i et a l . , 2 0 0 5 ) . F l o w c y t o m e t r y  c o n f i r m e d surface expression o f  exogenous p o d o c a l y x i n (above b a c k g r o u n d levels) i n a l l f i v e clones ( F i g u r e 4 - 7 ) . A g a i n , C r e was transfected transiently, and the e f f i c i e n c y w o u l d be i m p r o v e d u p o n sustained C r e expression in vivo. F u r t h e r m o r e , G F P expression was detected i n a l l c l o n e s , both b y R T P C R ( F i g u r e 4 - 8 ) , a n d by f l u o r e s c e n c e m i c r o s c o p y . B a s e d o n this e x p r e s s i o n data, as w e l l as c o l o n y m o r p h o l o g y , t w o c l o n e s were c h o s e n f o r generation o f c h i m e r i c m i c e ( 2 G 1 2 and 2 B 6 ) .  172  «  F i g u r e 4-6:  Podocalyxin  Expression of Podocalyxin m R N A in E S C s .  R T - P C R w a s used to detect p o d o c a l y x i n m R N A i n E S C s after transient transfection w i t h C r e , and in positive c o n t r o l ( F D C P - 1 ) cells.  173  2B6 (22%) C  x CD O  «i  4 ifs  080803 E S C J 13  080803 ESC.007  2A7  (14%)  080803 ESC .017  3E3 (22%) ^  090803ESC.021  =1  (45%)  3H7  3 ESC .025  if2'  o  •r  1)  "D O CL  5  2 G 1 2 (7%)  SV !«'  . J i FSC  21  •  080803 ESC .008  080803 ESC.012  ^  080803 ESC 020  si  SfoT  080803 ESC 016  ,...  11  •r""jai  o-  •  •r  Figure 4-7: Analysis of Podocalyxin Expression by Flow Cytometry.  The five clones expressed various levels of podocalyxin after Cre transfection. Rat IgGi was used as a negative control for podocalyxin staining.  174  GFP  •  Figure 4-8: Expression of G F P m R N A in transfected ESCs. Cre-induced expression of G F P was detected by R T - P C R in all transfected clones, but not i n parental R l E S C s .  175  4.7 Production of Chimeric Mice C h i m e r i c m i c e were generated w i t h the 2 B 6 c l o n e by C o r r i n n e L o b e ' s laboratory u s i n g the t e c h n i q u e o f E S C - m o r u l a a g g r e g a t i o n ( L o b e et a l . , 1999). A l t h o u g h aggregations were p e r f o r m e d repeatedly w i t h several c l o n e s , o n l y one e x p e r i m e n t led to p r o d u c t i o n o f chimeric mice, and unfortunately  n o n e o f the e x p e r i m e n t s r e s u l t e d i n g e r m l i n e  transmission o f the transgene.  4.8 Explanation for Lack of Germline Transmission in Transgenic Mice T h e E S C c l o n e s were then sent to J i l l L a h t i ' s l a b o r a t o r y f o r an a d d i t i o n a l attempt at generating c h i m e r i c m i c e . K a r y o t y p i n g is often c o m p l e t e d before generating transgenic m i c e w i t h c u l t u r e d E S C s , so t w o c l o n e s ( 2 G 1 2 a n d 3 E 3 ) w e r e k a r y o t y p e d  before  p r o c e e d i n g . U s i n g this t e c h n i q u e , it w a s d e t e r m i n e d that both c l o n e s were t r i s o m i c f o r c h r o m o s o m e eight, t h r o u g h c r e a t i o n o f w h a t appeared to be i s o c h r o m o s o m e s ( F i g u r e 4 - 9 ) . T r i s o m y eight is a f a i r l y c o m m o n c h r o m o s o m a l a b e r r a t i o n n o t i c e d i n c u l t u r e d E S C s , as it gives c e l l s a g r o w t h advantage ( L i u et a l . , 1997). U n f o r t u n a t e l y , although this genetic defect does not i n h i b i t p r o d u c t i o n o f c h i m e r i c m i c e , it does prevent g e r m l i n e t r a n s m i s s i o n o f the transgene because the a b n o r m a l c e l l s cannot f o r m an entire v i a b l e a n i m a l . S i n c e both tested c l o n e s h a d the same structural c h r o m o s o m a l a b e r r a t i o n , it is l i k e l y that the o r i g i n a l R l E S C s were also t r i s o m i c f o r c h r o m o s o m e eight. T h i s p r e c l u d e d further use o f these cells f o r generating transgenic a n i m a l s .  176  .1  I  )L << > < ><  II  d <« IMJ II »l M M •  Ci  7  tl 14  V «  II 15  »  if 16  10  ft 17  11  II  «  *  ttt  Figure 4-9: Karyotyping Uncovered the Presence of Trisomy Eight in Tested Clones.  177  4.9 Summary and Conclusions W e were interested i n generating transgenic m i c e c o n d i t i o n a l l y e x p r e s s i n g p o d o c a l y x i n as a tool f o r assessing p o d o c a l y x i n ' s role i n breast cancer, f o r i n v e s t i g a t i n g the effect o f a general loss o f a d h e s i o n i n n u m e r o u s c e l l t y p e s , and i n o r d e r to rescue p o d o c a l y x i n k n o c k o u t m i c e by s p e c i f i c a l l y expressing p o d o c a l y x i n i n k i d n e y s , where it is presumed to be essential f o r v i a b i l i t y o f m i c e . A transgenic construct w a s generated w h i c h e n a b l e d expression o f p o d o c a l y x i n f r o m a ubiquitous p r o m o t e r , o n l y after C r e - m e d i a t e d e x c i s i o n o f a spacer e l e m e n t . T h i s c o n s t r u c t w a s u s e d to generate E S C c l o n e s that e x h i b i t e d e x c e l l e n t e x p r e s s i o n o f the transgene before and after d i f f e r e n t i a t i o n , as assayed by (3galactosidase e x p r e s s i o n . S e v e r a l c l o n e s w e r e f o u n d to be acceptable f o r generation o f t r a n s g e n i c m i c e b a s e d o n m o r p h o l o g y as w e l l as e f f i c i e n t C r e - m e d i a t e d i n d u c i b l e e x p r e s s i o n o f p o d o c a l y x i n and G F P . H o w e v e r , i n i t i a l attempts at g e n e r a t i n g c h i m e r i c m i c e that propagated the transgene in the g e r m l i n e f a i l e d , and the E S C s were therefore e x a m i n e d f o r c h r o m o s o m a l aberrations. T w o c l o n e s were tested, and both were f o u n d to possess an a d d i t i o n a l c o p y o f c h r o m o s o m e eight. T h i s e x p l a i n s the l a c k o f g e r m l i n e t r a n s m i s s i o n , and necessitates the generation o f new E S C c l o n e s , b e g i n n i n g w i t h n e w , untransfected E S C s . In the m e a n t i m e , a more direct strategy was used to address one goal o f this study: the repair o f p o d o c a l y x i n - k n o c k o u t k i d n e y s w a s attempted u s i n g a k i d n e y s p e c i f i c p o d o c a l y x i n transgenic mouse (chapter 5).  178  CHAPTER 5 : REPAIR OF KIDNEY D E F E C T IN PODOCALYXIN-NULL MICE  5.1 Rationale L o s s - o f - f u n c t i o n e x p e r i m e n t s c a n p r o v i d e c o n s i d e r a b l e insights into protein f u n c t i o n , so p o d o c a l y x i n - n u l l m i c e had been generated p r e v i o u s l y ( D o y o n n a s et a l . , 2 0 0 1 ) . D e f e c t s w e r e e x p e c t e d i n tissues w h e r e p o d o c a l y x i n is e x p r e s s e d : p o d o c y t e s o f the k i d n e y , hematopoietic progenitor cells, megakaryocytes, vascular endothelia, mesothelial cells l i n i n g o r g a n s , and a subset o f n e u r o n a l c e l l s ( D o y o n n a s et a l . , 2 0 0 1 ; D o y o n n a s et a l . , 2 0 0 5 ; G a r c i a - F r i g o l a et a l . , 2 0 0 4 ; H a r a et a l . , 1 9 9 9 ; H o r v a t et a l . , 1 9 8 6 ; K e r j a s c h k i et a l . , 1 9 8 4 ; M c N a g n y et a l . , 1 9 9 7 ; M i e t t i n e n et a l . , 1 9 9 0 ; M i e t t i n e n et a l . , 1 9 9 9 ; V i t u r e i r a et a l . , 2 0 0 5 ) . H o w e v e r , deletion o f the podxl gene resulted i n perinatal lethality, p r e v e n t i n g a n a l y s i s o f defects in adult m i c e . F u r t h e r a n a l y s i s suggested that loss o f p o d o c a l y x i n i n podocytes o f the k i d n e y was the cause o f this dramatic phenotype. P o d o c a l y x i n - n u l l m i c e w e r e a n u r i c , e x h i b i t e d i n c r e a s e d b l o o d p r e s s u r e , a n d w e r e f o u n d to have  drastic  m o r p h o l o g i c a l a b n o r m a l i t i e s i n k i d n e y g l o m e r u l i u p o n a n a l y s i s by electron m i c r o s c o p y ( D o y o n n a s et a l . , 2 0 0 1 ) . In order to study defects i n adult m i c e l a c k i n g p o d o c a l y x i n , we a t t e m p t e d to r e s c u e p o d o c a l y x i n - n u l l specifically  in  podocytes.  The  first  m i c e by e c t o p i c a l l y e x p r e s s i n g strategy  involved  generating  podocalyxin podocalyxin  o v e r e x p r e s s i n g m i c e u s i n g the i n d u c i b l e s y s t e m d e s c r i b e d i n the p r e v i o u s chapter. H o w e v e r , due to d e l a y s in that strategy, it w a s d e c i d e d that a m o r e direct strategy w a s required. The  new  strategy  involved  generating a podocyte-specific  podocalyxin  transgene.  179  5.2 Transgenic Construct T h e p r o m o t e r f o r the NPHS1  gene, w h i c h encodes n e p h r i n (discussed in s e c t i o n 1.6.5.1,  and r e v i e w e d i n (Salant and T o p h a m , 2 0 0 3 ; T r y g g v a s o n , 1999)), w a s c h o s e n to d r i v e e c t o p i c e x p r e s s i o n o f p o d o c a l y x i n i n p o d o c y t e s . In the g l o m e r u l u s , n e p h r i n is f o u n d e x c l u s i v e l y i n the slit d i a p h r a g m r e g i o n o f p o d o c y t e s , and n e p h r i n and p o d o c a l y x i n are i n i t i a l l y expressed at a s i m i l a r t i m e point i n d e v e l o p m e n t ( K a w a c h i et a l . , 2 0 0 2 ; S c h n a b e l et a l . , 1989). T h e p I R E S 2 - E G F P e x p r e s s i o n vector w a s c h o s e n as the b a c k b o n e f o r the transgenic construct. T h i s vector i n c l u d e d a n e o m y c i n resistance cassette, as w e l l as a m u l t i p l e c l o n i n g site d o w n s t r e a m o f a u b i q u i t o u s promoter. M u r i n e p o d o c a l y x i n c D N A was c l o n e d into the m u l t i p l e c l o n i n g site, upstream o f GFP,  w h i c h w a s expressed u s i n g  an I R E S sequence. B e f o r e generating transgenic m i c e , the NPHS1 u p s t r e a m o f podxl, and the u b i q u i t o u s C M V  promoter was inserted  p r o m o t e r w a s deleted ( F i g u r e 5 - 1 ) . T h i s  enabled p o d o c a l y x i n and G F P expression s p e c i f i c a l l y i n podocytes.  180  NPHS1 promoter  Podocalyxin  IRES-GFP  Figure 5 - 1 : Schematic of Transgenic Construct. The transgenic construct enabled tissue-specific expression of podocalyxin and G F P from the podocyte-specific NPHSI promoter.  181  5.3 In Vitro Expression Analysis B e f o r e d e l e t i o n o f the u b i q u i t i o u s p r o m o t e r , the t r a n s g e n i c c o n s t r u c t w a s tested by transfection into C H O  cells. Transient transfection resulted in expression of  both  p o d o c a l y x i n and G F P , as detected by f l o w c y t o m e t r y ( F i g u r e 5 - 2 ) .  182  FBC-H  F S C + )  fSCM  Figure 5-2: Transiently Transfected CHO Cells Expressed Podocalyxin and GFP from the Transgenic Construct. Cells were labelled with anti-podocalyxin antibodies and assessed for podocalyxin and G F P expression by flow cytometry.  183  5.4 Generation of Transgenic Founders T h e t r a n s g e n i c construct w a s l i n e a r i z e d after s u c c e s s f u l l y testing it in vitro; the f i n a l construct c o n t a i n e d o n l y the NPHS1  p r o m o t e r u p s t r e a m o f podxl and GFP. T h i s D N A  f r a g m e n t w a s i n j e c t e d into the p r o n u c l e i o f f e r t i l i z e d eggs i n the B i o m e d i c a l R e s e a r c h C e n t r e ' s transgenic f a c i l i t y . A p p r o x i m a t e l y 4 0 0 i n j e c t i o n s were p e r f o r m e d , r e s u l t i n g i n 51 pups. G e n o t y p i n g results suggested that f i v e m i c e c o n t a i n e d the transgene, but o n l y one f o u n d e r p r o d u c e d consistently strong signals f o r the transgene by P C R .  5.5 Transgene Expression Analysis G e n e r a t i o n o f t r a n s g e n i c m i c e by o o c y t e m i c r o i n j e c t i o n often results i n i n s e r t i o n o f m u l t i p l e c o p i e s o f the transgene into a s i n g l e site. T r a d i t i o n a l l y , transgene c o p y n u m b e r has t h e r e f o r e been a s s e s s e d i n o r d e r to a p p r o x i m a t e e x p r e s s i o n l e v e l s .  However,  e x p r e s s i o n c a n also be affected by m a n y other factors. F o r e x a m p l e , the transgene m a y insert into a r e g i o n o f heterochromatin that c o u l d prevent its e x p r e s s i o n . A l t e r n a t i v e l y , it m a y be l o c a t e d n e x t to a strong p r o m o t e r , w h i c h c o u l d alter e x p r e s s i o n . In contrast, i n s e r t i o n a l m u t a g e n e s i s c o u l d lead to d i s r u p t i o n o f another g e n e ' s n o r m a l e x p r e s s i o n pattern or even creation o f a f u s i o n protein. T h e r e f o r e , w h e n generating transgenic m i c e , several f o u n d e r s s h o u l d be assessed to ensure that a n y phenotypes are the result o f the transgene itself, and not the result o f the transgene's insertion site. A l l f i v e f o u n d e r s were therefore i n i t i a l l y assessed f o r transgene e x p r e s s i o n .  184  5.5.1  Transgene Expression in Glomeruli of Founders' Kidneys  In that podocytes were the transgene's target c e l l s , the f o u n d e r s ' k i d n e y s were assessed f o r e x p r e s s i o n , after first i n s u r i n g that the transgene had been transmitted to subsequent pups. It is important to r e m e m b e r that the founders were otherwise n o r m a l a n i m a l s w i t h a d d i t i o n a l , ectopic expression o f p o d o c a l y x i n (as w e l l as G F P ) f r o m the transgene. T h u s , p o d o c a l y x i n e x p r e s s i o n c o u l d not be assessed in these a n i m a l s ; the f o r e i g n G F P protein w a s therefore used as a m a r k e r . G F P e x p r e s s i o n w a s v e r y f a i n t , and c o u l d o n l y be c o n v i n c i n g l y detected after i m m u n o l a b e l l i n g u s i n g an a n t i - G F P a n t i b o d y . T h e f o u n d e r w i t h the strongest transgene signal by P C R also had the brightest G F P s t a i n i n g i n k i d n e y g l o m e r u l i ( F i g u r e 5 - 3 ) . T h i s f o u n d e r was k n o w n as 3 - 3 as it w a s the third pup f r o m the third set o f injections. T w o other f o u n d e r s ( 1 - 9 and 3 - 1 0 ) also had reasonable l e v e l s o f G F P e x p r e s s i o n , w h i l e the last t w o ( 1 - 6 and 1-8) were e x t r e m e l y w e a k . A n a l y s i s at higher m a g n i f i c a t i o n s h o w e d a d i s t i n c t i v e s t a i n i n g pattern, as s h o w n i n the i m a g e o f a s i n gl e g l o m e r u l u s i n F i g u r e 5 - 4 . T h i s pattern is t y p i c a l o f a p i c a l m e m b r a n e l a b e l l i n g o f g l o m e r u l a r podocytes.  185  B6(wt)  1-9  3-3  Figure 5-3: GFP Expression in Glomeruli of Transgenic Founders. A f t e r perfusion with paraformaldehyde ( P F A ) , kidneys were frozen, sectioned, and i m m u n o l a b e l l e d w i t h a n t i - G F P a n t i b o d i e s , as d e s c r i b e d i n c h a p t e r 2 . B r i g h t staining  represents  G F P in g l o m e r u l i ,  while  yellow  staining  is  green  background  i m m u n o f l u o r e s c e n c e . T i s s u e s f r o m w i l d t y p e ( B 6 ) m i c e were used as negative controls f o r G F P s t a i n i n g . S c a l e bar: 5 0 p m .  186  Figure 5-4: Glomerulus from Founder with Highest GFP Expression Levels. A f t e r P F A perfusion, kidneys were frozen, sectioned, and immunolabelled  with  a n t i b o d i e s against G F P . P o s i t i v e s t a i n i n g c a n be seen i n the g l o m e r u l u s . T i s s u e s f r o m w i l d t y p e ( B 6 ) m i c e were used as negative controls f o r G F P staining. Scale bar: 2 0 u m .  187  5.5.2 Other Tissues Examined by Immunofluorescence for GFP Expression Demonstrate Lack of Non-Specific Expression A f t e r assessing transgene e x p r e s s i o n in k i d n e y , it was also necessary to i d e n t i f y sites o f inappropriate e x p r e s s i o n . O t h e r areas where p o d o c a l y x i n is n o r m a l l y e x p r e s s e d , such as h e m a t o p o i e t i c c e l l s and v a s c u l a r tissues, w e r e i m p o r t a n t to e x a m i n e because e c t o p i c e x p r e s s i o n i n these areas w o u l d prevent a n a l y s i s o f defects i n c e l l s l a c k i n g p o d o c a l y x i n w h e n crossed w i t h p o d o c a l y x i n - n u l l a n i m a l s . N o e x p r e s s i o n w a s detected in the l i v e r or l u n g , w h e r e v a s c u l a r e x p r e s s i o n w o u l d be n o t i c e a b l e ( F i g u r e 5 - 5 ) , or in the p e r i p h e r a l b l o o d ( F i g u r e 5 - 6 ) . A s p o d o c a l y x i n and nephrin can both be f o u n d i n a subset o f c e l l s i n the b r a i n ( G a r c i a - F r i g o l a et a l . , 2 0 0 4 ; Putaala et a l . , 2 0 0 0 ; P u t a a l a et a l . , 2 0 0 1 ; V i t u r e i r a et a l . , 2 0 0 5 ) , this organ was also e x a m i n e d , but G F P e x p r e s s i o n w a s not detected ( F i g u r e 5-7).  In s u m m a r y , a l t h o u g h a l l f i v e f o u n d e r s d e m o n s t r a t e d s o m e degree o f  GFP  e x p r e s s i o n i n k i d n e y , o n l y three had reasonable l e v e l s . Inappropriate e x p r e s s i o n o f the transgene was not detected ( T a b l e 5 - 1 ) .  188  Liver expression  Lung expression  B6 (wt)  3-3  F i g u r e 5-5: L a c k o f N o n - S p e c i f i c E x p r e s s i o n i n L i v e r a n d L u n g .  A f t e r P F A p e r f u s i o n , tissues were f r o z e n , sectioned, a n d i m m u n o l a b e l l e d w i t h antibodies against G F P . Transgene expression w a s not detected i n vascular endothelia. T i s s u e s f r o m w i l d t y p e ( B 6 ) m i c e w e r e u s e d as negative c o n t r o l s f o r G F P s t a i n i n g . K i d n e y sections f r o m f o u n d e r 3 - 3 were used as positive controls f o r G F P staining. Scale bar: 5 0 u.m.  189  IUU  10°  10  1  10  2  10 • 3  10  4  GFP Figure 5-6: Transgene Expression was Undetectable in Peripheral Blood. P e r i p h e r a l b l o o d w a s i s o l a t e d f r o m p r o g e n y o f o r i g i n a l f o u n d e r s a n d assessed f o r G F P e x p r e s s i o n by f l o w c y t o m e t r y . G F P e x p r e s s i o n w a s c o m p a r e d to expression i n peripheral b l o o d isolated f r o m w i l d t y p e ( B 6 ) a n d ubiquitous G F P - e x p r e s s i n g transgenics.  190  B6 (wt)  1-9  3-3  F i g u r e 5-7: G F P E x p r e s s i o n w a s N o t D e t e c t e d i n B r a i n s o f F o u n d e r s .  A f t e r P F A perfusion, brains were f r o z e n , sectioned, and i m m u n o l a b e l l e d for G F P . A l t h o u g h there appeared to be s o m e b a c k g r o u n d f l u o r e s c e n c e , there w a s n o s p e c i f i c e x p r e s s i o n i n b r a i n . T i s s u e s f r o m w i l d t y p e ( B 6 ) m i c e were used as negative controls f o r G F P s t a i n i n g . K i d n e y sections f r o m f o u n d e r 3 - 3 were used as p o s i t i v e c o n t r o l s f o r G F P staining. S c a l e bar: 5 0 p m .  191  E x p r e s s i o n Pattern Founder Liver  Lung  -  -  -  ++  -  nd  nd  3-10  +  -  nd  nd  1-8  weak  -  nd  nd  1-6  weak  -  nd  nd  Kidney  Brain  3-3  ++  1-9  Blood  Table 5 - 1 : S u m m a r y of Transgene Expression.  (nd: not determined)  192  5.6 Breeding Scheme and Expected Numbers of Rescued Mice I n d i v i d u a l c o l o n i e s were expanded f r o m the three most suitable founders ( 3 - 3 , 1-9, and 3 10) i n order to obtain m i c e f o r further breeding experiments. Podxl* *transgene* ' progeny 1  were then crossed w i t h podxl*''(transgene'')  1  m i c e to o b t a i n m i c e h e t e r o z y g o u s f o r both  the podxl k n o c k o u t a l l e l e and the n e w , r a n d o m l y integrated podxl transgene ( F i g u r e 5 - 8 ) . O b v i o u s l y , it w a s necessary to use m i c e h e t e r o z y g o u s f o r the podxl k n o c k o u t a l l e l e b e c a u s e o f the d e m o n s t r a t e d l e t h a l i t y o f h o m o z y g o t e s . transgene* ' a n i m a l s , 1  these  were  interbred  to o b t a i n  After obtaining  podxl* '  transgene-containing  1  mice  h o m o z y g o u s f o r the podxl k n o c k o u t a l l e l e . T h e e x p e c t e d f r e q u e n c y o f each potential g e n o t y p e f r o m these b r e e d i n g s is s h o w n i n T a b l e 5 - 2 . M i c e o f interest w e r e those e x p r e s s i n g n o e n d o g e n o u s p o d o c a l y x i n w h i l e also h a r b o u r i n g at least o n e c o p y o f the new transgene. T h e expected f r e q u e n c y o f these rescued m i c e w a s 3/16, as s h o w n i n blue in Table 5-2.  193  podxr' ^ transgene^'  i  /+/"  podxl  x podxl''  +/"  transgene  ;  transgene '  +1-  /+/"  x podxl  transgene  Rescued mice F i g u r e 5 - 8 : B r e e d i n g S c h e m e used to G e n e r a t e M i c e E x p r e s s i n g P o d o c a l y x i n o n l y i n Kidney.  podxl transgene transgene transgene  + / +  podxl  + /  podxl  +/+  1/16  2/16  1/16  +/-  2/16  4/16  2/16  -/-  1/16  2/16  1/16  T a b l e 5 - 2 : E x p e c t e d N u m b e r s o f A l l G e n o t y p e s R e s u l t i n g f r o m Podxt  Transgene  Crosses.  Blue represents the frequency of potentially rescued mice.  194  5.7 Transgenic Mice Still Die within 24 Hours of Birth A f t e r i n t e r c r o s s i n g podxt'~transgene '~ m i c e , a l l r e s u l t i n g pups that s u r v i v e d b e y o n d the +  first 2 4 hours were genotyped. A l t h o u g h n o r m a l M e n d e l i a n ratios were seen f o r  podxl  and podxt '  podxt'  1  t r a n s g e n e p o s i t i v e a n i m a l s , there w a s n o t a s i n g l e s u r v i v i n g  +l+  transgene" m o u s e ( T a b l e 5 - 3 ) . A l t h o u g h o n l y the first f i v e litters are s h o w n i n T a b l e 5 - 3 , all subsequent litters were also d e v o i d o f rescued pups. N o t a b l y , there was n o e v i d e n c e o f i n s e r t i o n a l m u t a g e n e s i s , as m i c e w i t h the transgene w e r e f o u n d i n s i m i l a r n u m b e r s to those l a c k i n g it w h e n m i c e heterozygous f o r e n d o g e n o u s podxl and the transgene w e r e crossed w i t h podxt ' a n i m a l s ( T a b l e 5 - 4 ) . 1  195  Genotype Litter  podxl  + , +  podxl  1  0  6  2  3  3  3  1  2  4  3  4  5  2  4  TOTAL  9  19  + l  0 0 0 0 0 0  Table 5-3: Actual Numbers of Transgene-Positive Mice of Each Genotype in the First Five Litters Resulting from Podxt'Transgene* ' Crosses Demonstrated a Lack 1  of Rescue of Podxt ' Mice. 1  196  transgene  Litter  podxl  + / +  podxl  transgene  +/  podxl  + /  + / +  podxl  + l  1  podxl  1  0  4  0  0  3  0  2  2  3  0  2  0  0  3  1  1  0  0  4  0  4  1  3  0  0  3  0  5  1  3  0  0  3  0  6  1  3  0  1  3  0  7  0  2  0  0  2  0  8  0  2  0  3  1  0  TOTAL  6  21  0  6  19  0  1  Table 5-4: Actual Numbers of Transgene-Positive and Transgene-Negative Mice in Eight Litters Resulting from Breeding of Podxt'Transgene* ' Mice with Podxl* ' 1  1  Transgene' ' Mice Demonstrated that Transgene-Integration was not Detrimental. 1  197  5.8 Potential Reasons for Lack of Rescue The  l a c k o f s u r v i v i n g pups w a s c l e a r l y d i s a p p o i n t i n g , a n d it w a s a l s o s o m e w h a t  surprising. T h e r e were, h o w e v e r , several possible explanations f o r this o u t c o m e : a)  P o d o c a l y x i n m a y not have been expressed f r o m the transgene. A l t h o u g h u n l i k e l y , it was possible that the G F P e x p r e s s i o n observed i n k i d n e y s o f transgenic a n i m a l s d i d not correlate w i t h ectopic p o d o c a l y x i n e x p r e s s i o n .  b)  P o d o c a l y x i n m a y have been expressed at the i n c o r r e c t t i m e d u r i n g d e v e l o p m e n t , thereby preventing proper maturation o f podocytes.  c)  P o d o c a l y x i n m a y have been e x p r e s s e d at inadequate l e v e l s . T h i s w a s also n o t p a r t i c u l a r l y l i k e l y because podxl ~ m i c e have w e l l - d e v e l o p e d , f u n c t i o n a l k i d n e y s +l  ( D o y o n n a s et a l . , 2 0 0 1 ) . d)  E v e n i f p o d o c a l y x i n w a s e x p r e s s e d i n the c o r r e c t c e l l s , it m a y h a v e  been  mislocalized. e)  T h e r e m a y be other lethal p o d o c a l y x i n - r e l a t e d defects.  5.9 Transgenic Podocalyxin Expression Analysis In order to address the p o s s i b i l i t i e s o u t l i n e d a b o v e , the e x p r e s s i o n o f p o d o c a l y x i n w a s first assessed. T h i s w a s a c c o m p l i s h e d by i s o l a t i n g k i d n e y s f r o m m i c e j u s t prior to b i r t h . A t this t i m e p o i n t , a l l genotypes were present i n expected ratios. F u r t h e r m o r e , e c t o p i c p o d o c a l y x i n expression c o u l d be a n a l y z e d b y c o m p a r i n g podxl' 'transgene* 1  podxl' 'transgene 1  kidneys with  k i d n e y s . R T - P C R w a s used to demonstrate the presence o f p o d o c a l y x i n  m R N A expressed f r o m the transgene ( F i g u r e 5 - 9 ) . K i d n e y s isolated f r o m w i l d t y p e a n d  198  p o d o c a l y x i n - n u l l m i c e l a c k i n g the transgene were used as positive and negative c o n t r o l s , r e s p e c t i v e l y . B o t h p o d o c a l y x i n - n u l l m i c e c o n t a i n i n g the transgene w e r e e x p r e s s i n g p o d o c a l y x i n m R N A , as s h o w n i n red i n F i g u r e 5 - 9 . I m m u n o h i s t o c h e m i s t r y w a s used to c o n f i r m p r o t e i n e x p r e s s i o n , as s h o w n i n F i g u r e 5 - 1 0 . A l t h o u g h there w a s o n l y a t h i n b r o w n layer o f p o d o c a l y x i n s t a i n i n g i n the podxl'transgene*  g l o m e r u l i (left p a n e l ) i n  c o m p a r i s o n to the w i d e s p r e a d expression observed i n w i l d t y p e a n i m a l s ( m i d d l e panel), it is i m p o r t a n t to r e m e m b e r that p o d o c a l y x i n i s a l s o n o r m a l l y expressed o n a l l v a s c u l a r e n d o t h e l i a l c e l l s . T y p i c a l vascular staining is s h o w n i n the right p a n e l , where P E C A M - 1 s t a i n i n g has been used to i n d i c a t e v a s c u l a t u r e . In contrast to the p o d o c a l y x i n s t a i n i n g seen i n the transgenic k i d n e y , a p i c a l s t a i n i n g w a s most notably absent f r o m the outer layer o f c e l l s i n the c o m p a c t g l o m e r u l u s l a b e l l e d w i t h antibodies against P E C A M - 1 .  At  h i g h m a g n i f i c a t i o n , it w a s also m o r e e v i d e n t that p o d o c a l y x i n w a s not o n l y e x p r e s s e d , but w a s c o r r e c t l y l o c a l i z e d to the a p i c a l surface o f p o d o c y t e s i n t r a n s g e n i c a n i m a l s ( F i g u r e 5 - 1 1 ) . T h u s , a l a c k o f appropriate p o d o c a l y x i n expression w a s not the reason f o r the l a c k o f rescue observed i n breeding experiments.  199  Endogenous Transgenic  +  +  +  +  -  +  Podocalyxin  Figure 5-9: Podocalyxin mRNA was Expressed from the Transgene during Development in Kidneys of Transgenic Mice. After isolation of m R N A from kidneys of E l 8 mice, R T - P C R was performed using / o a f r Z - s p e c i f i c p r i m e r s . K i d n e y s f r o m E l 8 p o d o c a l y x i n - n u l l m i c e were used as negative c o n t r o l s , a n d k i d n e y s f r o m E l 8 w i l d t y p e ( B 6 ) m i c e w e r e used as p o s i t i v e c o n t r o l s f o r expression of podocalyxin m R N A .  200  Podocalyxin staining  Podo KO + transgene F i g u r e 5-10:  PECAM-1 staining  Podo WT  Immunohistochemistry was used to Detect Podocalyxin Expression i n  K i d n e y G l o m e r u l i of Transgenic M i c e d u r i n g Development. K i d n e y s were isolated f r o m E l 8 m i c e , f r o z e n , s e c t i o n e d , f i x e d , a n d i m m u n o l a b e l l e d f o r p o d o c a l y x i n o r P E C A M - 1 , as a v a s c u l a r e n d o t h e l i a l c e l l - s p e c i f i c m a r k e r . N o t e the w i l d t y p e s a m p l e d i s p l a y e d p o d o c a l y x i n s t a i n i n g o n b l o o d vessels a n d p o d o c y t e s , w h i l e g l o m e r u l i f r o m podx/  1  m i c e e x p r e s s i n g the transgene d i s p l a y e d o n l y a t h i n l a y e r o f  p o d o c a l y x i n s t a i n i n g , i n d i c a t i v e o f a p i c a l l a b e l l i n g o f p o d o c y t e s . K i d n e y sections f r o m E 1 8 p o d o c a l y x i n - n u l l m i c e were used as negative controls f o r p o d o c a l y x i n s t a i n i n g , a n d rat I g G , w a s used as a negative c o n t r o l f o r P E C A M - 1 s t a i n i n g . R e p r e s e n t a t i v e o f t w o independent experiments. S c a l e bar: 5 0 p,m.  201  a - Podocalyxin  Isotype control  Figure 5-11: Apical Staining of Podocytes was Evident in High Magnification Images of Glomeruli from Podxt'' Transgene-Positive Mice. K i d n e y s were i s o l a t e d f r o m E l 8 m i c e , f r o z e n , s e c t i o n e d , f i x e d , a n d i m m u n o l a b e l l e d . A t h i n , d a r k l a y e r o f p o d o c a l y x i n s t a i n i n g w a s most o b v i o u s a r o u n d the outer edge o f the g l o m e r u l u s , w h i l e staining w a s absent i n the isotype c o n t r o l s a m p l e . S c a l e bar: 10 p m .  202  5.10Morphological Analysis of Kidneys A l t h o u g h l i g h t m i c r o s c o p y w a s used to demonstrate that p o d o c a l y x i n w a s e x p r e s s e d appropriately i n g l o m e r u l a r p o d o c y t e s , these i m a g e s c o u l d not be used to assess c e l l u l a r m o r p h o l o g y . T o address this issue, k i d n e y s were again isolated f r o m a n i m a l s j u s t before birth and a n a l y z e d by T E M .  P o d o c a l y x i n - n u l l mice exhibit t w o major morphological abnormalities in podocytes (reviewed  i n s e c t i o n 1.6.4.1) ( D o y o n n a s  et a l . , 2 0 0 1 ) . T h e y c o m p l e t e l y  l a c k the  i n t e r d i g i t a t i n g f o o t processes that n o r m a l l y surround c a p i l l a r i e s i n the g l o m e r u l u s , a n d n e i g h b o u r i n g p o d o c y t e s m a i n t a i n tight j u n c t i o n s between c e l l b o d i e s . D u r i n g n o r m a l maturation o f p o d o c y t e s , tight j u n c t i o n s are lost, and replaced b y slit d i a p h r a g m s between i n t e r d i g i t a t i n g f o o t processes ( r e v i e w e d i n s e c t i o n 1.6.1). T h e s e c h a r a c t e r i s t i c s w e r e therefore assessed b y electron m i c r o s c o p y i n k i d n e y s o f podxl'transgene* ' 1  morphologies previously  observed for wildtype and podocalyxin-null  animals. T h e animals were  c o n f i r m e d ( F i g u r e 5 - 1 2 ) . S t r i k i n g l y , p o d o c a l y x i n k n o c k o u t a n i m a l s e x p r e s s i n g the k i d n e y - s p e c i f i c transgene had noticeably i m p r o v e d podocyte m o r p h o l o g i e s . T h e r e was an absence o f tight j u n c t i o n s between p o d o c y t e s , a n d i n t e r d i g i t a t i n g f o o t processes w e r e very evident ( F i g u r e 5 - 1 2 ) . H i g h e r m a g n i f i c a t i o n images c l e a r l y s h o w e d the intricate f o o t processes ( F i g u r e 5 - 1 3 ) . S l i t d i a p h r a g m s between f o o t processes were also detectable under c l o s e e x a m i n a t i o n . T h i s c o n f i r m e d that p o d o c a l y x i n i s a b s o l u t e l y r e q u i r e d f o r m o r p h o g e n e s i s o f p o d o c y t e s , a n d that e c t o p i c e x p r e s s i o n o f p o d o c a l y x i n i n these c e l l s was s u f f i c i e n t f o r r e p a i r i n g the defect.  203  Podo WT  RBC  Absence of FP's  Podo KO  Podo KO + Transgene  EC body  FP's  Figure 5-12: TEM's of Podocytes from E18 Wildtype, Podocalyxin-Null, and Podocalyxin-Null/Transgene-Positive Double Transgenic Mice. K i d n e y s were i s o l a t e d f r o m E l 8 m i c e , f i x e d , s e c t i o n e d , a n d p r o c e s s e d f o r T E M . E a c h i m a g e s h o w s a c r o s s - s e c t i o n o f a c a p i l l a r y , a n d most i n c l u d e c i r c u l a t i n g r e d b l o o d c e l l s ( R B C ) . P o d o c y t e s w e r e situated o u t s i d e the c a p i l l a r y , e x t e n d i n g i n t e r d i g i t a t i n g f o o t processes ( F P ' s ) , w h i c h were visible i n wildtype and p o d o c a l y x i n knockout samples. S c a l e bar: 2 u m . I m a g i n g by D e r r i c k H o m e , B i o l m a g i n g F a c i l i t y , U n i v e r s i t y o f B r i t i s h Columbia.  204  Podo W T  Podo KO  Podo KO + Transgene  Cell junction Figure 5-13: High Magnification TEM's of Podocytes from E18 Wildtype, Podocalyxin-Null, and Podocalyxin-Null/Transgene-Positive  Double Transgenic  Mice. N o t e the i n t r i c a t e f o o t p r o c e s s e s i n w i l d t y p e a n d p o d o c a l y x i n  knockout/transgene-  p o s i t i v e s a m p l e s a n d the presence o f c e l l j u n c t i o n s i n the p o d o c a l y x i n k n o c k o u t s a m p l e . S c a l e bar: 3 p m . I m a g i n g by D e r r i c k H o m e , B i o l m a g i n g F a c i l i t y , U n i v e r s i t y o f B r i t i s h Columbia.  205  5.11 Summary and  Conclusions  P o d o c a l y x i n - n u l l a n i m a l s e x h i b i t perinatal lethality. T h i s phenotype w a s p r e s u m e d to be due to a p o d o c y t e - r e l a t e d k i d n e y defect since drastic m o r p h o l o g i c a l changes are observed i n these c e l l s i n the absence o f p o d o c a l y x i n ( D o y o n n a s et a l . , 2 0 0 1 ) . In an effort to rescue p o d o c a l y x i n k n o c k o u t m i c e to facilitate analysis o f defects i n adult a n i m a l s , p o d o c a l y x i n w a s e c t o p i c a l l y e x p r e s s e d u s i n g the p o d o c y t e - s p e c i f i c NPHS1  promoter.  Expression  a n a l y s i s demonstrated appropriate expression o f the transgene i n several f o u n d e r a n i m a l s , w i t h substantial expression i n one founder.  E c t o p i c k i d n e y - s p e c i f i c e x p r e s s i o n o f p o d o c a l y x i n d i d not prevent the perinatal lethality o b s e r v e d i n k n o c k o u t a n i m a l s , b u t it d i d r e p a i r the m o r p h o l o g i c a l d e f e c t s i n the p o d o c y t e s . It s h o u l d be n o t e d , h o w e v e r , that a l t h o u g h there w a s a d r a m a t i c d i f f e r e n c e i n p o d o c y t e m o r p h o l o g y b e t w e e n p o d o c a l y x i n k n o c k o u t a n i m a l s w i t h a n d w i t h o u t the t r a n s g e n e , it i s p o s s i b l e that the k i d n e y d e f e c t w a s n o t a d e q u a t e l y r e p a i r e d d u e to s o m e w h a t aberrant e x p r e s s i o n . C a r e f u l e x a m i n a t i o n suggests that f o o t processes m a y have been s l i g h t l y flatter and slit pores m a r g i n a l l y n a r r o w e r i n rescued m i c e c o m p a r e d to w i l d t y p e c o n t r o l s . It m a y be that e c t o p i c e x p r e s s i o n l e v e l s were s l i g h t l y l o w e r , or that e x p r e s s i o n w a s initiated a little later i n g l o m e r u l a r d e v e l o p m e n t than w o u l d be expected f r o m the e n d o g e n o u s podxl promoter. H o w e v e r , n e p h r i n a n d p o d o c a l y x i n are expressed at a l m o s t i d e n t i c a l t i m e s d u r i n g d e v e l o p m e n t (Putaala et a l . , 2 0 0 0 ; P u t a a l a et a l . , 2 0 0 1 ; S c h n a b e l et a l . , 1989). F u r t h e r m o r e , the s t r i k i n g m o r p h o l o g i c a l d i f f e r e n c e b e t w e e n p o d o c a l y x i n k n o c k o u t m i c e e x p r e s s i n g the transgene and those l a c k i n g it, i n c o m b i n a t i o n w i t h the c o n s i s t e n t l e t h a l i t y w i t h i n 2 4 hours o f b i r t h , suggests that p o d o c a l y x i n - n u l l  206  a n i m a l s m a y actually die as a result o f other p o d o c a l y x i n - r e l a t e d defects. T h i s hypothesis is supported by the fact that some transgenic m i c e l a c k k i d n e y s entirely but die w i t h i n 4 8 hours o f b i r t h , rather than 2 4 hours ( G u o et a l . , 2 0 0 2 ) . T h u s , a l t h o u g h k i d n e y f a i l u r e w o u l d e v e n t u a l l y l e a d to d e a t h , p o d o c a l y x i n - n u l l a n i m a l s m a y a c t u a l l y d i e e a r l i e r because o f an a d d i t i o n a l defect. T h i s is a very e x c i t i n g p o s s i b i l i t y and w i l l require further investigation.  207  CHAPTER 6 : CONCLUDING REMARKS  6.1  Summary  and  Discussion  P o d o c a l y x i n is a s i a l o m u c i n c l o s e l y related to C D 3 4 a n d e n d o g l y c a n ( N i e l s e n et a l . , 2 0 0 2 ) . C D 3 4 f a m i l y m e m b e r s have been proposed to f u n c t i o n i n e n h a n c i n g p r o l i f e r a t i o n , b l o c k i n g d i f f e r e n t i a t i o n , e s t a b l i s h i n g pol ari ty, and regulating c e l l a d h e s i o n p o s i t i v e l y a n d negatively ( B a u m h u e t e r et a l . , 1 9 9 3 ; C h e n g et a l . , 1 9 9 6 ; F a c k l e r et a l . , 1 9 9 5 ; F i e g e r et a l . , 2 0 0 3 ; M e d e r et a l . , 2 0 0 5 ; Sassetti et a l . , 1 9 9 8 ; T a k e d a et a l . , 2 0 0 0 ) . A l l three f a m i l y m e m b e r s have C - t e r m i n a l P D Z b i n d i n g m o t i f s that, i n the case o f p o d o c a l y x i n a n d e n d o g l y c a n , facilitate interaction w i t h N H E R F adapter proteins ( H e et a l . , 1 9 9 2 ; K e r s h a w et a l . , 1 9 9 7 a ; L i et a l . , 2 0 0 2 ; M c N a g n y et a l . , 1 9 9 7 ; N i e l s e n et a l . , 2 0 0 2 ; Sassetti et a l . , 2 0 0 0 ; S i m m o n s et a l . , 1 9 9 2 ; T a k e d a et a l . , 2 0 0 1 ; T a n et a l . , 2 0 0 6 ) . T h i s a s s o c i a t i o n is k n o w n to c o n n e c t p o d o c a l y x i n w i t h the actin c y t o s k e l e t o n v i a e z r i n , a l i n k a g e that is disrupted i n several disease m o d e l s ( O r l a n d o et a l . , 2 0 0 1 ; T a k e d a et a l . , 2 0 0 1 ) .  P o d o c a l y x i n is w i d e l y d i s t r i b u t e d o n v a s c u l a r e n d o t h e l i a l c e l l s ( H o r v a t et a l . , 1 9 8 6 ; K e r s h a w et a l . , 1997a). It is also expressed b y h e m a t o p o i e t i c progenitors, platelets a n d m e g a k a r y o c y t e s , a subset o f n e u r o n a l c e l l s , a n d m e s o t h e l i a l c e l l s l i n i n g m a n y organs ( D o y o n n a s et a l . , 2 0 0 1 ; D o y o n n a s et a l . , 2 0 0 5 ; M c N a g n y et a l . , 1 9 9 7 ; M i e t t i n e n et a l . , 1 9 9 9 ; V i t u r e i r a et a l . , 2 0 0 5 ) . S t r i k i n g l y , it is m o s t h i g h l y e x p r e s s e d o n the elaborate g l o m e r u l a r p o d o c y t e s o f k i d n e y , where it is vital f o r proper f o r m a t i o n o f elaborate f o o t p r o c e s s e x t e n s i o n s ( D o y o n n a s et a l . , 2 0 0 1 ; K e r j a s c h k i et a l . , 1 9 8 4 ) .  Importantly,  208  abnormal p o d o c a l y x i n expression is associated w i t h a n u m b e r o f p a t h o l o g i c a l c o n d i t i o n s . A l t h o u g h it has not yet been l i n k e d to k i d n e y d y s f u n c t i o n i n h u m a n , m i c e l a c k i n g p o d o c a l y x i n have p o o r l y d e v e l o p e d p o d o c y t e s , e x h i b i t a n u r i a , and die w i t h i n 2 4 hours o f b i r t h ( D o y o n n a s et a l . , 2 0 0 1 ) . In h u m a n , i n c r e a s e d p o d o c a l y x i n e x p r e s s i o n is an i n d e p e n d e n t p r e d i c t o r o f p o o r o u t c o m e i n breast c a n c e r , w h i l e m u t a t i o n s i n the p o d o c a l y x i n gene, podxl, are f o u n d i n patients w i t h h i g h l y a g g r e s s i v e prostate c a n c e r (Casey et a l . , 2 0 0 6 ; S o m a s i r i et a l . , 2 0 0 4 ) . P o d o c a l y x i n is also l i n k e d w i t h several other types o f m a l i g n a n c i e s .  6.1.1  Podocalyxin's Role in Cell Morphogenesis  In order to gain insights into p o d o c a l y x i n ' s role i n disease and in n o r m a l d e v e l o p m e n t , it was overexpressed i n M C F - 7 breast c a r c i n o m a c e l l s (chapter 3). T h i s b l o c k e d f o r m a t i o n of confluent  m o n o l a y e r s , and cells instead e x h i b i t e d  decreased  cell-substratum  interactions. E c t o p i c expression also induced a dramatic increase in  microvillus  f o r m a t i o n . P o d o c a l y x i n e x p r e s s i o n o n c e l l s w i t h c o m p l e x m e m b r a n e e x t e n s i o n s is a c o m m o n t h e m e u n d e r p h y s i o l o g i c a l c o n d i t i o n s . M o s t n o t i c e a b l y , it is e s s e n t i a l f o r podocyte foot process f o r m a t i o n ( D o y o n n a s et a l . , 2 0 0 1 ; S c h n a b e l et a l . , 1989). H o w e v e r , it is a l s o e x p r e s s e d on a subset o f n e u r o n s , and f o r m a t i o n o f c e l l u l a r e x t e n s i o n s by p o d o c y t e s and neurons d i s p l a y m a n y s i m i l a r i t i e s ( r e v i e w e d i n ( K o b a y a s h i et a l . , 2 0 0 4 ) ) . A l t h o u g h these t w o c e l l types are f u n c t i o n a l l y v e r y d i f f e r e n t , the a c t i v i t i e s o f both are c r i t i c a l l y dependent on intricate architectures. T h e s e h i g h l y b r a n c h e d c e l l s require an extensive n e t w o r k o f c y t o s k e l e t a l constituents. M i c r o t u b u l e s and intermediate f i l a m e n t s support m a j o r processes o f p o d o c y t e s and n e u r o n a l a x o n s and d e n d r i t e s , w h i l e a c t i n  209  f i l a m e n t s f o r m the b a c k b o n e o f f o o t processes and d e n d r i t i c spines. F u r t h e r m o r e , m a n y o f the s a m e c y t o s k e l e t a l p r o t e i n s are f o u n d i n p o d o c y t e s a n d n e u r o n s , i m p l y i n g that s i m i l a r m e c h a n i s m s underlie the f o r m a t i o n o f c e l l u l a r extensions i n both c e l l types.  M o r e o v e r , p o d o c a l y x i n is also expressed by megakaryocytes, w h i c h extend  long  p r o c e s s e s w h e n d i f f e r e n t i a t i n g i n t o platelets ( M i e t t i n e n et a l . , 1 9 9 9 ; V i t u r e i r a et a l . , 2 0 0 5 ) . O v e r a p e r i o d o f 4 - 1 0 h o u r s , the entire m e g a k a r y o c y t i c c y t o p l a s m is c o n v e r t e d i n t o m i c r o t u b u l e - b a s e d p r o p l a t e l e t e x t e n s i o n s ( r e v i e w e d i n ( P a t e l et a l . , , 2 0 0 5 ) ) . In a d d i t i o n to f o r m a t i o n o f these l o n g extensions, the ends o f proplatelets b e n d and bifurcate i n a n a c t i n - d e p e n d e n t p r o c e s s t e r m e d e n d a m p l i f i c a t i o n ; this f a c i l i t a t e s generation o f t h o u s a n d s o f platelets f r o m each m e g a k a r y o c y t e . T h e e x a c t m e c h a n i s m s i n v o l v e d i n f o r m a t i o n o f p o d o c y t e f o o t processes a n d d e n d r i t i c spines, as w e l l as the b i f u r c a t i o n o f proplatelets are u n k n o w n , but they are a l l actin-based processes and they all o c c u r i n cells e x p r e s s i n g the a c t i n - a s s o c i a t e d p r o t e i n , p o d o c a l y x i n . T h u s , I b e l i e v e that a g l o b a l f u n c t i o n o f p o d o c a l y x i n is to f a c i l i t a t e the g e n e r a t i o n o f m o r p h o l o g i c a l l y  distinct  structures.  In support o f this h y p o t h e s i s , C h e n g et al recently p u b l i s h e d that p o d o c a l y x i n is required f o r t u b u l o g e n e s i s in vitro ( C h e n g et a l . , 2 0 0 5 ) . M D C K c e l l s are d e r i v e d f r o m k i d n e y t u b u l a r c e l l s a n d c a n be i n d u c e d to f o r m t u b u l e s in vitro.  Depletion of endogenous  p o d o c a l y x i n b y s i R N A b l o c k s tubulogenesis. A l t h o u g h this system is s o m e w h a t a r t i f i c i a l since k i d n e y tubules d o not n o r m a l l y express p o d o c a l y x i n , the results m a y be relevant to other tissues where p o d o c a l y x i n is t y p i c a l l y expressed ( D e k a n et a l . , 1 9 9 0 ; T a k e d a et a l . ,  210  2 0 0 1 ) . F o r e x a m p l e , p o d o c a l y x i n in m a m m a r y epithelial c e l l s ( S o m a s i r i et a l . , 2 0 0 4 ) and v a s c u l a r e n d o t h e l i a ( H o r v a t et a l . , 1986) m a y f a c i l i t a t e r e m o d e l l i n g and a n g i o g e n e s i s , respectively.  P o d o c a l y x i n is c o m p r i s e d o f an e x t e n s i v e m u c i n - l i k e e x t r a c e l l u l a r r e g i o n as w e l l as a w e l l - c o n s e r v e d c y t o p l a s m i c t a i l , w h i c h interacts w i t h the a c t i n c y t o s k e l e t o n  through  N H E R F proteins and e z r i n . V a r i o u s p o d o c a l y x i n mutants were generated and expressed in vitro i n order to study p o d o c a l y x i n ' s m e c h a n i s m o f a c t i o n . S u r p r i s i n g l y ,  although  i n t e r a ct io n w i t h the a c t i n c y t o s k e l e t o n is l i k e l y i n v o l v e d i n f o r m a t i o n o f p o d o c a l y x i n i n d u c e d c e l l u l a r extensions, deletion o f the majority o f p o d o c a l y x i n ' s c y t o p l a s m i c tail d i d not a f f e c t m i c r o v i l l u s f o r m a t i o n . In contrast, a n d m o r e e x p e c t e d l y , t u b u l o g e n e s i s i n M D C K c e l l s does require p o d o c a l y x i n ' s i n t r a c e l l u l a r d o m a i n ( C h e n g et a l . , 2 0 0 5 ) . T h e apparently c o n t r a d i c t o r y e v i d e n c e suggests that alternative m e c h a n i s m s m a y be u t i l i z e d under certain c o n d i t i o n s , or that the t w o types o f m o r p h o g e n e t i c alterations o c c u r v i a s o m e w h a t different pathways.  T h e requirement f o r the m u c i n - l i k e d o m a i n , h o w e v e r , is supported by a l l a v a i l a b l e data. P o d o c a l y x i n - d e p e n d e n t m i c r o v i l l u s f o r m a t i o n w a s c o m p l e t e l y ablated u p o n d e l e t i o n o f the extracellular d o m a i n , f o o t process architecture is d r a s t i c a l l y affected by neutralization o f p o d o c a l y x i n ' s negative c h a r g e , and t u b u l o g e n e s i s is m u c h less e x t e n s i v e w h e n the m u c i n d o m a i n is r e m o v e d , w h e t h e r g e n e t i c a l l y or by treatment w i t h i n h i b i t o r s o f O l i n k e d g l y c o s y l a t i o n (see chapter 3) ( A n d r e w s , 1 9 7 9 ; C h e n g et a l . , 2 0 0 5 ; S e i l e r et a l . , 1977). P o d o c a l y x i n m a y stimulate the f o r m a t i o n o f c e l l u l a r extensions in an attempt to  211  separate its bulky, negatively charged mucin domains from each other. Once generated, however, the stability of such protrusions would likely be dependent on reorganization of the actin cytoskeleton.  v Although it is clear that podocalyxin can induce actin reorganization (Figure 3-23 and (Schmieder et al., 2004)), it is surprising that direct interaction with N H E R F 1 or ezrin was not required in M C F - 7 cells. The most plausible explanation is that podocalyxin can associate with another integral membrane protein (via its extracellular domain) that itself interacts  with the actin cytoskeleton  or transduces  a signal to instigate actin  reorganization (Figure 3-25). Although this has not been proven, there is also no evidence to definitively refute it. For example, in rats treated with protamine sulfate or sialidase to neutralize podocalyxin's negative charge, there is a decrease in the amount of ezrin and N H E R F 2 that co-immunoprecipitate with podocalyxin in glomerular extracts (Takeda et al., 2001). This has been explained by postulating that reduction of podocalyxin's extracellular negative charge leads to an intracellular conformational change, which disrupts the interaction between N H E R F 2 and podocalyxin. However, it is also possible that an additional integral membrane protein that interacts with  podocalyxin's  extracellular domain is normally found in this protein complex and that protamine sulfate and sialidase treatments remove an extracellular protein recognition site, thereby preventing the interaction of podocalyxin with the rest of the complex. Thus, the idea of an extracellular binding partner for podocalyxin is an intriguing possibility that I would like to investigate.  212  6.1.2  Podocalyxin's Role in Cell Adhesion  A l o n g w i t h its role i n c e l l m o r p h o l o g y , there is c l e a r l y m o u n t i n g e v i d e n c e to i m p l i c a t e p o d o c a l y x i n , a l o n g w i t h C D 3 4 , in d e c r e a s i n g c e l l u l a r a d h e s i o n . Interestingly, it appears that C D 3 4 and p o d o c a l y x i n m a y e m p l o y several m e c h a n i s m s to act as g l o b a l a n t i adhesins. T h e y have been s h o w n to decrease c e l l - s u b s t r a t u m a d h e s i o n , as w e l l as c e l l - c e l l a d h e s i o n , both by interfering w i t h c e l l - c e l l j u n c t i o n s and by b l o c k i n g aggregation o f c e l l s i n s u s p e n s i o n (chapter 3) ( D o y o n n a s et a l . , 2 0 0 1 ; D r e w et a l . , 2 0 0 5 ; E c o n o m o u et a l . , 2 0 0 4 ; S o m a s i r i et a l . , 2 0 0 4 ; T a k e d a et a l . , 2 0 0 0 ) . D e c r e a s e d c e l l - c e l l a d h e s i o n between m u l t i p l e p o d o c a l y x i n or C D 3 4 e x p r e s s i n g c e l l s i n s u s p e n s i o n c a n e a s i l y be e x p l a i n e d by steric h i n d r a n c e or charge r e p u l s i o n between s i a l o m u c i n s o n nearby c e l l s . E x p r e s s i o n o f p o d o c a l y x i n at the tips o f m i c r o v i l l i , also generated in a p o d o c a l y x i n - d e p e n d e n t m a n n e r , w o u l d further increase the a n t i - a d h e s i o n p h e n o t y p e , as m i c r o v i l l i are the f i r s t point o f contact between c e l l s .  S i m i l a r l y , c e l l - c e l l j u n c t i o n s m a y s i m p l y be affected by r e l o c a l i z a t i o n o f p o d o c a l y x i n or C D 3 4 t o w a r d the j u n c t i o n s , thereby f o r c i n g n e i g h b o u r i n g c e l l s further and further apart. In support o f this m o d e l , loss o f c e l l - c e l l j u n c t i o n s between adjacent k i d n e y p o d o c y t e s during development coincides precisely with p o d o c a l y x i n expression and migration t o w a r d the basal c e l l surface ( S c h n a b e l et a l . , 1989). A g a i n , m i c r o v i l l u s f o r m a t i o n near the a p i c a l s u r f a c e , b e t w e e n these c e l l s w o u l d f u r t h e r serve to i n c r e a s e the s p a c i n g between c e l l s .  213  D e c r e a s e d c e l l - s u b s t r a t u m interactions are m o r e d i f f i c u l t to e x p l a i n because C D 3 4 and p o d o c a l y x i n are g e n e r a l l y a p i c a l m e m b r a n e p r o t e i n s . In the u n u s u a l H G E C  culture  s y s t e m , w h e r e s o m e p o d o c a l y x i n is a b e r r a n t l y l o c a l i z e d to the b a s o l a t e r a l s u r f a c e , decreased c e l l - s u b s t r a t u m i n t e r a c t i o n s c a n be e x p l a i n e d by steric h i n d r a n c e or charge r e p u l s i o n , where the n e g a t i v e l y c h a r g e d m u c i n - l i k e d o m a i n o f p o d o c a l y x i n is expressed i n the v i c i n i t y  o f the s i m i l a r l y c h a r g e d l a m i n i n o r c o l l a g e n e x t r a c e l l u l a r  matrix  c o m p o n e n t s . In most c e l l s , t h o u g h , this is not the case. H o w , then, does an a p i c a l protein affect adhesion at the basal.surface? A n i n t r i g u i n g p o s s i b i l i t y is that p o d o c a l y x i n - i n d u c e d actin recruitment f o r generation o f a p i c a l c e l l surface structures m a y deplete actin f r o m the b a s a l s u r f a c e , t h e r e b y d e c r e a s i n g the s t a b i l i t y o f i n t e g r i n - m e d i a t e d  adhesion  c o m p l e x e s at the basal surface ( F i g u r e 6 - 1 ) . In support o f this i d e a , cells transfected w i t h p o d o c a l y x i n mutants that i n d u c e d m i c r o v i l l u s f o r m a t i o n also d i s p l a y e d decreased c e l l a d h e s i o n , whereas the e x t r a c e l l u l a r deletion mutant d i d not i n d u c e m i c r o v i l l u s f o r m a t i o n or decrease c e l l a d h e s i o n . S i m i l a r l y , p o d o c a l y x i n m a y w e a k e n c e l l - c e l l j u n c t i o n s  by  titrating actin a w a y f r o m these j u n c t i o n a l proteins as w e l l . M o r e o v e r , since podocyte f o o t processes c o n t a i n an e x t e n s i v e n e t w o r k o f a c t i n f i l a m e n t s , p o d o c a l y x i n m a y a f f e c t m u l t i p l e aspects o f p o d o c y t e architecture by i n d u c i n g actin r e o r g a n i z a t i o n . T h u s , loss o f p o d o c a l y x i n m a y i n d i r e c t l y a f f e c t c y t o s k e l e t a l i n t e r a c t i o n s w i t h proteins o f the slit d i a p h r a g m c o m p l e x i n a d d i t i o n to those on the basal c e l l surface. T h i s w i l l require further i n v e s t i g a t i o n o f the i n t e r p l a y b e t w e e n a c t i n r e c r u i t m e n t by a p i c a l a n d b a s o l a t e r a l proteins.  214  Stable cell-cell and cell-substrate interactions | + Podocalyxin Apical actin recruitment and microvillus formation  Weakened cell-cell and cell-substrate interactions due to junctional actin loss  Decreased cell adhesion  *  t T :  f-actin  cell junctions  I I  III  integrins  microvilli  Figure 6-1: Model Demonstrating a Possible Mechanism for Decreased CellSubstrate Adhesion Induced by Apical Podocalyxin Expression. A p i c a l recruitment o f f - a c t i n b y p o d o c a l y x i n m a y titrate a c t i n a w a y f r o m basal integrins and c e l l - c e l l j u n c t i o n a l proteins, thereby w e a k e n i n g these interactions.  215  6.1.3  Podocalyxin-Dependent NHERF Localization  P o d o c a l y x i n ' s role in a p i c a l recruitment o f N H E R F 1 is another s i g n i f i c a n t result o f this w o r k . P o d o c a l y x i n is k n o w n to interact w i t h the N H E R F f a m i l y o f adapter proteins through its C - t e r m i n a l P D Z r e c o g n i t i o n sequence ( L i et a l . , 2 0 0 2 ; T a k e d a et a l . , 2 0 0 1 ; T a n et a l . , 2 0 0 6 ) . S i n c e N H E R F f a m i l y m e m b e r s can l i n k proteins indirectly to the actin cytoskeleton through E R M domain-dependent interactions with cytoskeletal linker m o l e c u l e s , it had been p r o p o s e d that N H E R F  p r o t e i n s are r e s p o n s i b l e f o r a p i c a l  l o c a l i z a t i o n o f p o d o c a l y x i n . In support o f t h i s , p o d o c a l y x i n mutants l a c k i n g the P D Z b i n d i n g site are subtly m i s l o c a l i z e d ( C h e n g et a l . , 2 0 0 5 ; L i et a l . , 2 0 0 2 ; M e d e r et a l . , 2 0 0 5 ; S c h m i e d e r et a l . , 2 0 0 4 ) . H o w e v e r , m o r e i n t r i g u i n g l y , I have n o w s h o w n that p o d o c a l y x i n is a c t u a l l y r e s p o n s i b l e f o r l o c a l i z a t i o n o f N H E R F 1 i n M C F - 7 c e l l s (see chapter 3). W h i l e o n l y about 2 0 % o f p o d o c a l y x i n is m i s l o c a l i z e d i n the absence o f interaction w i t h N H E R F , v i r t u a l l y a l l N H E R F 1 is dispersed throughout the c y t o p l a s m , rather than at the a p i c a l s u r f a c e , i n c e l l s e c t o p i c a l l y e x p r e s s i n g p o d o c a l y x i n mutants i n c a p a b l e o f i n t e r a c t i n g w i t h N H E R F proteins. T h i s , i n c o m b i n a t i o n w i t h the a p i c a l l o c a l i z a t i o n o f both p o d o c a l y x i n and N H E R F e a r l y i n c e l l p o l a r i z a t i o n , suggests that p o d o c a l y x i n , not N H E R F 1 , is the m a j o r p l a y e r i n the p r o c e s s o f a p i c a l d o m a i n establishment and targeting.  A p i c a l recruitment o f N H E R F proteins by p o d o c a l y x i n also has i m p o r t a n t i m p l i c a t i o n s f o r the n u m e r o u s a c t i v i t i e s o f these s c a f f o l d i n g p r o t e i n s . N H E R F 1 and N H E R F 2 are c a p a b l e o f i n t e r a c t i n g w i t h a w i d e v a r i e t y o f m o l e c u l e s , i n c l u d i n g i o n transporters, s i g n a l l i n g p r o t e i n s , and receptors, s u c h as e p i d e r m a l g r o w t h f a c t o r receptors and |3 2  216  adrenergic receptors ((Lazar et al., 2004), and reviewed in (Shenolikar et al., 2004)). Thus, podocalyxin-dependent localization may play an essential role in regulation of many cellular processes.  6.1.4 Ectopic Podocyte-Specific Podocalyxin Expression Repairs Foot Process Architecture but Fails to Rescue PodocalyxinNull Mice Although podocalyxin overexpression has been assessed in vitro (chapter 3), it has yet to be investigated in vivo. Another major goal of my thesis was therefore to attempt to generate transgenic mice overexpressing podocalyxin in an inducible manner (chapter 4). I cloned podxl into a transgenic vector for use in the Cre-loxP inducible system, and produced E S C s that specifically expressed podocalyxin and G F P in a Cre recombinasedependent manner. Unfortunately, although chimeric mice were generated, the transgene was never transmitted in the germline. Karyotyping of two E S C clones revealed the existence of a chromosomal aberration, likely also present in the parental R l E S C s , which precluded generation of viable, fully transgenic animals.  One of the main reasons for generating conditional podocalyxin overexpressing transgenic mice (chapter 4) was to generate animals specifically expressing podocalyxin in podocytes. These mice were then to be crossed with podxt ' mice, with the intention of 1  generating podxt' mice expressing transgenic podocalyxin specifically in podocytes. Since podxt'  animals die within 24 hours of birth, apparently as a result of a kidney  217  d e f e c t , e x p r e s s i o n i n p o d o c y t e s w a s e x p e c t e d to rescue these m i c e , thereby e n a b l i n g analysis o f adult m i c e l a c k i n g p o d o c a l y x i n i n other tissues.  D u e to delays i n p r o d u c t i o n o f c o n d i t i o n a l p o d o c a l y x i n e x p r e s s i n g m i c e (chapter 4 ) , a n e w strategy w a s i n i t i a t e d i n o r d e r to express p o d o c a l y x i n s p e c i f i c a l l y i n p o d o c y t e s . Podxl NPHSJ  w a s inserted i n t o an e x p r e s s i o n v e c t o r d o w n s t r e a m o f the p o d o c y t e - s p e c i f i c promoter, and transgenic m i c e were generated u s i n g this construct (chapter 5 ) .  T h e transgene w a s expressed s p e c i f i c a l l y i n p o d o c y t e s , so podxt' transgene '~ +  c r o s s e d w i t h podxt'transgene' ' 1  +  m i c e were  m i c e , a n d these w e r e i n t e r b r e d to generate  mice  e x p r e s s i n g p o d o c a l y x i n o n l y i n k i d n e y . S t r i k i n g l y , p o d o c y t e s i n these m i c e d i s p l a y e d r e l a t i v e l y n o r m a l f o o t p r o c e s s e s a n d l a c k e d aberrant c e l l - c e l l j u n c t i o n s , w h i c h are inappropriately  retained i n p o d o c a l y x i n - d e f i c i e n t  mice. This provided  conclusive  e v i d e n c e that p o d o c a l y x i n is both necessary and s u f f i c i e n t f o r p o d o c y t e m o r p h o g e n e s i s , again demonstrating p o d o c a l y x i n ' s role i n generation o f c e l l surface projections.  H o w e v e r , although p o d o c y t e architecture was repaired i n these transgenic m i c e , they still s u f f e r e d perinatal l e t h a l i t y , s u g g e s t i n g that p o d o c a l y x i n m a y p l a y a d d i t i o n a l essential roles i n other tissues. L o s s o f p o d o c a l y x i n m a y cause v a s c u l a r defects, such as decreased permeability f o r transendothelial migration, increased global stickiness of vascular e n d o t h e l i a l c e l l s to h e m a t o p o i e t i c c e l l s , o r c o l l a p s e o f b l o o d vessels d u e to interactions between opposing membranes in their lumens. Platelets l a c k i n g p o d o c a l y x i n m a y generate clots i n a p p r o p r i a t e l y , or they m a y be decreased i n n u m b e r due to m e g a k a r y o c y t e a b n o r m a l i t i e s . L o s s o f p o d o c a l y x i n f r o m t h e l i n i n g s o f b o d y c a v i t i e s m a y l e a d to  218  i n c r e a s e d o r g a n d a m a g e due to i n a p p r o p r i a t e adhesion o f organs to o n e another. T h e r e m a y even be neuronal defects i n p o d o c a l y x i n - n u l l a n i m a l s . T h u s , the absence o f rescued m i c e is a c t u a l l y a m o r e f a s c i n a t i n g o u t c o m e than that w h i c h w a s p r e d i c t e d . It w i l l be i n t e r e s t i n g to assess other d e v e l o p m e n t a l defects that m a y be c a u s e d b y l o s s o f podocalyxin.  6.2 Significance of Results E x p r e s s i o n o f p o d o c a l y x i n i n a variety o f tissues, i n c l u d i n g p o d o c y t e s , vasculature, s o m e h e m a t o p o i e t i c c e l l s , boundary elements separating organs, and a subset o f neuronal c e l l s , a l o n g w i t h altered e x p r e s s i o n i n n u m e r o u s m a l i g n a n t situations, m a k e s it a p o t e n t i a l l y important protein f o r further analysis. T h e absolute requirement f o r p o d o c a l y x i n e x p r e s s i o n i n order f o r m i c e to s u r v i v e b e y o n d the first d a y o f l i f e m a k e s it even m o r e intriguing.  6.2.1 Podocalyxin in Normal Development Adhesion  and morphogenesis  are b o t h e x t r e m e l y  important activities  throughout  d e v e l o p m e n t . A d h e s i o n , o r l a c k thereof, regulates p r o p e r m i g r a t i o n o f c e l l s , w h i l e m o r p h o g e n e s i s i s c r i t i c a l f o r the generation o f m a n y s p e c i a l i z e d c e l l types. P o d o c a l y x i n is c l e a r l y i m p o r t a n t i n f a c i l i t a t i n g f o r m a t i o n o f intricate p o d o c y t e f o o t processes, but it m a y a l s o p l a y a r o l e i n g e n e r a t i o n o f n e u r o n a l p r o c e s s e s a n d the m e g a k a r y o c y t i c extensions i n v o l v e d i n platelet p r o d u c t i o n . In a d d i t i o n , it m a y be i n v o l v e d i n g l o b a l c e l l m i g r a t i o n and m o v e m e n t o f hematopoietic cells across endothelial barriers  when  219  c o l o n i z i n g new n i c h e s . F u r t h e r m o r e , the c o a t i n g o f p o d o c a l y x i n o n m e s o t h e l i a l c e l l s l i n i n g b o d y c a v i t i e s l i k e l y protects organs f r o m d a m a g e . T h i s is e s p e c i a l l y e v i d e n t i n podocalyxin omphaloceles  knockout late  in  animals, which development.  are i n e f f i c i e n t at r e t r a c t i n g  Thus,  podocalyxin  expression  physiologic throughout  d e v e l o p m e n t and i n the adult m a y regulate a d h e s i o n and c e l l m o r p h o g e n e s i s i n a variety o f tissues.  6.2.2  Podocalyxin in Cancer Progression  P o d o c a l y x i n has also been associated w i t h a w i d e variety o f cancers. It is upregulated i n h e p a t o c e l l u l a r c a r c i n o m a and a subset o f t e s t i c u l a r c a n c e r s , and it is a l s o f o u n d i n l e u k e m i a s ( C h e n et a l . , 2 0 0 4 ; K e l l e y et a l . , 2 0 0 5 ; S c h o p p e r l e et a l . , 2 0 0 3 ) . Importantly, it is u p r e g u l a t e d or mutated i n h i g h l y a g g r e s s i v e breast c a n c e r s , W i l m s ' t u m o u r s , and prostate cancers ( C a s e y et a l . , 2 0 0 6 ; S o m a s i r i et a l . , 2 0 0 4 ; S t a n h o p e - B a k e r et a l . , 2 0 0 4 ) . A l t h o u g h the result o f p o d o c a l y x i n d y s r e g u l a t i o n i n these m a l i g n a n t situations is not yet c l e a r , its a s s o c i a t i o n w i t h the m o s t a g g r e s s i v e cases and its role i n r e g u l a t i n g c e l l adhesion suggest that it m a y facilitate metastasis. M o r e o v e r , s o l i d t u m o u r s , w h i c h require increased nutrients, and therefore increased b l o o d f l o w , m a y b e c o m e more aggressive as a result of p o d o c a l y x i n - i n d u c e d  a n g i o g e n e s i s . D e t e r m i n a t i o n o f the  underlying  m e c h a n i s m s b e h i n d i n c r e a s e d p o d o c a l y x i n e x p r e s s i o n and its effects i n these disease c o n d i t i o n s is therefore particularly important.  220  6.3 Future Directions A l t h o u g h I have g a i n e d some important insights into the f u n c t i o n s o f p o d o c a l y x i n , the m e c h a n i s m s b e h i n d its a c t i v i t i e s , its roles i n d e v e l o p m e n t , a n d the c o n s e q u e n c e s o f aberrant e x p r e s s i o n , this w o r k has generated m a n y m o r e questions. In m y o p i n i o n , the most interesting avenues  to p u r s u e  include understanding  where  p o d o c a l y x i n is  absolutely essential throughout d e v e l o p m e n t , h o w it affects cancer p r o g r e s s i o n , and h o w it can i n duc e m o r p h o l o g i c a l changes i n the absence o f the majority o f its c y t o p l a s m i c t a i l .  6.3.1 Understanding Podocalyxin Mechanistically F i n d i n g an in vitro m o d e l system f o r assessing p o d o c a l y x i n f u n c t i o n (chapter 3) w a s a m a j o r step f o r w a r d , a n d the i n d u c t i o n o f m i c r o v i l l u s f o r m a t i o n c a n n o w be u s e d to elucidate m e c h a n i s m s o f p o d o c a l y x i n - i n d u c e d m o r p h o l o g i c a l changes. D e l e t i o n o f a l l but six j u x t a m e m b r a n e a m i n o a c i d s o f p o d o c a l y x i n ' s i n t r a c e l l u l a r d o m a i n w a s e x p e c t e d to c o m p l e t e l y b l o c k interactions w i t h a l l k n o w n c y t o p l a s m i c interaction partners, i n c l u d i n g N H E R F proteins and e z r i n . W h i l e N H E R F certainly does not interact w i t h this d e l e t i o n m u t a n t ( F i g u r e 3 - 1 5 ) , absence o f p o d o c a l y x i n / e z r i n  i n t e r a c t i o n s m u s t be c o n f i r m e d .  A l t h o u g h t w o o f the three a m i n o a c i d s k n o w n to be i n v o l v e d i n d i r e c t l y b i n d i n g e z r i n were retained i n the p o d o c a l y x i n A t a i l mutant, I C A M - 3 , w h i c h also interacts directly w i t h e z r i n , f a i l s to b i n d w h e n o n l y the first eight a m i n o acids are retained i n the c y t o p l a s m i c tail ( S c h m i e d e r et a l . , 2 0 0 4 ; Serrador et a l . , 2 0 0 2 ) . T h u s , the R E H Q R S G S sequence o f I C A M - 3 is i n s u f f i c i e n t f o r i n t e r a c t i o n w i t h e z r i n , s u g g e s t i n g that the p o d o c a l y x i n t a i l mutant that i n c l u d e d o n l y C C H Q R F ( A t a i l ) w o u l d also be u n l i k e l y to b i n d . H o w e v e r , to  221  f o r m a l l y p r o v e t h i s , G S T - p u l l d o w n e x p e r i m e n t s w i l l be p e r f o r m e d u s i n g  GST-tagged  N ' t e r m i n a l ezrin and lysates f r o m c e l l s e x p r e s s i n g p o d o c a l y x i n mutants.  A s s u m i n g that the A t a i l mutant cannot interact w i t h e z r i n , it is p o s s i b l e that an a d d i t i o n a l protein interacts w i t h the extracellular or transmembrane regions o f p o d o c a l y x i n i n order to l i n k it to the actin c y t o s k e l e t o n , or at least to transduce the s i g n a l r e q u i r e d f o r a c t i n reorganization and m i c r o v i l l u s f o r m a t i o n . A l t h o u g h m u c h o f p o d o c a l y x i n ' s e x t r a c e l l u l a r d o m a i n is not c o n s e r v e d , the j u x t a m e m b r a n e stalk retains about 7 0 % s i m i l a r i t y across s p e c i e s , so this is a l i k e l y c a n d i d a t e r e g i o n f o r a p r o t e i n - p r o t e i n i n t e r a c t i o n d o m a i n . A l t h o u g h f i n d i n g an e x t r a c e l l u l a r b i n d i n g partner f o r p o d o c a l y x i n m a y be d i f f i c u l t , the question o f whether or not there is one is an important one to address.  Specific  deletions  of p o d o c a l y x i n ' s  t r a n s m e m b r a n e d o m a i n , the  juxtamembrane  e x t r a c e l l u l a r s t a l k , the c y s t e i n e - b o n d e d g l o b u l a r d o m a i n , a n d p o r t i o n s o f the m u c i n d o m a i n s h o u l d be generated in an effort to p i n p o i n t regions o f i m p o r t a n c e . In a d d i t i o n , reagents c o u l d be used to m o d i f y p o d o c a l y x i n ' s m u c i n d o m a i n in vitro. F o r e x a m p l e , s i a l i c a c i d residues c o u l d be r e m o v e d u s i n g n e u r a m i n i d a s e , or O - g l y c o s y l a t i o n c o u l d be b l o c k e d entirely. T h u s , m a n y e x p e r i m e n t s c o u l d be p e r f o r m e d i n order to p i n p o i n t the m e c h a n i s m by w h i c h p o d o c a l y x i n regulates c e l l m o r p h o l o g y .  6.3.2  Podocalyxin's Role in Development  T h e u n e x p e c t e d l a c k o f rescue o f p o d o c a l y x i n k n o c k o u t m i c e e x p r e s s i n g t r a n s g e n i c p o d o c a l y x i n s p e c i f i c a l l y i n k i d n e y was quite i n t r i g u i n g . If the k i d n e y defect w a s r e a l l y  222  the o n l y cause o f perinatal lethality i n p o d o c a l y x i n - d e f i c i e n t a n i m a l s , r e i n t r o d u c t i o n o f the protein i n t o p o d o c y t e s s h o u l d have repaired the defect and rescued the m i c e . T h u s , either p o d o c a l y x i n w a s e x p r e s s e d i n a d e q u a t e l y i n t r a n s g e n i c a n i m a l s , o r the k i d n e y defect is not the o n l y m a j o r a b n o r m a l i t y i n p o d o c a l y x i n - n u l l m i c e . T h e transgene d i d s e e m to be e x p r e s s e d s u f f i c i e n t l y to r e p a i r the d e f e c t , as d r a m a t i c m o r p h o l o g i c a l differences were noted between podxl' ' a n i m a l s w i t h and w i t h o u t the transgene, a l t h o u g h 1  it w a s p o s s i b l e that there w e r e some slight d i f f e r e n c e s between these m i c e and w i l d t y p e littermates (chapter 5). T h u s , a l t h o u g h p o d o c y t e - s p e c i f i c e x p r e s s i o n o f p o d o c a l y x i n d i d not enable a n a l y s i s o f defects i n adult a n i m a l s , w i t h the appropriate assays it m a y still be possible to f i n d other defects in p o d o c a l y x i n - d e f i c i e n t a n i m a l s shortly before birth.  6.3.3  P o d o c a l y x i n ' s R o l e in C a n c e r P r o g r e s s i o n  W h i l e p o d o c a l y x i n has been associated w i t h numerous types o f cancer, c o n f i r m a t i o n that it is a causal f a c t o r i n cancer p r o g r e s s i o n has yet to be obtained. T h i s w i l l be investigated u s i n g t w o approaches. F i r s t , M C F - 7 c e l l s e c t o p i c a l l y e x p r e s s i n g p o d o c a l y x i n (chapter 3) w i l l be i n j e c t e d into i m m u n o c o m p r o m i s e d m i c e . M C F - 7 c e l l s are k n o w n to generate t u m o u r s , and o v e r e x p r e s s i o n o f p o d o c a l y x i n m a y enhance metastasis o f these c e l l s . If d i f f e r e n c e s are n o t e d b e t w e e n c a n c e r p r o g r e s s i o n i n m i c e i n j e c t e d w i t h p o d o c a l y x i n transfected c e l l s and v e c t o r c o n t r o l c e l l s , then the e x p e r i m e n t w i l l be repeated u s i n g M C F - 7 c e l l s e c t o p i c a l l y e x p r e s s i n g p o d o c a l y x i n mutants (chapter 3). T h i s w i l l a l l o w us to g a i n insights into e x a c t l y h o w p o d o c a l y x i n affects cancer p r o g r e s s i o n , m e c h a n i s t i c a l l y . T h u s , the c r i t i c a l d o m a i n s r e s p o n s i b l e f o r c a n c e r p r o g r e s s i o n w i l l be i d e n t i f i e d , and p o t e n t i a l i n t e r a c t i n g proteins w i l l be e x a m i n e d . I m p o r t a n t l y , the p o d o c a l y x i n b i n d i n g  223  p r o t e i n , e z r i n is essential f o r numerous s i g n a l l i n g p a t h w a y s i m p l i c a t e d i n metastasis and p o o r o u t c o m e i n various cancers (Casey et a l . , 2 0 0 6 ) .  T h e s e c o n d strategy f o r assessing p o d o c a l y x i n ' s i n v o l v e m e n t i n c a n c e r aggressiveness i n v o l v e s o v e r e x p r e s s i o n o f p o d o c a l y x i n i n m a m m a r y tissue, and perhaps other sites, i n transgenic m i c e . T h e C r e - l o x P strategy d i s c u s s e d i n chapter 4 is b e i n g repeated u s i n g fresh R l E S C s . P o d o c a l y x i n expression w i l l therefore be i n d u c i b l e i n any tissue, or at any t i m e p o i n t , as l o n g as a C r e m o u s e is a v a i l a b l e . F o r e x a m p l e , c r o s s i n g p o d o c a l y x i n i n d u c i b l e transgenic m i c e to w h e y a c i d i c protein ( W A P ) - C r e m i c e ( W a g n e r et a l . , 1997) w i l l specifically induce podocalyxin  e x p r e s s i o n i n the m a m m a r y g l a n d . T u m o u r  f o r m a t i o n w i l l then be i n d u c e d i n these m i c e u s i n g the m o u s e m a m m a r y t u m o u r v i r u s ( M M T V ) , and c a n c e r p r o g r e s s i o n w i l l be c o m p a r e d b e t w e e n these m i c e and those i n w h i c h p o d o c a l y x i n e x p r e s s i o n was not i n d u c e d . T h u s , these t w o m o d e l s w i l l be used to address p o d o c a l y x i n ' s role in cancer progression. 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