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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 I N A D H E S I O N A N D C E L L M O R P H O G E N E S I S by J U L I E S U S A N N E N I E L S E N B . S c , The Univers i ty of V i c t o r i a , 2 0 0 0 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( M e d i c a l Genet ics) T H E 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 Augus t 2006 © Ju l ie Susanne N i e l s e n , 2 0 0 6 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 podocalyxin 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 i i p o d o c a l y x i n expression in selected tissues. For example , podoca l yx in overexpression in m a m m a r y t issue was intended to fac i l i tate evaluat ion of p o d o c a l y x i n ' s role in breast cancer progression. S i m i l a r l y , these mice cou ld be used to select ively rescue defects in p o d o c a l y x i n - d e f i c i e n t mice . Unfor tunate ly , chromosomal abnormal i t ies in the parental embryon ic stem cells temporar i ly prevented complet ion of this study. A s an alternative strategy, we attempted to selectively rescue the k idney defects observed in p o d o c a l y x i n - n u l l mice by creation of mice wi th a k i d n e y - s p e c i f i c podxl t ransgene. Surpr is ing ly , although transgenic podoca lyx in was appropriately expressed, and podocyte morpho logy appeared relat ively normal in contrast to p o d o c a l y x i n - n u l l m ice , transgenic m i c e s t i l l d ied pe r ina ta l l y . T h i s suggests the presence o f other se r ious , as yet undetermined, abnormal i t ies in podoca lyx in -def ic ient animals . Cont inued assessment o f these defects and podoca lyx in ' s role in cancer progression is underway. In summary , this thes is revea ls a new m e c h a n i s t i c ro le f o r p o d o c a l y x i n in the process o f c e l l morphogenes is and suggests that in addi t ion to its v i ta l role in k idney deve lopment , podoca lyx in may play an essential role in other aspects of m a m m a l i a n development. i i i TABLE OF CONTENTS ABSTRACT « TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xii ACKNOWLEDGEMENTS xvii CHAPTER 1 : INTRODUCTION 1 1.1 Podocalyxin is a Member of the CD34 Family of Sialomucins 1 1.1.1 Protein Structure 2 1.1.2 Genomic Organization and Alternative Splicing 5 1.1.3 Expression Pattern 9 1.2 Cloning of Podocalyxin 10 1.3 Transcriptional Regulation of Podocalyxin 13 1.3.1 Positive Regulation by WT 1 13 1.3.2 Negative Regulation by p53 15 1.3.3 Positive Regulation by Ets-1 16 1.4 Intracellular Binding Partners for Podocalyxin 16 1.4.1 Ezrin 17 1.4.2 NHERF Proteins 18 1.4.3 Other CD34-Family Binding Proteins 23 1.5 Proposed Functions of CD34 Family Members 24 1.5.1 Enhancing Proliferation and Blocking Differentiation 25 1.5.2 Pro-Adhesion 28 1.5.3 Anti-Adhesion 32 1.5.4 Establishing Polarity 39 1.5.5 Podocalyxin and NHERF: A Role in Asymmetric Cell Division? 41 1.6 Podocalyxin in Kidney Development : 45 1.6.1 Overview of Glomerular Development 46 1.6.2 Glomerular Diseases and Model Systems 50 1.6.3 Early Studies of Podocalyxin 51 1.6.4 Lessons from the Podocalyxin Knockout 56 1.6.4.1 Podocalyxin is Essential for Podocyte Morphogenesis 56 1.6.4.2 The Kidney Defect in Podocalyxin-Null Mice is the Apparent Cause of Perinatal Lethality in these Animals 61 1.6.5 Other Molecules Involved in Glomerular Development 63 1.6.5.1 Nephrin 64 1.6.5.2 WT1 65 1.7 Podocalyxin in the Hematopoietic System 67 1.7.1 Overview of Hematopoiesis 67 1.7.2 Podocalyxin's Role in the Hematopoietic System 68 1.7.3 Hematopoiesis in the Podocalyxin Knockout 71 1.7.4 Podocalyxin in the Vasculature 74 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 78 1.10.1 Podocalyxin in Breast Cancer 78 1.10.2 Podocalyxin in Prostate Cancer 81 1.10.3 Podocalyxin in Testicular Cancer 82 1.10.4 Podocalyxin in Leukemia 83 1.10.5 Podocalyxin in Hepatocellular Carcinoma 84 1.10.6 Podocalyxin in Wilms' Tumours 85 1.11 Thesis Objectives 86 CHAPTER 2 : MATERIALS AND METHODS 88 2.1 Cloning and Mutagenesis of Podocalyxin 88 2.1.1 Conditional Podocalyxin Overexpression Transgenic Construct 88 2.1.2 Podocyte-Specific Podocalyxin Transgenic Construct 88 2.1.3 Murine Podocalyxin Expression Vector 89 2.1.4 Chicken Podocalyxin Expression Vectors 89 2.1.5 Chicken Podocalyxin Mutants • 90 2.2 Cell Culture 93 2.2.1 Culture Conditions 93 2.2.2 Transfection Techniques 94 2.3 Expression Analysis 95 2.3.1 Antibodies 95 2.3.2 Flow Cytometry and Cell Sorting 99 2.3.3 Western blotting 100 2.3.4 RT-PCR •. 100 2.4 Additional Characterization of ESC Clones for Conditional Podocalyxin Overexpressing Transgenic 101 2.4.1 (3-galactosidase Assay 101 2.4.2 PCR to Detect Transgene : 101 2.5 Adhesion Assays 102 2.6 Confocal Microscopy 102 2.7 Scanning Electron Microscopy (SEM) 104 2.8 Transmission Electron Microscopy (TEM) 104 2.8.1 T E M of Cell Lines 104 2.8.2 T E M of Kidneys 105 2.9 Immunofluorescence of Tissue Sections 105 2.9.1 Sample Preparation 105 2.9.2 Tissue Staining 106 2.10 Immunohistochemistry 106 2.11 Mice 107 CHAPTER 3 : OVEREXPRESSION OF WILDTYPE AND MUTANT PODOCALYXIN IN EPITHELIAL CELLS 108 3.1 Rationale 108 v 3.2 Expression of Full-Length Podocalyxin in MCF-7 and M D C K Cells I l l 3.2.1 Expression Analysis I l l 3.2.2 Podocalyxin Decreases Cell Adhesion in Epithelial Cells 113 3.2.3 Podocalyxin Recruits NHERF1 to the Apical Surface of Cells 117 3.2.4 Morphological Changes: Podocalyxin Induces Microvillus Formation in Epithelial Cells... 119 3.3 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 NHERF Binding. 148 3.4 Discussion 152 3.4.1 Summary 152 3.4.2 Podocalyxin's Role in Determining Cell Morphology 152 3.4.3 Significance of Recruitment of NHERF by Podocalyxin 157 3.4.4 Podocalyxin and Microvilli in Adhesion / Anti-Adhesion 159 CHAPTER 4 : GENERATION OF TRANSGENIC MICE CONDITIONALLY OVEREXPRESSING PODOCALYXIN 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 184 5.5.1 Transgene Expression in Glomeruli of Founders' Kidneys 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 : CONCLUDING REMARKS 208 6.1 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 Significance of Results 219 6.2.1 Podocalyxin in Normal Development 219 6.2.2 Podocalyxin in Cancer Progression 220 6.3 Future Directions 221 6.3.1 Understanding Podocalyxin Mechanistically 221 6.3.2 Podocalyxin's Role in Development 222 6.3.3 Podocalyxin's Role in Cancer Progression 223 CHAPTER 7 : REFERENCES 225 vn LIST OF TABLES Tab le 1-1: Frequency of podxl+l+, podxP1', and podxl1' A n i m a l s dur ing Deve lopment . . . . 62 Tab le 2 - 1 : P r imary Ant ibod ies used in this Thesis 97 Tab le 2 - 2 : Secondary Ant ibod ies used in this Thesis 98 Tab le 5 - 1 : S u m m a r y of Transgene Express ion 192 Tab le 5 - 2 : Expected Numbers o f A l l Genotypes Resul t ing f r o m PodxT'Transgene*1' Crosses 194 Tab le 5 - 3 : A c t u a l Numbers of Transgene-Pos i t ive M i c e of Each Genotype in the First F i ve Litters Resul t ing f r o m PodxP''Transgene+l' Crosses Demonstrated a L a c k of Rescue of Podxl'1 M i c e 196 Tab le 5 - 4 : A c t u a l Numbers of Transgene-Posi t ive and Transgene-Negat ive M i c e in Eight Litters Resul t ing f r o m Breed ing of PodxT''Transgene+'~ M i c e wi th PodxP1' Transgene'' M i c e Demonstrated that Transgene-Integration was not Detr imental . 197 v i i i LIST OF FIGURES Figure 1-1: Schematic of 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 Organizat ion of C D 3 4 F a m i l y M e m b e r s 7 Figure 1-3: A l ternat ive 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 igure 1-4: Proposed Funct ions for C D 3 4 F a m i l y : Enhanc ing Prol i ferat ion and B l o c k i n g Dif ferent iat ion 26 Figure 1-5: Proposed Funct ion for C D 3 4 F a m i l y : L -se lect in Mediated Adhes ion 30 Figure 1-6: Proposed Funct ion for 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 33 Figure 1-7: T w o M o d e l s of Podoca lyx in -Dependent A s y m m e t r i c C e l l D i v i s i o n 4 4 Figure 1-8: Schematic of M o l e c u l e s Related to Podocyte Archi tecture 48 Figure 1-9: E lectron M i c r o g r a p h of a N o r m a l G lomeru la r Podocyte with Extended M a j o r Processes and Foot Processes Surrounding a B l o o d Vesse 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 59 Figure 1-11: Podoca lyx in is Expressed in Hematopoiet ic T issues throughout Deve lopment 69 Figure 1-12: Steady State Leve ls of A l l Hematopoiet ic L ineages in E15 Fetal L i v e r in Podoca l yx in ( 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: Surv iva l Rates for Patients Diagnosed wi th Breast Tumours S h o w n to be Express ing V a r y i n g Leve ls of P o d o c a l y x i n 80 Figure 3 - 1 : Endogenous H u m a n P o d o c a l y x i n Express ion in Breast C a r c i n o m a L i n e s . . 110 Figure 3 - 2 : Ectopic M u r i n e P o d o c a l y x i n Express ion in Transfected M C F - 7 Breast Ep i the l ia l Ce l l s 112 Figure 3 - 3 : Transfected M C F - 7 C e l l s Ec top ica l l y Express ing M u r i n e Podoca lyx in Exh ib i ted Decreased Ce l l -Subs t ra tum Interactions 114 Figure 3 -4 : Podoca lyx in Decreases C e l l Substrate A d h e s i o n 115 Figure 3 - 5 : Podocalyx in - Induced A l terat ion of C e l l - C e l l Junct ions 116 Figure 3 - 6 : Confoca l Images Demonstrated A p i c a l Recrui tment of N H E R F 1 by Podoca lyx in 118 Figure 3 - 7 : Podoca lyx in Induced M i c r o v i l l u s Format ion in M C F - 7 C e l l s 121 Figure 3 - 8 : Podoca lyx in A l s o Induced M i c r o v i l l u s Format ion in M D C K Epi the l ia l Ce l l s . 122 Figure 3 - 9 : T E M ' s of Ep i the l ia l C e l l s Transfected wi th E m p t y V e c t o r or Vec to r 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 123 Figure 3 - 1 0 : Schematic of P o d o c a l y x i n Mutants 125 Figure 3 - 1 1 : Podoca lyx in Express ion Af te r M u l t i p l e Rounds of C e l l Sort ing 128 Figure 3 - 1 2 : Western B lo ts Demonstrated Express ion of P o d o c a l y x i n Mutants of the Expected Mo lecu la r Weights in C l o n a l Populat ions 130 Figure 3 - 1 3 : Western B lots Demonstrated Express ion of P o d o c a l y x i n in C l o n a l Populat ions Used for Subsequent Exper iments 132 Figure 3 - 1 4 : Podoca lyx in was Expressed at Comparab le Leve ls at the C e l l Surface in C l o n a l Populations 133 Figure 3 - 1 5 : Confoca l A n a l y s i s of 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 of N H E R F 1 and Podoca lyx in in Ver t i ca l S l ices of C o n f o c a l Stacks 137 Figure 3 - 1 7 : Podoca lyx in Induced M i c r o v i l l u s Format ion in a N H E R F - I n d e p e n d e n t M a n n e r 139 Figure 3 - 1 8 : Delet ion of the Major i t y of Podoca lyx in ' s Ext racel lu lar 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 Figure 3 - 1 9 : M i c r o v i l l u s Counts for Transfected C e l l s 143 Figure 3 - 2 0 : Confoca l A n a l y s i s of Podoca lyx in and Ez r in at the A p i c a l Surface of Transfected M C F - 7 C e l l s 146 Figure 3 - 2 1 : Ver t i ca l Sections of C o n f o c a l Stacks Demonstrated Increased A p i c a l Recru i tment of Ez r in in C e l l s Transfected with Podoca l yx in 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 Show that f : A c t i n was Recrui ted in Podocalyx in -Transfected Ce l l s 149 Figure 3 - 2 3 : Ver t i ca l Sections of Confoca l Stacks Demonstrated A p i c a l Recrui tment o f f-A c t i n in Podocalyx in -Transfected C e l l s 150 Figure 3 - 2 4 : A c t i n F i laments were V i s i b l e in Indiv idual 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 Podocalyx in - Induced M i c r o v i l l u s Format ion 155 Figure 3 -26 : M o d e l of Podoca lyx in ' s R o l e in the Funct ion of N H E R F F a m i l y M e m b e r s . 158 Figure 4 - 1 : Schematic of C r e - M e d i a t e d Recombinat ion 164 Figure 4 - 2 : Schematic of Transgenic Construct Before and A f t e r C r e - M e d i a t e d Recombinat ion 166 Figure 4 - 3 : C r e - M e d i a t e d Recombinat ion Induced Podoca lyx in Express ion in N S O C e l l s Transfected wi th the Construct 168 Figure 4 - 4 : Overv iew of Select ion Process 170 Figure 4 - 5 : P C R - B a s e d Detect ion of Transgene in G e n o m i c D N A of E S C s 171 Figure 4 - 6 : Expression of Podoca lyx in m R N A in E S C s 173 Figure 4 - 7 : A n a l y s i s of Podoca lyx in Express ion by F l o w Cytometry 174 Figure 4 - 8 : Express ion of G F P m R N A in transfected E S C s 175 Figure 4 - 9 : Karyo typ ing Uncovered the Presence of T r i s o m y E ight in Tested C lones . . 177 Figure 5 - 1 : Schematic of Transgenic Construct 181 Figure 5 - 2 : Transient ly Transfected C H O Ce l l s Expressed P o d o c a l y x i n and G F P f r o m the Transgenic Construct 183 Figure 5 - 3 : G F P Expression in G l o m e r u l i of Transgenic Founders 186 F igure 5 -4 : G lomeru lus f r o m Founder wi th Highest G F P Express ion Leve ls 187 Figure 5 - 5 : L a c k of N o n - S p e c i f i c Expression in L i v e r and L u n g 189 Figure 5 -6 : Transgene Express ion was Undetectable in Peripheral B l o o d 190 F igure 5 -7 : G F P Expression was Not Detected in Brains of Founders 191 Figure 5 -8 : Breed ing Scheme used to Generate M i c e Express ing P o d o c a l y x i n on ly in K i d n e y 194 Figure 5 - 9 : Podoca l yx in m R N A was Expressed f rom the Transgene dur ing Deve lopment in K i d n e y s of Transgenic M i c e 2 0 0 Figure 5 - 1 0 : Immunohistochemistry was used to Detect P o d o c a l y x i n Express ion in K i d n e y G l o m e r u l i o f Transgenic M i c e dur ing Deve lopment 201 F igure 5 - 1 1 : A p i c a l Staining of Podocytes was Ev ident in H i g h M a g n i f i c a t i o n Images of G l o m e r u l i f r o m Podxl'1' T ransgene-Posi t ive M i c e 2 0 2 x Figure 5 - 1 2 : T E M ' s of Podocytes 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 Podoca lyx in -Nu l l /T ransgene -Pos i t i ve Doub le 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 Podocytes f r o m E 1 8 Wi ld t ype , P o d o c a l y x i n -N u l l , and Podoca lyx in -Nu l l/T ransgene -Pos i t i ve D o u b l e Transgenic M i c e 205 Figure 6 - 1 : M o d e l Demonstrat ing a Poss ib le M e c h a n i s m for Decreased Cel l -Substrate 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 Express ion 215 xi LIST OF ABBREVIATIONS aa amino acid A G M aorta-gonad-mesonephros A L L acute l ymphoblast ic leukemia A M L acute mye lo id leukemia A P C a l lophycocyan in bp base pair B M bone marrow B S A bovine serum a lbumin B 6 C 5 7 B L / 6 wi ldtype mouse strain P -gal P -galactosidase p > A R (^-adrenergic receptor C F T R cyst ic f ibrosis transmembrane regulator C F U co lony f o r m i n g unit ch ch icken C h I P chromatin immunoprecip i tat ion C H O Chinese hamster ovary C K I I casein kinase II D A B d iaminobenz id ine D A P I 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 D S D e n y s - D r a s h syndrome D K O p o d o c a l y x i n / C D 3 4 double knockout A D T H L podoca lyx in mutant lack ing C - te rmina l D T H L A E C podoca lyx in mutant lack ing most of the extracel lular domain Atai l podoca lyx in mutant lack ing cy top lasmic tail E embryon ic day E B P 5 0 ezrin b ind ing phosphoprotein of 50 k D a E C L enhanced chemi luminescence E D T A ethylenediaminetetraacetic ac id E G T A ethylene-bis(oxyethylenenitr i lo)tetraacetic acid E G F P enhanced green f luorescent protein E L A M endothelial leukocyte adhesion molecule E R estrogen receptor E R M ezr in - rad ix in -moes in E S C embryon ic stem cel l E 3 K A R P N H E 3 kinase A regulatory protein F A C S f luorescence activated cel l sort ing F B S fetal bovine serum F C f l o w cytometry F T L / F L fetal l iver G B M glomerular basement membrane G F P green f luorescent protein G L E P P glomerular epithel ial protein G l y C A M glycosylat ion-dependent cel l adhesion molecule j' G R K G protein -coupled receptor kinase H C C hepatocellular carc inoma H E V high endothelial venule H G E C human glomerular epithelial cel ls H R P horse radish peroxidase H S C hematopoietic stem cel l H U V E C human umbi l i ca l vein endothelial cel l I C A M intracel lular adhesion molecule IF immunof luorescence Ig immunog lobu l in I H C immunohistochemistry I L - 6 inter leukin 6 ip immunoprecip i tat ion I R E S internal r ibosome entry site k D a k i loDa l ton K O knockout L I F leukemia inhibitory factor L S K L i n S c a - l + c - K i t + L T R long term repopulating M A d C A M mucosal addressin cel l adhesion molecule M D C K M a d i n - D a r b y canine kidney M E P M y b - E t s transformed progenitor ms mouse nd not determined N H E N a 7 H + exchanger x i v N H E R F N a 7 H + exchanger regulatory factor N S G C T nonseminomatous germ cell tumour pAb polyclonal antibody P A G E polyacrylamide gel electrophoresis P A N puromycin aminonucleoside PB peripheral blood PBS phosphate buffered saline PC post coitum P C R polymerase chain reaction P D G F R platelet-derived growth factor receptor P D Z PSD-95 /Dlg /ZO- l P E phycoerythrin P F A paraformaldehyde P K C protein kinase C P M S F phenylmethanesulfonyl fluoride Podo podocalyxin Po lyA/pA polyadenylation PP post partum PS protamine sulfate P 2 Y 1 R purinergic receptor rb , rabbit R N A i R N A interference R T - P C R reverse transcriptase-polymerase chain reaction S A streptavidin S C F stem cel l factor S D S sod ium dodecyl sulfate S E M scanning electron microscopy s i R N A smal l interfering R N A S P L spleen T B S Tr is buffered saline T B S - T T r i s buffered saline plus 0.05 % T w e e n 20 T E M transmission electron microscopy T E R transepithelial resistance U T R untranslated region W T wi ldtype W T 1 W i l m s ' tumour 1 Y S yo lk sac xv i ACKNOWLEDGEMENTS It has been quite a journey , and I have had the opportunity to w o r k wi th many great people over the past few years. I a m very grateful to Dr . K e l l y M c N a g n y for g i v ing me the chance to carry out my studies in his lab and for a lways be l iev ing in me ; his support has really made all this possible . I want to thank everyone in the M c N a g n y lab, past and present, for the advice and especial ly for the laughs. I part icularly thank Shier ley C h e l l i a h for countless hours of help, a lways with a smi le . I also thank He len M e r k e n s , Dr . ' s Reg is Doyonnas and Sebastian Furness, Jamie H a d d o n , Steve M a l t b y , and Poh T a n , who have al l contributed in many ways and also made the M c N a g n y lab an enjoyable place to be. T h e B i o m e d i c a l R e s e a r c h Ce nt re is a f a b u l o u s e n v i r o n m e n t f o r research and co l laborat ion , and I am lucky to have been a part of this group. I wou ld part icular ly l i ke to thank Dr . W i l f Jefferies and Mat t F in lay for generating my k idney -spec i f i c transgenic m i c e . In add i t i on , 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 tener , F a b i o R o s s i , John Schrader , M u r i e l D a v i d , D o u g C a r l o w , Pete Schubert , M a y a K o t t u r i , Jason Grant , Stephane C o r b e l , B r o c k G r i l l , and Gary M c L e a n , as we l l as P h i l O w e n , W o o s e o k Seo , Bernhard Lehner tz , Jef f Duenas, and M i k e W i l l i a m s for adv ice and reagents. I am also grateful to A n d y Johnson for hours of sort ing ce l ls , and to the amaz ing core staff at the B R C , part icular ly 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 also grateful to many people outside of the B R C . D r . ' s C a r o l y n B r o w n , Pau l ine Johnson, and R o b K a y of my supervisory committee have a lways provided me with good xvii advice and kept me on track, and I real ly appreciate their c r i t i ca l rev iews of my thesis. O n e o f the most product ive and enjoyable aspects o f my thesis research has been my co l labora t ion w i t h Dr . C a i R o s k e l l e y , Dr . 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 am truly grateful for al l their contr ibut ions to the m i c r o v i l l i project. I a lso appreciate the expert electron microscopy assistance f r o m Der r i ck H o m e , as we l l as adv ice f r o m Garnet Mar tens , 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 Dr . Robert N a b i ' s confocal microscope, and contr ibutions f r o m the Department of M e d i c a l Genet ics , especial ly those of Chery l B i s h o p . I a m also grateful to Dr . Cor r inne L o b e and R a y m o n d W o n g at Sunnybrook Heal th Sc iences Centre and Dr . J i l l L a h t i at St. Jude C h i l d r e n ' s Research Hospi ta l for assistance wi th the inducib le podoca lyx in expression project, and to Dr . ' s D a v i d Kershaw and Atsush i M i y a j i m a for invaluable reagents. I a m also thankful for fund ing f r o m N S E R C and U B C and travel funding f r o m the Stem C e l l Network . None of this wou ld have been possible without the tremendous support f r o m my f a m i l y and fr iends. A t U B C , I espec ia l l y want to thank M a r c i a M c C o y , Jasmeen M e r z a b a n , N i c o l e V o g l m a i e r , Jul ie W o n g , and Jane C ippo lone for their constant encouragement; the many frustrations of research are so much easier to deal w i th when you have good fr iends w h o are in s imi la r situations. In add i t ion , my parents have a lways been an incred ib le source of encouragement for me, and I cannot thank them enough. M y grandparents, w h o cont inue to l i v e l i fe to the fu l les t every day , have a lso been a constant source o f inspi rat ion fo r me. L a s t l y , there are no words to express how m u c h I appreciate the unwaver ing support of m y husband, Chr ist ian N ie lsen . I a m except ional ly grateful for his encouragement, every single day throughout my doctoral studies. x v i i i 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 ce l l surface protein essential fo r k idney deve lopment (Doyonnas et a l . , 2001) and impl icated in a wide range of cancers (Casey 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 ; Schopper le et a l . , 2 0 0 3 ; Somasi r i et a l . , 2 0 0 4 ; S tanhope -Baker et a l . , 2004) . It was first ident i f ied by Dr. M a r i l y n Farquhar 's group and termed " p o d o c a l y x i n " as it is the major c o m p o n e n t of the g l y c o c a l y x of k i d n e y g l o m e r u l a r podocytes (Ker jaschk i et a l . , 1984). It has also been ca l led p o d o c a l y x i n - l i k e protein 1 ( P C L P - 1 ) , th rombomuc in , M y b - E t s transformed progenitor ( M E P ) - 2 1 , and gp 135, and it is encoded by the podxl gene (Kershaw et a l . , 1995; M c N a g n y et a l . , 1992; M c N a g n y et a l . , 1997; M e d e r et a l . , 2005)*. • Some figures in this chapter have been publ ished in 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 mur ine hematopoiet ic stem cel ls and embryon ic erythroid cel ls . 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 models to study the t ranscr ipt ional contro l of hematopoiet ic l ineage c o m m i t m e n t and to ident i fy l ineage -speci f ic 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 . , Prent ice , L. , G i l k s , C . B . , C h i a , S . K . , G e l m o n , K . A . , K e r s h a w , D . B . , H u n t s m a n , D . G . , M c N a g n y , K . M . and R o s k e l l e y , C D . (2004) Overexpression of the anti -adhesin podoca lyx in is an independent predictor of breast cancer progression. Cancer Res, 64, 5 0 6 8 - 5 0 7 3 . 1 1.1.1 P ro te in S t ruc ture Podoca l yx in is a type I transmembrane protein wi th a predicted mass, based on its protein backbone, of approx imately 55 k D a (Kershaw et a l . , 1997a; M c N a g n y et a l . , 1997). Its ser ine - th reon ine -pro l ine r ich ext race l lu lar d o m a i n is ex tens ive ly O - g l y c o s y l a t e d and s ia l y la ted , resu l t ing in an actual mass o f 1 4 0 - 1 7 0 k D a and charac te r i z ing it as a s ia lomuc in (Doyonnas et a l . , 2 0 0 1 ; H i lkens et a l . , 1992; Ker jaschk i et a l . , 1984; Kershaw et a l . , 1997a; M c N a g n y et a l . , 1997; Mie t t inen et a l . , 1999; Or lando et a l . , 2 0 0 1 ; Sassetti et a l . , 1998; Takeda et a l . , 2000) . The extracel lular domain also contains several potential sites o f N - l i n k e d g l ycosy la t ion , a g lobular domain cons is t ing of four cysteine residues, and a juxtamembrane stalk region (Doyonnas et a l . , 2 0 0 1 ; Kershaw et a l . , 1997a; Or lando et a l . , 2 0 0 1 ) . A 2 6 a m i n o a c i d (aa) h y d r o p h o b i c reg ion encodes a s ing le pass t ransmembrane d o m a i n , w h i c h is f o l l o w e d by 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 ta i l (Kershaw et a l . , 1997a; M c N a g n y et a l . , 1997; Sassetti et a l . , 1998; Takeda et a l . , 2000) . T h i s intracel lu lar d o m a i n contains putative phosphory lat ion sites for protein kinase C ( P K C ) and casein kinase II ( C K I I ) as we l l as the C - t e r m i n a l P D Z - b i n d i n g moti 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 . , 1997a; M c N a g n y et a l . , 1997). L i k e other s ia lomucins , the extracel lular domain of podoca lyx in demonstrates a very low degree of sequence conservat ion (<33%) (Kershaw et a l . , 1997a; O r l a n d o et a l . , 2 0 0 1 ; T a k e d a et a l . , 2000) . In contrast, the 75 aa cy top lasmic tai l exh ib i ts a h igh level o f aa sequence identity (~95 % between rat, rabbit , and h u m a n , and s l ight l y less in compar i son to ch icken) , indicat ive of an important, conserved funct ion (Kershaw et a l . , 1997a; L i et a l . , 2 0 0 2 ; M c N a g n y et a l . , 1997; Miet t inen et a l . , 1999). 2 A m i n o ac id sequence, protein structure, genomic organizat ion, and patterns of alternative sp l i c ing (described be low) suggest that podoca lyx in is most c losely related to C D 3 4 and endog lycan ( M c N a g n y et a l . , 1997; N ie lsen et a l . , 2 0 0 2 ; Sassetti et a l . , 2000) . Each of these proteins conta ins a h igh l y g l ycosy la ted and s ia ly lated ex t race l lu la r d o m a i n , a g lobu la r d o m a i n , and a stalk f o l l o w e d by a s ingle pass membrane spanning d o m a i n (F igure 1-1) ( B r o w n et a l . , 1991; He et a l . , 1992; Kershaw et a l . , 1997a; K rause et a l . , 1996; K rause et a l . , 1994; M c N a g n y et a l . , 1997; N a k a m u r a et a l . , 1993; N ie lsen 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). The cy top lasmic tails contain the highest degree of s imi lar i ty across species and between f a m i l y members , wi th sequences suggest ive o f roles in ce l l s i g n a l i n g or ce l lu la 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 conta in consensus phosphory lat ion sites, as we l l as a C -terminal P D Z - b i n d i n g mot i f ( B r o w n et a l . , 1991; Krause et a l . , 1996; M c N a g n y et a l . , 1997; N i e l s e n et a l . , 2 0 0 2 ; Sassetti et a l . , 1998; 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 CD34 Family Members. B l u e : m u c i n domains , b lack c i rc les : potential N - l i n k e d carbohydrates, hor izonta l l ines : potential O - l i n k e d carbohydrates, tr iangles: potential s ia l ic ac id residues, green: g lobular m o t i f s , y e l l o w : stalk, orange: t ransmembrane d o m a i n s , red: c y t o p l a s m i c ta i l s , large c i rc les : potential phosphory la t ion sites. Reproduced w i t h k i n d permiss ion of S . K a r g e r A G , Base l (N ie lsen et a l . , 2002) . There are, however , several notable differences between these three s ia lomucins . F i rs t ly , the muc in domains vary in length, wi th C D 3 4 being the shortest protein overal l (Krause et a l . , 1994; Sassetti et a l . , 1998; Sassetti et a l . , 2000) . Second ly , in the g lobular region p o d o c a l y x i n and C D 3 4 have f o u r and s ix cys te ine res idues , r e s p e c t i v e l y , w h i l e endoglycan has two, with an addit ional unpaired juxtamembrane cysteine, l ike ly invo lved in d imer izat ion ( B r o w n et a l . , 1991; Fieger et a l . , 2 0 0 3 ; Kershaw et a l . , 1997a; Sassetti et a l . , 2000) . T h i r d l y , whi le podoca lyx in and endoglycan share the C - t e r m i n a l D T H L moti f , C D 3 4 ' s C - t e r m i n a l sequence is sl ight ly altered: it is D T E L (He et a l . , 1992; Kershaw et a l . , 1997a; M c N a g n y et a l . , 1997; 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 , endog lycan has a unique N - te rmina l region r ich in g lutamic ac id residues; wh i le this mot i f is found in many intracel lular regulators of transcript ion, it is rarely a characteristic o f ext racel lu lar domains (Sassetti et a l . , 2000) . T h u s , a l though this f a m i l y is c lose ly related, the three proteins do have some unique, and potential ly important, features. 1.1.2 Genomic Organization and Alternative Splicing T h e g e n o m i c o rgan i za t ions o f podxl, cd34, and endoglycan s t rongly suggest an evolut ionary relationship (F igure 1-2) ( L i et a l . , 2 0 0 1 ; N ie lsen et a l . , 2002) . Each protein is encoded by eight exons, and , across the f a m i l y , i nd i v idua l exons encode equivalent prote in mot i fs and are very s i m i l a r in s ize ( 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). Throughout the f a m i l y , intronic distances are also st r ik ingly s imi la r , and s p l i c i n g to an addit ional exon between exons seven and eight generates a longer transcript encoding a protein l a c k i n g much o f the c y t o p l a s m i c tai l (Figure 1-3) (Kershaw et a l . , 1997a; L i et a l . , 2 0 0 1 ; M c N a g n y et a l . , 1997; N a k a m u r a et a l . , 1993; 5 Nie lsen et a l . , 2 0 0 2 ; Sassetti et a l . , 2 0 0 0 ; Suda et a l . , 1992). Thus , these three s ia lomucins have been grouped into a single f a m i l y based on protein structure, genomic organizat ion, and patterns of alternative sp l i c ing . 6 1 11.0 0.5 2 3 cd34 • m i 44.0 1.2 podxl | • I 1 1 10.0 7.5 endgl M I I I 1 1 5'UTR & M u c i n 13 7.9 ! 0.3 0.3 | : 0.8 4 /\ 5: A 6 A :7: A . 8 • 0.3: 2.2 1.1 : 7.0 Signal peptide d o m a i n C - C d o m a i n 0.3 0.2 V i 0.5 2.2 J b 0=1 Stalk T M : d o m a i n 1.3 0.7 Cyt. tail 3'UTR 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 \ stop Truncated forms | |TM| [ |^ 1 ' 7 8b 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 level of p o d o c a l y x i n expression on the surface of renal g lomeru lar epithel ia l cel ls (podocytes) enabled its init ial isolat ion and characterization f r o m these cel ls in 1984 (Ker jaschk i et a l . , 1984). P o d o c a l y x i n is also expressed on the lumina l face of vascular endothel ia in a wide variety of vessels, by hematopoiet ic stem cel ls and progenitors, and even on hemangioblasts, wh ich are the precursors of hematopoietic and endothel ial ce l ls (De l ia et a l . , 1993; Doyonnas et a l . , 2 0 0 5 ; Hara et a l . , 1999; Horvat et a l . , 1986; Kershaw et a l . , 1997a; M c N a g n y et a l . , 1997; Miet t inen et a l . , 1990; Sassetti et a l . , 1998). In more mature hematopoiet ic ce l l s , it is on ly expressed on platelets, as we l l as their precursors, m e g a k a r y o c y t e s ( M c N a g n y et a l . , 1997 ; M i e t t i n e n et a l . , 1999). O u t s i d e o f the hematopoiet ic system, podoca lyx in is expressed by mesothel ial cel ls l i n ing many organs (Doyonnas et a l . , 2 0 0 1 ; M c N a g n y et a l . , 1997), and dur ing development, it is expressed in al l three germ layers , as w e l l as e m b r y o n i c stem ce l ls and a subset of neurons (Doyonnas et a l . , 2 0 0 5 ; V i tu re i ra et a l . , 2005) . L i k e p o d o c a l y x i n , C D 3 4 is expressed on v a s c u l a r endothe l ia l ce l l s as w e l l as hematopoiet ic progenitors and stem cells (Andrews et a l . , 1989; Baumhueter et a l . , 1994; Berenson et a l . , 1988; E m a et a l . , 1990; F i n a et a l . , 1990; Sato et a l . , 1999; Y o u n g et a l . , 1995). There has, however , been considerable controversy about expression of C D 3 4 on long term repopulat ing hematopoiet ic stem cel ls ( L T R - H S C s ) . It is now bel ieved that C D 3 4 expression f luctuates depending on deve lopmenta l stage and ce l l act ivat ion : in older adult mice C D 3 4 is expressed only on activated H S C s , whereas expression is more widespread dur ing development (Dao 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 . , 1999; 9 O g a w a , 2 0 0 2 ; O s a w a et a l . , 1996; Sato et a l . , 1999; T a j i m a et a l . , 2 0 0 0 ; Zan jan i et a l . , 2003) . Thus , it may be that the most pr imit ive and quiescent H S C s are C D 3 4 . T h e n , in order fo r cel ls to contr ibute to engraftment, C D 3 4 may be upregulated. T h i s cou ld be f o l l o w e d by a decrease in expression as the bone marrow achieves steady state levels o f hematopoiet ic cel ls . A d d i t i o n a l l y , C D 3 4 is expressed on mature murine mast ce l ls , but it is not found on platelets (Drew et a l . , 2002) . There is therefore considerable over lap between podocalyx in and C D 3 4 expression, but there are some notable dif ferences. A l t h o u g h much less is k n o w n about endog lycan , its express ion pattern d isp lays some s imi lar i ty to those of C D 3 4 and podoca lyx in . Some hematopoiet ic ce l ls , i n c l u d i n g bone mar row macrophages and T o l l - l i k e receptor -act ivated B ce l l s , as w e l l as a subset of neuronal cel ls and vascular smooth muscle cel ls al l express endoglycan ( (N ie lsen et a l . , 2 0 0 2 ; Sassetti et a l . , 2000) , and He len Merkens and K e l l y M c N a g n y unpubl ished) . There is also some evidence for expression on endothel ial ce l ls 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 in i t ia l l y ident i f ied and character ized in rat in the 1980's (Ker jaschk i et a l . , 1984), it was not unti l 1995 that it was f i rst c loned us ing a rabbit g lomeru lar c D N A l ibrary (Kershaw et a l . , 1995). A bacterial c D N A l ibrary express ion system was screened wi th monoc lona l ant ibodies generated against a p o d o c a l y x i n - l i k e p ro te in expressed in g l o m e r u l i . S i x pos i t i ve c l o n e s were i d e n t i f i e d , and these cor responded to a 5.5 kb transcript h igh ly expressed in 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 ac id sequence demonstrated that it contained many characterist ics of p o d o c a l y x i n , so the m o l e c u l e was n a m e d " p o d o c a l y x i n - l i k e protein 1 ( P C L P 1 ) " . Subsequent ly , rabbit c D N A e n c o d i n g p o d o c a l y x i n was used to screen a human renal cortex c D N A l ib rary , and human P C L P 1 was c loned (Kershaw et a l . , 1997a). c D N A cor respond ing to the c y t o p l a s m i c tai l of human P C L P 1 was then used to probe the genomic D N A of a variety of species. H o m o l o g o u s sequences were found in monkey , rat, mouse , d o g , c o w , rabbit , and c h i c k e n , but not yeast, demonstrat ing the remarkable conservation of this region of the gene in vertebrates (Kershaw et a l . , 1997a). C o n c u r r e n t l y , the c h i c k e n h o m o l o g o f p o d o c a l y x i n was c l o n e d and n a m e d " t h r o m b o m u c i n " ( M c N a g n y et a l . , 1997) . T o beg in w i t h , p r i m i t i v e hematopo ie t i c progenitors der ived f r o m ch icken e m b r y o y o l k sac were transformed with 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 rus , E 2 6 ; these ce l ls were termed M y b - E t s -transformed progenitors , or M E P s (Graf et a l . , 1992). A panel of monoc lona l antibodies was generated against surface antigens on these cel ls by using them to i m m u n i z e mice ( M c N a g n y et a l . , 1992) . O n e o f the ant ibod ies , M E P 2 1 , spec i f i ca l l y recogn ized an antigen present on M E P s and platelets, but not on other mature hematopoiet ic cel ls . The M E P 2 1 antigen cou ld on ly be pur i f ied in very low quantit ies, so nanoelectrospray mass spectrometry was used to sequence the protein , and degenerate o l igonuc leot ides were used to c lone the M E P 2 1 -encod ing c D N A s ( M c N a g n y et a l . , 1997). Three dist inct types of c lones were obtained. T h e f i rst encoded a 5 2 4 base pair (bp) 5 ' untranslated region ( U T R ) , a 1347 bp 3 ' U T R , and an open reading f rame cod ing for 571 aa, w h i c h inc luded a putative N - t e r m i n a l s ignal pept ide, a m u c i n d o m a i n , a g lobular d o m a i n , a stalk, a 11 putative transmembrane d o m a i n , and a cy top lasmic tai l conta in ing consensus P K C and C K I I phosphorylat ion sites. The second type of c lone was very s imi lar , but it encoded an insert ion of 18 addit ional amino acids in the jux tamembrane region of the c y t o p l a s m i c ta i l . T h e third type of c lone contained a novel sequence beginning at the same site as the insert ion in type two c lones, and it encoded a shorter cy top lasmic tail w i th an earl ier stop c o d o n . T h i s was the f i rs t ev idence of a l ternat ive s p l i c i n g to generate a t runcated cy top lasmic tail in podoca lyx in . Express ion of this m u c i n on platelets, or thrombocytes , led to it being cal led thrombomucin . Subsequent ly , rat podxl was c loned using c D N A l ibrar ies f r o m k idney . It was part ia l ly c loned using rabbit c D N A as a probe (Miet t inen et a l . , 1999); it was later comple te l y c loned us ing pr imers designed f r o m homologous sequences in the c y t o p l a s m i c tai ls o f rabbit and human p o d o c a l y x i n ( Takeda et a l . , 2 0 0 0 ) . In terest ing ly , a l though d o g podoca l yx in (gp 135) has been used as an apical marker o f M a d i n - D a r b y canine k idney ( M D C K ) epi thel ia l ce l ls for many years (O jak ian and S c h w i m m e r , 1988), it was on ly recently recognized as podocalyx in (Meder et a l . , 2005) . It was prev iously k n o w n s imply as " g p l 3 5 , " but after p u r i f i c a t i o n and i d e n t i f i c a t i o n o f t r y p t i c pept ides by nanoelect rospray tandem mass spect rometry , it became c lear that it was , in fact , p o d o c a l y x i n . E S T ' s corresponding to podoca lyx in have now been ident i f ied in human, dog , rat, mouse, squirrel , ch icken , f rog , and zebrafish. 12 1.3 Transcriptional Regulation of Podocalyxin The process of transcriptional regulation of CD34 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 CD34, while 160 kb is, suggesting that considerable upstream, downstream, and intronic sequences are required for tissue-specific expression of CD34 (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 Positive Regulation by WT1 W T l 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 . , 2001) , and although it was in i t ia l ly ident i f ied as a tumour suppressor it may also funct ion as an oncogene (reviewed in (Loeb and Sukumar , 2002)) . W T 1 is normal ly expressed in the developing g lomerulus , as wel l as in the urogenital r idge, mesothe l ium, spleen, brain , and spinal cord dur ing embryogenesis and in the uterus, ov iduct , granulosa cel ls of the ovary , Sertol i cel ls of the testes, hematopoiet ic progenitor or stem ce l l s , and kidney podocytes in adult ( reviewed in (Discenza and Pel let ier , 2004)) . Several studies have impl icated W T 1 in regulation of podoca lyx in express ion. S p l i c i n g generates two major variants of W T 1 whereby a th ree -amino ac id ( K T S ) sequence between the third and fourth z inc f ingers is either omit ted or inc luded (Pa lmer et a l . , 2 0 0 1 ; W a n g et a l . , 2001) . T h e ( - K T S ) variant binds D N A and acts as a t ranscr ipt ional regulator, wh i le the ( + K T S ) variant is a poor t ranscr ipt ional regulator but can instead interact with sp l ic ing machinery (Roberts, 2005) . W i t h i n hours of induc ib le expression of the ( - K T S ) i so fo rm in rat embryonic k idney cel l l ines, a dramatic increase in podoca lyx in is detected at both the m R N A and protein levels (Pa lmer et a l . , 2001) . T h i s effect is revers ib le upon remova l of W T 1 . A d d i t i o n a l exper iments ident i f ied a W T 1 ( - K T S ) responsive domain in the podxl promoter (Palmer et a l . , 2001) , and an independent group used chromat in immunoprec ip i tat ion (ChIP) to demonstrate that W T 1 does, in fact , b ind the p romoter ( S t a n h o p e - B a k e r et a l . , 2 0 0 4 ) . Fu r the rmore , in a t ransgenic mouse e x p r e s s i n g a mutant f o r m of W T 1 , there is a s tat is t ica l l y s i g n i f i c a n t decrease in podoca lyx in expression in newborn k idneys , although normal levels are achieved wi th in several months of birth (Gao et a l . , 2004) . M o r e o v e r , p o d o c a l y x i n express ion in the d e v e l o p i n g g lomeru lus , and in other ce l l types, corresponds to that o f W T 1 . In the 14 g lomeru lus , W T 1 is expressed dur ing the renal ves ic le and S -shaped body stages of development, and it is restricted to v isceral epithelial cel ls (podocytes) of deve lop ing and mature g lomeru l i ( A r m s t r o n g et a l . , 1993; P a l m e r et a l . , 2001) . P o d o c a l y x i n is f i rst evident at a s imi lar stage o f g lomerular development, and expression is greatest in mature podocytes (Pa lmer et a l . , 2 0 0 1 ; Pr i tchard - Jones et a l . , 1990). In add i t i on , W T 1 and podoca lyx in are co-expressed in mesothel ium and hematopoietic precursors (Doyonnas et a l . , 2 0 0 1 ; Doyonnas et a l . , 2 0 0 5 ; E l l i s e n et a l . , 2 0 0 1 ; Pr i tchard-Jones et a l . , 1990). T h u s , W T 1 is the most conc lus ive regulator of podoca lyx in expression to date. 1.3.2 Negative Regulation by p53 In contrast, there is some evidence to suggest that p53 represses podoca lyx in expression. U s i n g c D N A microar ray ana lys is , p o d o c a l y x i n was ident i f ied as a p53 target in the W i l m s ' tumour mode l c e l l l i ne , W i T 4 9 (S tanhope -Baker et a l . , 2004) . A luc i ferase reporter construct was then used to c o n f i r m the considerable 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 phys ica l l y interact and modulate the funct ions o f one another in some si tuat ions. W h i l e p53 can alter the t ranscr ipt ional regulatory act iv i ty of W T 1 , W T 1 can stabi l ize p53 , alter its act iv i ty , and prevent p 5 3 -induced apoptosis (Maheswaran et a l . , 1995; Maheswaran et a l . , 1993). H o w e v e r , in the W i T 4 9 ce l l l ine , 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 p53 's effects on W T 1 : a m i n i m a l podxl promoter conta in ing the W T l - r e p o n s i v e port ion is not suff ic ient 15 to decrease podoca lyx in expression upon activation of p53 (Stanhope-Baker et a l . , 2004) . Thus , p53 can modulate podoca lyx in ' s expression in a VVT1-independent manner. 1.3.3 Positive Regulation by Ets-1 T h e on ly addit ional in format ion regarding control o f podoca lyx in expression comes f r o m a study based on c D N A microar ray analysis of human u m b i l i c a l vein endothel ia l cel ls ( H U V E C ) infected with an adenovirus encoding the Ets -1 transcription factor (Teruyama et a l . , 2001) . E ts -1 expression in endothel ial cel ls is k n o w n to promote angiogenesis . Its expression in H U V E C s leads to a 3 . 3 - f o l d upregulation of podoca lyx in in compar ison to n u l l - v i r u s infected ce l l s , a l though there is no ev idence to suggest that p o d o c a l y x i n expression is direct ly regulated by this factor. There is therefore considerable work sti l l to be done in unravel ing the complex transcriptional regulation of podocalyx in . 1.4 Intracellular Binding Partners for Podocalyxin C l u e s regard ing the func t ion o f novel proteins can often be obtained by ident i f y ing b i n d i n g partners w i th k n o w n funct ions . The h igh degree of sequence conservat ion in p o d o c a l y x i n ' s c y t o p l a s m i c tai l imp l ies that int racel lu lar b ind ing partners might ex ist ; these potential interactors are thus the focus of much research. 16 1.4.1 Ezrin T h e f irst ind icat ion o f an intracel lu lar b ind ing partner for p o d o c a l y x i n came f r o m Dr . M a r i l y n Farquhar 's group in 2001 (Or lando et a l . , 2001) . They had prev ious ly noticed that apical podoca lyx in local izat ion in podocytes coincides with that o f ezr in , a member o f the ez r in - rad ix in -moes in ( E R M ) f a m i l y of cytoskeletal l inker proteins (Kur ihara et a l . , 1995). E R M proteins contain a C - te rmina l actin b inding mot i f and an N - termina l F E R M protein modu le , thought to anchor them to membrane proteins (Ch isht i et a l . , 1998; T u r u n e n et a l . , 1994). T h e y regulate ce l l adhes ion and m o r p h o g e n e s i s , i n c l u d i n g format ion of m i c r o v i l l i and membrane ruff les ( reviewed in (Mangeat et a l . , 1999)). T h e importance of p o d o c a l y x i n in ma in ta in ing podocyte foot process integr i ty (d iscussed b e l o w ) , 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 poss ib le l i n k to the act in c y t o s k e l e t o n . F o r these reasons, this g roup assessed the potent ia l a s s o c i a t i o n o f podoca lyx in wi th ezrin and actin (Or lando 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 ezr in in podocytes was c o n f i r m e d by immunof luo rescence and dual i m m u n o g o l d labe l l ing of u l t ra - th in c ryosect ions o f rat k idney (Or lando et a l . , 2001) . B o t h proteins are concentrated a long the apical p lasma membrane of podocyte cel l bodies and foot processes above the level of slit d iaphragms, and they f o r m a stable , 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 co -sedimentat ion assays in p o d o c a l y x i n -t ransfected M D C K ce l l s demonstrate that a s ign i f i cant por t ion o f p o d o c a l y x i n is associated wi th actin f i laments and that this interaction is dependent on the interaction of podoca lyx in wi th ezr in. 17 Subsequent experiments ident i f ied an ezrin b ind ing site in the juxtamembrane region of p o d o c a l y x i n ' s c y t o p l a s m i c ta i l (Schmieder et a l . , 2004) . T h e H Q R I S sequence o f podoca lyx in is s imi la r to the H Q R S found in intracel lu lar adhesion molecu le ( I C A M ) - 3 (Serrador et a l . , 2002). The hist idine, arginine, and several serine residues are required for the recogni t ion o f I C A M - 3 by ezr in (Serrador et a l . , 2 0 0 2 ) , so these residues were mutated in a H is - tagged p o d o c a l y x i n tail construct in order to assess b ind ing to G S T -tagged N - termina l ezr in (Schmieder et a l . , 2004) . Mutat ion of serine to alanine, hist idine and arginine to alanine, or al l three residues to alanine, or deletion of the 12 N - te rmina l residues of the cy top lasmic tai l altogether decreases b ind ing by 2 2 , 4 6 , 9 5 , or 100 % , respect ively . These results strongly suggest that ezr in and podoca lyx in are capable o f direct interaction. 1.4.2 NHERF Proteins There is also increasing evidence that podoca lyx in interacts wi th members of the N a 7 H + exchanger ( N H E ) regulatory factor ( N H E R F ) f a m i l y of scaf fo ld ing proteins ( reviewed in ( D o n o w i t z et a l . , 2 0 0 5 ; Sheno l ikar 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 , 2001) ) . N H E R F 1 / E B P 5 0 (ezr in b i n d i n g phosphoprote in o f 50 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 th podoca lyx in (d iscussed be low) , w h i l e the more distant ly related and recently d i scovered f a m i l y members 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 ind p o d o c a l y x i n . N H E R F molecules al l have mul t ip le protein-protein interaction modules . N H E R F 1 and N H E R F 2 each have two tandem P S D - 9 5 / D l g / Z O - l ( P D Z ) domains and a 18 C - t e r m i n a l E R M domain . In contrast, N H E R F 3 and N H E R F 4 both lack E R M domains , but each have four P D Z domains. E R M domains faci l i tate interaction w i th E R M f a m i l y members and indirect l inkage to the act in cy toske le ton , w h i l e P D Z d o m a i n s , w h i c h represent one of the most c o m m o n modular domains in the human genome, recognize spec i f ic sequences, general ly at the C - te rminus o f proteins. There are several classes of P D Z domains , but the second P D Z domains of both N H E R F 1 and N H E R F 2 recognize the consensus sequence X- (S/T) -X - ( I/V/L/M) where X represents any amino ac id . N H E R F proteins have the capacity to homod imer i ze or heterodimerize w i th each other, and their mul t ip le protein- interaction domains cou ld enable the format ion of large protein complexes connected to the actin cytoskeleton. Ear ly studies of this f a m i l y also suggested that N H E R F proteins were invo lved in apica l membrane loca l i zat ion of other proteins, but recent data has raised doubts about this proposed funct ion . N H E R F f a m i l y members are thought to be invo lved in a wide variety of b io log ica l processes, inc lud ing t ra f f i ck ing , transport, and s ignal l ing , based on their interaction with over 30 target proteins. They can interact w i t h ion transporters, such as N H E 3 , the cyst ic f ibrosis transmembrane regulator ( C F T R ) , and the Na + - phosphate transporter (Npt2) , and G p ro te in -coup led receptors, i n c l u d i n g |3 2 -adrenergic receptors ( f3 2 -AR) , the pur inerg ic receptor P 2 Y 1 R , and the K -o p i o i d receptor. They can also associate w i th s igna l l ing proteins, scaf fo lds , and nuclear proteins, such as platelet-derived growth factor receptor ( P D G F R ) , phosphol ipase C(31, 2 , and 3 , the G prote in -coupled receptor k inase ( G R K ) , and |3-catenin. It must be noted, however , that many of these interactions have not yet been conf i rmed in vivo. 19 Short ly after ident i fy ing ezr in as a podoca l yx in b ind ing protein, Dr. M a r i l y n Farquhar 's group also ident i f ied N H E R F 2 as a b i n d i n g partner fo r podoca l yx in (Takeda et a l . , 2001)) . U s i n g the c y t o p l a s m i c tai l of p o d o c a l y x i n as bait, N H E R F 2 was detected in a yeast t w o - h y b r i d screen o f a rat g lomeru la r c D N A l ibrary . Furthermore, in G S T p u l l -down assays, in vitro translated N H E R F 2 binds strongly to G S T - t a g g e d podoca lyx in ta i l . T h i s assay a lso demonstrates that the related m o l e c u l e , N H E R F 1 can interact wi th podoca lyx in . W h e n podoca lyx in and N H E R F f a m i l y members are co-transfected in vitro, podoca lyx in is co - immunoprec ip i ta ted w i th both N H E R F 1 and N H E R F 2 . The interaction o f podoca lyx in w i th these two P D Z - c o n t a i n i n g proteins is not surpr is ing in that it has a conserved P D Z b ind ing mot i f , D T H L , at its C - t e r m i n u s ; delet ion of this mot i f prevents b ind ing in transfected ce l ls (Takeda et a l . , 2001) . A d d i t i o n a l yeast two -hybr id and G S T p u l l - d o w n exper iments us ing in vitro translated f ragments of N H E R F l a n d N H E R F 2 showed that p o d o c a l y x i n interacts wi th the second P D Z domain of both molecu les , but not wi th the f irst d o m a i n , and that the interaction between podoca lyx in and N H E R F 2 is much stronger than the interaction wi th N H E R F 1 . In k idney , N H E R F 1 is expressed in p rox imal tubules, but not g lomeru l i , wh i le N H E R F 2 is f o u n d a l o n g the ap ica l p l a s m a m e m b r a n e o f podocyte foot processes, where 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 and ez r in ( Takeda et a l . , 2001) . In add i t i on , c o -i m m u n o p r e c i p i t a t i o n ( co - ip ) exper iments f r o m g lomeru la r extracts demonstrate that p o d o c a l y x i n can interact w i t h both N H E R F 2 and e z r i n , but not w i t h N H E R F 1 , in g lomeru l i (Or lando et a l . , 2 0 0 1 ; T a k e d a et a l . , 2001) . S ince 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 addi t ion 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 N H E R F 2 , 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 (Li et al., 2002) . 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, completely abolishes binding. Co-immunoprecipitation and immunofluorescence 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 podocalyxin/NHERF 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 hematopoiet ic speci f ic interactors, an early hematopoiet ic c D N A expression l ibrary ( M c N a g n y et a l . , 1996) was screened with a biot iny lated peptide cor responding to the cy top lasmic tail of podoca lyx in (Tan et a l . , 2006) . Th i s screen ident i f ied N H E R F 1 as a p o d o c a l y x i n interact ing protein in the hematopoiet ic system (Tan et a l . , 2 0 0 6 ) . T h e p o d o c a l y x i n peptide was also used to pur i fy interact ing proteins f r o m hematopoiet ic progenitor cel l extracts; mass spectrometry conf i rms the associat ion of podoca l yx in 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 recognit ion sequence o f p o d o c a l y x i n . T h e s e two prote ins a lso c o l o c a l i z e and 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 in hematopo ie t i c p rogen i to rs . In te res t ing ly , s t rong c o l o c a l i z a t i o n corresponds to ce l ls in w h i c h podoca l yx in is capped to one po le ; ce l l s e x p r e s s i n g u n i f o r m leve ls o f p o d o c a l y x i n over the entire surface do not d i s p l a y c o l o c a l i z a t i o n o f the two proteins. Important ly , essent ia l l y a l l hematopo ie t i c c e l l s , 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 markers i n d i c a t i v e o f s tem c e l l s , express N H E R F 1 , w h i c h suggests that it may be an important l igand for p o d o c a l y x i n in these cel ls (Tan et a l . , 2006) . 1.4.3 Other CD34-Family Binding Proteins T o date, there has been on ly one study invest igat ing potent ial in t race l lu la r b i n d i n g partners for endoglycan. U s i n g the hematopoietic c D N A l ibrary screen descr ibed above, the c y t o p l a s m i c tai l of endog lycan , l ike that of p o d o c a l y x i n , was also shown to b ind N H E R F 1 (Tan et a l . , 2 0 0 6 ) . S i m i l a r l y , a f f in i t y p u r i f i c a t i o n f r o m h e m a t o p o i e t i c progenitor lysates indicates that N H E R F 1 can associate wi th a peptide corresponding 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 same exper iments reveal a lack o f 23 interact ion of 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 contain a C - t e r m i n a l P D Z recogn i t ion sequence, it d i f fe rs f r o m both p o d o c a l y x i n and endog l ycan , w h i c h are ident ica l : p o d o c a l y x i n contains a D T H L mot i f , w h i l e the cor responding sequence in C D 3 4 is D T E L (Kershaw et a l . , 1995; Suda et a l . , 1992). Th is difference l ike ly conveys the speci f ic i ty for interaction of podoca lyx in and endoglycan wi th N H E R F proteins, and suggests that w h i l e the three proteins may have some over lapp ing funct ions , there are probably some key differences. A l t h o u g h C D 3 4 does not appear to interact w i t h N H E R F f a m i l y m e m b e r s , in hematopoietic progenitor ce l ls it has been shown to interact wi th C r k L , a member of the C r k f a m i l y of adapter proteins (Fe lschow et a l . , 2001) . These proteins l ink proteins that do not possess kinase act iv i ty to intracel lu lar s igna l ing cascades, thereby enabl ing them to indirect ly transmit s ignals. A l t h o u g h the exact b ind ing site for 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 dependent on a h i g h l y conserved in t race l lu la r 10 aa juxtamembrane sequence present in both isoforms of C D 3 4 . The intracel lular sequences of p o d o c a l y x i n and endog lycan are more s i m i l a r to each other than to C D 3 4 , so the existence of diverse b ind ing partners is not surpr is ing (He et a l . , 1992; Ke rshaw et a l . , 1997a; M c N a g n y et a l . , 1997; 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 though there are over 12 0 0 0 C D 3 4 - r e l a t e d publ icat ions to date, the funct ions o f C D 3 4 and its f a m i l y members remain to be ascertained. A var iety of potent ia l f u n c t i o n s 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 in e n h a n c i n g 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 fe rent ia t ion , ac t ing as adhes ive l i gands , p revent ing ce l l adhesion, establ ishing polar i ty , and regulating asymmetr ic ce l l d i v i s ion . 1.5.1 Enhancing Proliferation and Blocking Differentiation There are two reasons for C D 3 4 ' s hypothes ized role in enhanc ing p ro l i fe ra t ion or b l o c k i n g d i f ferent iat ion. First , its expression on mult ipotent hematopoiet ic progenitors and progressive downregulat ion on more mature cel ls suggests a role in maintenance of the stem ce l l phenotype ( rev iewed in (Krause et a l . , 1996)). S e c o n d , in one strain of C D 3 4 knockout ( K O ) an imals , there are less progenitor ce l l s in e m b r y o n i c and adult t issues, and adult -der ived progenitors appear to have a prol i ferat ion defect (F igure 1 -4A) ( 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 ignif icant decrease in total co lony fo rming unit ( C F U ) progenitor cel ls in adult as we l l as a reduction in fetal l i ver -der ived erythroid and m y e l o i d progenitors, despite the fact that there is no clear defect in absolute numbers of mature cel ls in the hematopoiet ic system of adult animals . A l t h o u g h this phenotype cou ld also be expla ined by decreased surv ival of progenitors or a decl ine in their retention in the bone marrow, the fact that there is no increase in these ce l ls in the per iphery, and the observat ion that a l though progenitors surv ive in vitro, there is l itt le expansion in compar ison to w i ld t ype -de r i ved ce l l s , both favour the init ial idea. 25 wild type CD34 knockout B IL-6 Myeloblast • t Macrophage | 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 C D 3 4 - d e p e n d e n t b l o c k in d i f fe ren t ia t ion is supported by one set o f in vitro overexpress ion exper iments ( F a c k l e r et a l . , 1995). T h e C D 3 4 + mur ine m y e l o b l a s t s leukemia ce l l l ine , M l can be induced to terminal ly differentiate into macrophages upon treatment w i th in te r leuk in -6 ( I L -6 ) or leukemia inhib i tory factor ( L IF ) , at w h i c h point C D 3 4 is downregulated (Suda et a l . , 1992). W i t h i n 24 hours o f L I F or I L - 6 treatment, there is a m a x i m a l reduct ion in C D 3 4 m R N A levels , f o l l o w e d by d i f ferent iat ion into morpho log ica l l y mature, funct ional ly active macrophages wi th in three days. W h e n C D 3 4 is e c t o p i c a l l y expressed in M l c e l l s , it appears to b l o c k I L - 6 and L I F i n d u c e d di f ferent iat ion: cel ls are morpho log ica l l y immature as they display m i n i m a l vacuolat ion , a h igh nucleus to cytop lasm ratio, open chromat in , and prominent nuc leo l i , and they are v i r tua l l y non -phagocy t i c (F igure 1 -4B) (Fack le r et a l . , 1995). N o t a b l y , the natural ly occur r ing spl ice variant of C D 3 4 is incapable of b l o c k i n g di f ferent iat ion. Thus , there is evidence to suggest that C D 3 4 may be invo lved in b lock ing differentiat ion or enhancing progenitor prol i ferat ion. H o w e v e r , there is also mount ing evidence to indicate that C D 3 4 f a m i l y proteins are not invo lved in enhancing prol i feration or b lock ing differentiat ion. Regard ing the C D 3 4 - n u l l phenotype, there is a second strain of mice lack ing C D 3 4 , and these mice do not display detectable defects in progenitor cel l populations (Suzuki et a l . , 1996). Total bone marrow ce l l number and relative ratios of T e r l l 9 , B 2 2 0 , M a c - 1 , G r - 1 , C D 3 , c - K i t , and S c a - 1 expressing cel ls are al l normal , as are numbers of platelets, red b lood cel ls , white b lood ce l l s , and white b lood ce l l subpopulat ions in peripheral b lood . Important ly , culture of bone marrow cel ls w i th stem cel l factor ( S C F ) , I L - 3 , and erythropoietin in vitro produces 27 normal numbers of progenitors, as measured by co lony assays. Furthermore, when mast cel ls (the only mature C D 3 4 expressing hematopoiet ic cel ls) f r o m these mice are cultured in vitro, there is no difference in kinetics of prol i ferat ion, di f ferent iat ion, or degranulation (Drew et a l . , 2 0 0 2 ; D rew et a l . , 2005) . M o r e o v e r , in contrast to the overexpress ion of C D 3 4 in M l cel ls , ectopic expression in two other cel l l ines fa i ls to b lock differentiat ion (Fackler et a l . , 1995). A l s o wi th respect to the M l exper iments, it should be kept in m i n d that two characteristics used to measure differentiat ion were adhesion and phagocytosis , w h i c h cou ld both potent ial ly be affected 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 ie lsen et a l . , 2002) (discussed be low) . T h u s , it is un l ike l y that C D 3 4 f a m i l y members play an important role in enhanc ing pro l i ferat ion or b l o c k i n g d i f ferent iat ion. Instead, the observed phenotypes may be the result of other C D 3 4 - r e l a t e d funct ions. 1.5.2 Pro -Adhes ion There is conv inc ing evidence showing that C D 3 4 f a m i l y members can act in an adhesive manner for recruitment o f l ymphocytes . Leukocy tes are constant ly recruited f r o m the b lood into secondary l y m p h o i d organs and sites o f chronic i n f l a m m a t i o n in a mult i -s tep process i n v o l v i n g low aff ini ty b ind ing (known as ro l l ing) f o l l o w e d by integr in -mediated f i r m arrest and transendothelial migrat ion (reviewed in (Butcher and P icker , 1996; L a s k y , 1992)). T h e init ial step, wh ich requires adhesion of leukocytes to endothel ial cel ls under condit ions of vascular b lood f l o w , depends upon associat ion o f selectin molecu les wi th their l igands. Recrui tment of l ymphocytes into peripheral l y m p h nodes requires b ind ing o f L - s e l e c t i n on l ymphocy tes to par t icu lar carbohydrate m o d i f i c a t i o n s on proteins expressed on the specia l i zed postcapi l lary venules k n o w n as h igh endothel ia l venules 28 ( H E V ) . These sulfated and s ialy lated O - l i n k e d carbohydrates are presented to L -se lect in by m u c i n - l i k e g lycoprote ins , i n c l u d i n g g lycosy lat ion-dependent cel l adhesion molecule ( G l y C A M ) - l , mucosal addressin ce l l adhesion molecule ( M A d C A M ) - l , and potential ly endomuc in ( (Samulowi tz et a l . , 2002) , and reviewed 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 endog lycan can a l l act in a pro -adhes ive manner as l igands for L -se lect in on H E V (Figure 1-5). The role of C D 3 4 as an L -se lect in l igand was f irst demonstrated in 1993, when it was shown 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 (Baumhueter et a l . , 1993) . S i m i l a r l y , podoca lyx in is also mod i f i ed with the appropriate glycosylat ions for L -se lect in b inding in H E V , and in vitro it supports L -se lec t in dependent tethering and ro l l ing of l ymphocytes under condi t ions of f l o w (Sassetti et a l . , 1998). Endog lycan can also act as a l igand for L -se lect in , i f expressed in con junct ion w i th the necessary enzymes for appropriate post-t ranslat ional m o d i f i c a t i o n (F ieger et a l . , 2003) . E n d o g l y c a n b i n d i n g does, however , invo lve alternative g lycosy lat ions f r o m those present in C D 3 4 and podoca lyx in . Thus , al l three molecules can funct ion as adhesive l igands for L -se lect in . 29 f CD34 e (-) charge HEV-specific glycosylation L-selectin Figure 1-5: Proposed Function for CD34 Family: L-selectin Mediated Adhesion. CD34 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 ev idence to support an adhesive func t ion fo 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 un iversa l ro le . O p t i m a l recogn i t ion o f g l y c o p r o t e i n s by L - s e l e c t i n requires exqu is i te l y s p e c i f i c m o d i f i c a t i o n s , i n c l u d i n g s ia l y la t ion , fucosy la t ion , and sul fat ion (Rosen et a l . , 1997). In the case of C D 3 4 and p o d o c a l y x i n , the epitope recognized by L -se lect in is termed M E C A - 7 9 ; this epitope is not found on C D 3 4 or podoca lyx in expressed on other vascular endothel ia l cel ls or on p o d o c a l y x i n on g lomeru lar podocytes (Baumhueter et a l . , 1993 ; M i c h i e et a l . , 1993; Sassett i et a l . , 1998; S e g a w a et a l . , 1997). M o r e o v e r , I have demonst rated that podoca lyx in and C D 3 4 expressed on fetal l i ver ce l ls do not interact wi th L - s e l e c t i n , or any other c o m m o n selectin molecules (Doyonnas et a l . , 2005) . O f note, it has been shown that t w o sulfotransferases that can m o d i f y C D 3 4 have d is t inct express ion patterns ( B i s t r u p et a l . , 1999). In vitro exp ress ion o f these t w o e n z y m e s , a l o n g w i t h a fucosyltransferase, facil itates modi f icat ion of C D 3 4 for L -se lect in recognit ion (Bist rup et a l . , 1999). A b s e n c e of ind iv idua l sulfotransferases does not prevent product ion of L -selectin l igands, but when both enzymes are present, they synergize to produce a l igand with much greater aff in i ty for L - se lec t in . Importantly , w h i l e the Ga l -6 -su l fo t ransferase displays a w ide tissue distr ibut ion, 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 igh ly restricted to h igh endothel ial cel ls . Thus , the expression pattern of this , or other m o d i f y i n g enzymes, may exp la in the interaction of L -se lect in wi th C D 3 4 f a m i l y members on ly in H E V . T h e question, then, is what the alternative funct ion is for these s ia lomucins in other tissues. 31 1.5.3 Anti-Adhesion A proposed podoca lyx in funct ion that is gaining 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 adhesion molecu les , such as integrins, as we l l as anti -adhesins. Ant i -adhes ins are often cel l surface associated muc ins (Wesse l ing et a l . , 1996). M u c i n s are heav i l y s ia ly lated, O - l i n k e d g lycoprote ins that can block adhesion either by steric hindrance or by charge repuls ion, both properties conveyed by their b u l k y , negatively charged extracel lu lar domains . S ince p o d o c a l y x i n and the related f a m i l y members have this type o f extracel lular d o m a i n , they have been proposed to act in this capacity (Figure 1-6). S ia lomuc ins are often found on the lumina l surface of vessels where they prevent b lockage of the lumen caused by weak , n o n -speci f ic interaction of molecules on opposing lumina l membranes (H i lkens et a l . , 1992). S i m i l a r l y , expression by mal ignant tumours can decrease adhesion and prevent immune recogn i t ion . P o d o c a l y x i n express ion on c i r c u l a t i n g platelets may a lso prevent their inappropriate adhesion to vessel wal ls ( M c N a g n y et a l . , 1997). Thus , it seems plausible that the C D 3 4 f a m i l y may have global anti -adhesive funct ions. 32 ttt ttt , t t t f podocalyxin e I (-) charge integrin ligand F i g u r e 1-6: P r o p o s e d F u n c t i o n f o r CD34 F a m i l y : B l o c k i n g A d h e s i o n . 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 CD34 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 (TER) 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). Of interest, RhoGDIa ' 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 f32-adrenergic receptors and 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 (HGEC) , 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 CD34 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-p1, integrin antibodies blocks adhesion. 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 block podocalyxin's anti-adhesive effects, and adhesion increases. 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 CD34 signalling (Cheng et al., 1996), but capping of CD34 may expose integrins and provide an alternative explanation for the increase in adhesion. Most experiments involving overexpression of CD34 family members therefore suggest an anti-adhesive function for these proteins. Expression of CD34 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 CD34 knockout animals demonstrates that CD34 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 CD34 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, CD43, is a much more effective anti-adhesin: loss of CD34 leads to aggregation of 16 ± 7 % of cells, while 70 ± 20 % of CD43-null cells form aggregates. 37 Further ev idence for the ant i -adhes ive effects of p o d o c a l y x i n and C D 3 4 have been prov ided by in vivo repopulat ion studies. In the first set of exper iments , short - term homing assays were performed with wi ldtype , podoca l yx in -nu l l , C D 3 4 - n u l l , and double -def ic ient fetal l iver cel ls (Doyonnas et a l . , 2005) . S ingle knockout cel ls are less eff icient at h o m i n g to bone marrow, and cel ls lack ing both molecules display an addit ive defect. A l t h o u g h this cou ld be expla ined by a loss of spec i f ic homing molecu les , there is no decrease in b inding to the only known receptors for these molecules , the selectins. Thus, it is more l ike ly that the loss of ant i -adhesion molecules results in non-spec i f ic adhesion to endothelial cells of vessels en route to the bone marrow. T h e second experiment invo lved repopulation of the peritoneal cavity by mast cel ls after their ablat ion by inject ion of water into wi ld type , C D 3 4 - n u l l , C D 4 3 - n u I I , and double -def ic ient mice (Drew et a l . , 2005) . Doub le -de f i c ien t mice d isplay delayed mast cel l repopulation kinetics. W h i l e ind iv idual loss of C D 3 4 also appears to delay repopulation, the results are not stat ist ical ly s igni f icant . In another system, w i ld type and C D 3 4 - or C D 4 3 - d e f i c i e n t bone marrow cel ls were injected into sub- lethal ly i rradiated mast cel l de f i c ient W / W v m i c e in c o m p e t i t i v e r e p o p u l a t i o n e x p e r i m e n t s , and mast c e l l reconstitut ion was assessed after 11 -12 weeks. Notab ly , almost a l l mast cel ls in mice injected w i t h a combinat ion of w i ld type and double -def ic ient ce l l s are der ived f r o m wi ldtype bone marrow cel ls . A g a i n , although the trend is the same with single knockout ce l ls , the results are not statist ical ly s ignif icant . S t r ik ing ly , in the same mice , there is a s ign i f i cant decrease in C D 3 4 - d e f i c i e n t hematopoiet ic progeni tor ce l l engraftment, whereas C D 4 3 ' cel ls engraft normal ly . A g a i n , loss of C D 3 4 may lead to increased non-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 CD34 expression is downregulated upon IL-1 induced upregulation of the adhesion molecules ICAM-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 CD34 may inhibit adhesive functions of vascular endothelial cells, and downregulation (Delia et al., 1993) or relocalization may enable adhesion when necessary. As discussed, there is therefore mounting evidence supporting an anti-adhesive 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 of ce l ls ( M e d e r et a l . , 2005) . A l t h o u g h loca l i za t ion o f numerous ap ica l and basolateral markers was assessed, al l proteins d isplay un i fo rm expression patterns wi th the exception of E -cadher in , wh ich is enriched at ce l l - ce l l contact sites, and g p l 3 5 , w h i c h is ap ica l l y loca l i zed on s ingle cel ls wi th in one hour. A t the t ime, the identity of g p l 3 5 was unknown, but its unique expression exc lus ive ly on the free surface of cel ls prompted further study; as d iscussed in section 1.2, it is now k n o w n to be canine p o d o c a l y x i n (Meder et a l . , 2005) . The early polar ized distr ibution of podoca lyx in , in contrast to other typ ical markers , suggests that it may play a role in this process. T h i s was investigated further by deplet ing endogenous podoca lyx in expression by R N A interference ( R N A i ) . Po la r i za t ion , as assessed by marker distr ibut ion on M D C K monolayers , was delayed in cel ls lack ing podocalyx in (Meder et a l . , 2005). A model often used to assess polar izat ion in M D C K cel ls is that o f cyst fo rmat ion in a co l lagen matr ix . In this assay, s ingle cel ls grow and f o r m mul t ice l lu lar , po lar ized cysts each wi th a central lumen. S t r i k i n g l y , many podoca lyx in -dep le ted M D C K cel ls f o r m cysts wi th mul t ip le lumens, or cysts complete ly devo id of lumens ( M e d e r et a l . , 2005) . A l t h o u g h the 2005 publ icat ion by M e d e r et al (Meder et a l . , 2005) was the first to suggest a role for podocalyx in in establ ishing polarity , apical expression of g p l 35 (later identif ied as podoca lyx in ) was noted in M D C K cel ls l a c k i n g ce l l junct ions in a paper publ ished over a decade earl ier (O jakian and S c h w i m m e r , 1988). In addi t ion , removal of surface expressed g p l 3 5 by t rypsin izat ion is f o l l o w e d by rapid reinsertion of podoca lyx in into the p lasma membrane, but only at the apical surface (O jak ian et a l . , 1990). Thus , recent 40 results suggest the exc i t ing poss ib i l i t y that p o d o c a l y x i n in invo l ved in regulat ing ce l l polarity . T h e same group addressed 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 c e l l po la r i za t ion ( M e d e r et a l . , 2005) . S ince 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 isp lays s l ight ly less restricted l o c a l i z a t i o n , it was hypothes ized that N H E R F proteins cou ld also play a role in ear ly ce l l po la r i za t ion . M D C K cel ls were transfected w i th a green f luorescent protein ( G F P ) - t a g g e d N H E R F 2 fus ion protein in order to f o l l o w N H E R F 2 loca l i za t ion dur ing M D C K ce l l po lar i zat ion . L o c a l i z a t i o n of this protein is s imi la r to that of endogenous p o d o c a l y x i n throughout a l l stages o f early polar izat ion, and the two proteins f o r m a co - immunoprec ip i tab le complex that appears to strengthen as cel ls become more polar ized. S ince N H E R F proteins contain tandem P D Z domains w i th d i f fe r ing b i n d i n g spec i f ic i t ies , they are able to d imer i ze and c r o s s - l i n k mul t ip le l igands (Shenol ikar et a l . , 2004). In the case of epithel ia l po lar i zat ion , perhaps N H E R F proteins can l ink mul t ip le l igands wi th the E R M f a m i l y of cytoskeletal adapters in order to establ ish a pre -apica l membrane scaf fo ld for d i rect ing junct ion format ion and membrane t raf f ick ing (Meder 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 of podocalyx in in asymmetr ic cel l d i v i s ion is based on very l i t t le ev idence , but it is an exc i t ing poss ib i l i t y wor th cons ide r ing . A s y m m e t r i c ce l l d i v i s i o n has recently gained much attention in the hematopoiet ic system, as stem cel ls must d iv ide in this manner in order to produce daughter cel ls wi th d i f fe r ing properties. 41 Maintenance o f the stem ce l l pool requires that one daughter ce l l remain a stem c e l l , wh i le the other begins to populate the hematopoiet ic system by virtue of its increased progress a long the differentiat ion pathway (reviewed in (Ho , 2 0 0 5 ; W i l s o n and T r u m p p , 2006)) . There are two models that address the mechanisms under ly ing asymmetr ic d i v i s ion . The first suggests that external st imul i f o l l o w i n g cel l d i v i s ion provide cues that determine cel l fate. T h i s is based on the idea of a stem cel l niche and d i v i s ion a long a plane in w h i c h one daughter ce l l remains in contact wi th the niche, and therefore remains an H S C whi le the other does not. T h e second 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 redistr ibuted unequal ly before d i v i s i o n , such that those required for m a i n t a i n i n g an immature c e l l are on one side w h i l e determinants of a more mature ce l l are on the opposite side. T h e expression of podoca lyx in in early hematopoietic progenitor ce l ls , but perhaps not in the most immature hematopoiet ic stem cel ls , and its associat ion wi th the actin cytoskeleton and the free surface of cel ls suggests that it cou ld play a role in either situation (F igure 1-7). In the f irst case, podoca lyx in ' s association wi th the free surface o f ce l l s may enable an immature H S C to adhere to the niche v i a adhes ion molecu les segregated f r o m the p o d o c a l y x i n - c o n t a i n i n g d o m a i n . T h e n , after d i v i s i o n , the podoca lyx in - conta in ing cel l w i l l be destined to become more mature, w h i l e the adherent c e l l w i l l remain in contact w i th the n iche , thereby rece iv ing the s ignals required for ma in ta in ing the immature state. O n the other hand, perhaps p o d o c a l y x i n ' s interact ion w i th N H E R F 1 faci l i tates segregation of a mult i tude of fate determining proteins on one side of the ce l l before cel l d i v i s ion . The fact that podoca lyx in and N H E R F 1 co loca l i ze to 42 one side of ce l ls upon I L - 3 st imulat ion of a hematopoiet ic progenitor cel l l ine provides some evidence for re local izat ion prior to cel l d i v i s ion (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 Niche-derived molecules factors N H E R F 1 Cell-fate determinants F i g u r e 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 . (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 endoglycan are related s ia lomucins w i th over lapp ing expression patterns and l i ke l y over lapp ing funct ions. A l t h o u g h a role in enhanc ing pro l i ferat ion or b l o c k i n g d i f ferent iat ion was in i t ia l l y p roposed, there is more ev idence in support o f alternative funct ions. They can al l act as adhesive tethers for L - se lec t in in H E V but are not appropriately mod i f i ed for this funct ion in other tissues. G l o b a l l y , it is more l i ke l y that p o d o c a l y x i n and C D 3 4 , at least, act as b lockers of adhes ion . In add i t ion , there is recent ev idence to suggest that podoca lyx in may regulate ce l l polar i ty , and it is possible that the C D 3 4 f a m i l y cou ld also be invo l ved in regulat ion of asymmetr i c ce l l d i v i s i o n . These funct ions a l l rely on a l ink to the act in 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 establ ished to occur v ia interact ions w i t h ezr in and N H E R F proteins. 1.6 Podocalyxin in Kidney Development W h i l e much informat ion can be gained through overexpression exper iments and in vitro a n a l y s i s , it is a lso necessary to invest igate 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 prote in 's expression in tissues where it is normal ly found. In the case o f p o d o c a l y x i n , it was first ident i f ied in the g lomerulus o f rat k idney , and its expression in this tissue is not only abundant, but also essential for podocyte development (Doyonnas et a l . , 2 0 0 1 ; Ker jaschki et a l . , 1984). 45 1.6.1 Overview of Glomerular Development G l o m e r u l i are responsible for b lood f i l t rat ion and urine product ion in the kidney (Gao et a l . , 2004) . They are composed of capi l lary loops l ined with fenestrated endothel ial ce l l s , support ing mesangial ce l l s , a g lomerular basement membrane ( G B M ) , and g lomeru lar epi thel ia l ce l ls ca l led podocytes (Gao et a l . , 2004) . W h i l e vascular endothel ia l ce l l s prov ide the f i rst barrier in product ion of the g lomeru lar f i l t rate, endothel ia l fenestrae provide an exit route for smal l molecules (Gelberg et a l . , 1996). T h e G B M consists of a ne twork of c o l l a g e n I V , l a m i n i n , heparan sul fate p ro teog l ycans , and f i b r o n e c t i n ( E c o n o m o u et a l . , 2 0 0 4 ) ; its s i ze - and charge -se lect iv i ty p rov ide the main select ive barrier enabl ing retention of macromolecu les in the c i rcu lat ion ( rev iewed in (Farquhar, 1975; K a n w a r , 1984)). Podocytes have a unique architecture wi th three main parts. The cel l body contains the majority of the ce l l ' s cy top lasmic organelles, inc lud ing the nucleus, mi tochondr ia , rough and smooth endop lasmic re t i cu lum, 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 in f o r m a t i o n and degradat ion o f the G B M c o m p o n e n t s ( T a k e d a , 2 0 0 3 ) . Project ions, termed major processes, extend f r o m the cel l body and surround the capi l lary loops (Andrews , 1979). Smal le r projections, cal led foot processes, extend f r o m the major processes, interdigitate wi th foot processes f r o m neighbour ing podocytes, and connect the basal surface of podocytes to the G B M , m a i n l y through a 3(3, in tegr in -mediated foca l contacts (Ad ler , 1992; A n d r e w s , 1979). Foot processes lack cy top lasmic organel les, and instead contain a dense network o f act in f i l aments , w h i c h s tabi l i ze the foot process structure through adhesion to the G B M and by f o r m i n g connect ions wi th proteins of the 46 sl i t d iaphragm c o m p l e x (F igure 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 iaphragms are tenuous br idges between adjacent foot processes through w h i c h the g lomerular fi ltrate must pass (Kur ihara et a l . , 1992). Recent evidence suggests that, a long w i t h the G B M , sl it d iaphragms also play a role in f i l t rat ion ( rev iewed in (Salant and T o p h a m , 2 0 0 3 ; T r y g g v a s o n , 1999)). The apical surface of podocytes , w h i c h faces the ur inary space, is coated by a s ia l i c a c i d - r i c h g l y c o c a l y x designated the ep i the l ia l p o l y a n i o n , and now k n o w n to be main ly composed of podoca l yx in (Ker jaschk i et a l . , 1984; M i c h a e l et a l . , 1970). 47 GBM Figure 1-8: Schematic of Molecules Related to Podocyte Architecture. T h i s d i a g r a m shows t w o adjacent podocyte foot processes b r idged by a c o m p l e x o f proteins that f o r m the sl it d iaphragm. Proteins f o u n d i n the basal d o m a i n , such as a 3 p , in tegr in ( a 3 p , ) , interact w i t h the ac t in c y t o s k e l e t o n and l i n k p o d o c y t e s w i t h the g l o m e r u l a r b a s e m e n t m e m b r a n e ( G B M ) . P o d o c a l y x i n in teracts w i t h the ac t in cytoskeleton through N H E R F 2 and ez r in , and molecu les o f the slit d iaphragm c o m p l e x a lso interact w i t h the cytoskeleton . Other abbrev iat ions inc lude ct -act4: a - a c t i n i n - 4 , a -D G : a - d y s t r o g l y c a n , | 3 - D G : | 3 -dys t rog lycan , P: p a x i l l i n , P - c a d : P - c a d h e r i n , S y n p o : synaptopodin , T : ta l in , and V : v i n c u l i n . Reproduced w i t h k i n d permiss ion 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 Shank land , 2002) . 48 D u r i n g g lomeru lar development , w h i c h proceeds v ia a series o f loosely def ined stages, m e s e n c h y m a l ce l l s are induced to condense and undergo mesenchymal to ep i the l ia l transit ion ( G u o et a l . , 2002) . They in i t ia l l y f o r m a cluster of cel ls k n o w n as the renal ves ic le ( G u o et a l . , 2 0 0 2 ; Reeves et a l . , 1978). T h e renal ves ic le is invag inated by mesenchymal ce l l s , and the cluster of cel ls present at this stage rearranges and matures v ia the c o m m a - and S -shaped body stages to f o r m a central lumen, 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 -shaped body stage, mesangial ce l ls deve lop f r o m the mesenchymal cleft , wh i le the cel ls on one side of the cleft begin to f o r m the glomerular epi thel ium and those on the other side differentiate to f o r m the p rox imal tubule and capi l lary endothel ium (Reeves et a l . , 1978). T h e glomerular ep i the l ia l ce l l s in i t ia l l y have tight junct ions at their ap ices , but upon express ion of podoca lyx in the junct ions begin to migrate laterally toward the G B M . Throughout the next stage, k n o w n as the deve lop ing capi l la ry loop stage, g lomeru la r epithel ia l ce l ls (podocytes) prol i ferate, and movement of the c e l l - c e l l junct ions enlarges the in t race l lu la r spaces, w h i c h are cont inuous w i th B o w m a n ' s space (Reeves et a l . , 1978). Extens ive morpholog ica l rearrangements occur whereby foot processes extend and j u n c t i o n s redist r ibute between foot processes. S l i t d iaphragms later replace these junct ions (Reeves et a l . , 1978; Schnabel et a l . , 1989). Podoca l yx in redistr ibution f o l l o w s that of the junct ions : it is a lways found a long the apical surface of podocyte ce l l bodies and foot processes above the leve l of the slit d iaphragms ( K e r j a s c h k i et a l . , 1984; K u r i h a r a et a l . , 1992; Schnabel et a l . , 1989). The matur ing g lomeru lus stage invo lves further maturat ion of podocytes, maturation of the G B M into .a c o m p l e x structure with 4 9 several layers, and fenestration of endothel ial ce l ls to f o r m the f ina l g lomerular structure (Reeves e t a l . , 1978). 1.6.2 Glomerular Diseases and Model Systems Maintenance of the intricate architecture of g lomerular podocytes is essential for opt imal kidney funct ion ; numerous human diseases and an imal models o f g lomerular mal funct ion invo l ve d isrupt ion of these unique structures ( E c o n o m o u et a l . , 2004) . L o s s of foot 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 wi th nephrotic syndrome (Farquhar et a l . , 1957). D iabet i c nephropathy, a leading cause of chron ic k idney fa i lure and end-stage renal d isease, also invo l ves broadening of foot processes and is accompanied by a decrease in g lomerular s ia l ic ac id content (Cardenas e t a l . , 1991; Pagtalunan et a l . , 1997). P u r o m y c i n aminonuc leos ide ( P A N ) nephrosis and protamine sulfate (PS) perfusion are two rodent models o f g lomerular disease (Farquhar and Pa lade , 1961; Kur iha ra et a l . , 1992). S i a l y l a t i o n of p o d o c a l y x i n is reduced in P A N nephros is , and PS neutral izes podoca lyx in ' s negative charge (Ker jaschk i et a l . , 1985; Sei ler et a l . , 1977). Thus , in both models , the negative charge on p o d o c a l y x i n is decreased, foot process architecture is d isrupted, f i l t rat ion slits are reduced in number , and sl it d iaphragms are d isplaced or comple te l y replaced by l e a k y , d i scont inuous t ight j u n c t i o n s ; o v e r a l l , the ep i the l ium resembles that of immature g lomeru l i (Gao et a l . , 2 0 0 4 ; K u r i h a r a et a l . , 1992). Injection of sial idase intraperitoneally causes s imi la r morpholog ica l alterations as a result of loss of s ial ic acid residues f r o m the surface of podocytes (Ge lberg et a l . , 1996). Features of these 50 models inc lude decreased g lomerular f i l t rat ion and a perhaps counterintuit ive increase in protein in the urine (proteinuria) (Bohrer et a l . , 1977; Kur ihara et a l . , 1992). Reduct ion in g lomerular charge select iv i ty results in an increase in G B M permeabi l i ty and subsequent proteinuria. H o w e v e r , there is an overal l decl ine in glomerular f i l t rat ion as the col lect ive s l i t pore area, and thereby the exit path for f i l t rate , decreases (Bohre r et a l . , 1977 ; K u r i h a r a et a l . , 1992). B l o c k a g e of the exit path may also lead to a reactive elevat ion in g lomeru la r b lood pressure and a subsequent dramat ic increase in permeabi l i t y of the g l o m e r u l a r f i l t e r ( G e l b e r g et a l . , 1996) . P o d o c y t e s , their ex tens ive express ion o f p o d o c a l y x i n , and alterations in disease states have therefore been the focus of m u c h research for 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 ident i f ied unti l 1984, the importance of the "ep i the l ia l p o l y a n i o n " in maintenance o f podocyte m o r p h o l o g y was noted more than ten years earl ier (Ker jaschk i et a l . , 1984; M i c h a e l et a l . , 1970). A reduction in foot process number in associat ion wi th loss of the epithel ial po lyanion was first proposed in 1970 ( M i c h a e l et a l . , 1970). T h i s was conf i rmed by experiments in w h i c h rat k idneys were perfused wi th po lycat ions to neutral ize the epithel ia l po l yan ion . T h i s leads to d istort ion of podocyte m o r p h o l o g y : s l i t pores are narrower or absent and foot processes are fused together (Sei ler et a l . , 1977). In contrast, perfusion with po lyanions and neutral molecu les has no effect, a l though perfusion of an ion ic molecu les after perfus ion of cat ion ic molecu les part ial ly reverses their disruptive effects. The phenotypes observed upon cation perfusion are analogous to those observed in proteinuric condi t ions, where podocyte morphology is 51 s i m i l a r to that observed in immature g l o m e r u l i (Se i ler et a l . , 1977). T h u s , it is not surpr is ing that appearance of the epithel ial po lyan ion is associated with extension o f foot processes dur ing development : it was demonstrated in 1978 by Reeves et al that the epithel ial po lyan ion , as detected by co l lo ida l i ron staining, f irst appears on the surface of g lomerular epithel ial cel ls just before format ion of foot processes and slit pores (Reeves e t a l . , 1978). A major contr ibutor to the negative charge on the epithel ial po lyan ion is its h igh s ia l ic ac id content ( M i c h a e l et a l . , 1970; M o h o s and S k o z a , 1969). T h i s was demonstrated by the observed reduct ion in c o l l o i d a l i ron s ta in ing of podocytes after treatment w i th neuramin idase , w h i c h removes s ia l i c a c i d residues. S i m i l a r to the effects seen wi th po l yca t ion per fus ion , neuraminidase treatment also disrupts foot process architecture ( A n d r e w s , 1979). Foot processes fuse together, s l i t d iaphragms 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 du r ing deve lopment , reappear. Fur thermore , it has been suggested that the defects observed in P A N models o f nephrosis m a y be caused by interference wi th s ia l ic ac id metabo l i sm in podocytes and a corresponding loss of the sial ic acid content in the g lycocalyx . It was at this stage that the molecu lar identity of the epi thel ia l po l yan ion was f ina l l y determined (Ker jaschk i et a l . , 1984). Several characteristics of the epithel ia l po lyan ion had p rev ious l y been demonstrated, and these features, a long w i t h it be ing the most abundant membrane g lycoprote in in the g lomeru lus , enabled its i so lat ion . F i rs t l y , it is h igh ly negatively charged, as demonstrated by its recognit ion by cat ionic h is tochemical 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). Secondly , neuraminidase treatment alters its s ta in ing propert ies, thereby demonstrat ing its extensive s ia l i c ac id content. T h i r d l y , it is recognized by wheat germ agglut in in before neuraminidase treatment and peanut agglut inin afterwards (Holthofer et a l . , 1981). U s i n g these characterist ics, a single 140 k D a prote in , composed of approx imate ly 20 % hexose and 4.5 % s ia l i c ac id by weight , was iso lated, character ized, and named podoca l yx in (Ker jaschk i et a l . , 1984). T h e s ia l ic acid r ich nature of podocalyx in can be fu l l y appreciated when it is real ized that it contains the majority of the protein-bound s ia l ic acid of the g lomerulus . T h e presence o f O - l i n k e d g l ycosy la t ions was i m p l i e d based on the b ind ing of peanut lect in after neuraminidase treatment; the s imultaneous presence of some N - l i n k e d o l igosacchar ide chains was also suggested by the b ind ing of concanaval in A . Cons iderable analys is has been undertaken to begin to character ize the o l igosacchar ide chains of p o d o c a l y x i n ' s m u c i n - l i k e domain , inc lud ing their s ialylat ions and sulfations (Dekan et a l . , 1991). M u c i n domains are predicted to be r i g id , extended structures, so presumably p o d o c a l y x i n ' s ext racel lu lar d o m a i n funct ions as an attachment site fo r the many negat ively charged moiet ies that contribute to the anionic g l ycoca lyx of podocytes ( (Kershaw et a l . , 1997a), and r e v i e w e d in (Jentoft , 1990)) . T h u s , 15 years after detect ion of the ep i the l ia l po lyan ion , its molecular nature was determined. T h e ident i f i ca t ion o f p o d o c a l y x i n and generation of spec i f i c m o n o c l o n a l ant ibodies fac i l i tated a detai led assessment of its expression pattern (Ker jaschk i et a l . , 1984). In adult, podoca lyx in is h ighly expressed on the apical surface of podocytes and at 5 - 1 0 fo ld lower levels on endothel ial cel ls (Dekan et a l . , 1990; Ker jaschk i et a l . , 1984). It covers al l 53 surfaces of podocytes fac ing the urinary space, inc lud ing the apical surface of cel l bodies and foot processes above the leve l of sl it d iaphragms; it is not expressed on the basal surface of podocytes or on the abluminal face of endothelial cel ls (Ker jaschki et a l . , 1984; S a w a d a et a l . , 1986) . T h e s e deta i led studies a lso p r o v i d e d some ins ights into podoca lyx in ' s funct ion. It had previously been proposed that the purpose of its prominent negat ive charge was to p rov ide a charge -se lect i ve barr ier fo r g lomeru la r f i l t r a t i o n . H o w e v e r , the main barrier per forming this funct ion is located at the level of the G B M , where p o d o c a l y x i n express ion is l a c k i n g , so it is instead postulated to be i n v o l v e d in maintenance of structural integrity of foot processes (Farquhar, 1975; Ker jaschk i et a l . , 1984; Sawada e t a l . , 1986). P o d o c a l y x i n express ion and loca l i za t ion dur ing deve lopment has also been f o l l o w e d carefu l l y in rat (Schnabel et a l . , 1989). Rodent k idneys prov ide an excel lent mode l for s tudy ing k idney development for two reasons. F i rst , throughout development new renal vesic les are cont inual ly fo rmed at the periphery of the k idney cortex, w i th more mature nephrons near the cor t ica l -medul lary junct ion , so al l stages of maturation are v is ib le in a s ingle k idney . A n d second, this process is not completed unti l one week after birth in rodents, in contrast to the situation in humans, so g lomerular development can easi ly be investigated in newborn rodents (Reeves et a l . , 1978). The earliest detectable podocalyx in is expressed on cap i l la ry endothel ia l ce l ls in the mesenchymal t issue sur rounding the renal ves ic le (Schnabel et a l . , 1989). Subsequent ly , the f irst podoca l yx in expression on ce l ls that eventual ly become podocytes is detected as the p r e - B o w m a n ' s space lumen 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 ap ica l surface o f these g lomeru la r 54 epithel ial ce l l s , but it then moves a long the lateral surface toward the basal surface as c e l l - c e l l junct ions migrate in a s imi la r fash ion . P o d o c a l y x i n is a lways expressed on the entire apical surface and a long the lateral membrane to the posit ion immediate ly above tight junct ions . Its express ion extends a long the tops o f foot processes, but it is never expressed on the basal surface fac ing the G B M . U p o n replacement o f tight junct ions with slit d iaphragms, p o d o c a l y x i n express ion is mainta ined on the entire podocyte surface above this l e v e l . M o r e o v e r , in immature podocytes p o d o c a l y x i n is detected in the endoplasmic ret icu lum, G o l g i apparatus, and carrier vesicles o f the biosynthesis pathway demonstrating that it is synthesized at a h igh rate in these cel ls . S ince neutra l izat ion o f the epi the l ia l p o l y a n i o n (podoca l yx in ) leads to alterat ions in podocyte m o r p h o l o g y , d isease m o d e l s were used to ga in fur ther ins ights into the mechanisms i n v o l v e d . F o r example , in P A N nephros is , act in f i lament reorganizat ion occurs (Whi tes ide et a l . , 1993); s ince 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 ezr in 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 . , 2001) , c o -immunoprec ip i tat ion and sequential detergent extract ion experiments were performed in order to investigate potential disruptions of this complex in disease states (Takeda et a l . , 2 0 0 1 ) . In P A N n e p h r o s i s , the re is l i t t l e , i f a n y , d i s r u p t i o n o f 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 overa l l amount o f phosphory lated (active) ezr in decreases and the entire c o m p l e x dissociates f r o m the act in cy toske leton . U p o n protamine sulfate perfusion, N H E R F 2 and ezr in remain bound to act in, but podoca lyx in dissociates f r o m the c o m p l e x . S ia l idase treatment leads to d issoc iat ion of podoca l yx in 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 act in. T h u s , in a l l three 55 models , the associat ion of podoca lyx in wi th the actin cytoskeleton is disrupted, p rov id ing a possible explanat ion for the morpho log ica l changes noted in podocyte foot processes. H o w exactly an alteration in the surface charge of podoca lyx in affects the complex is not k n o w n , but it may be that neutral ization of the charge induces a conformational change in the cy top lasmic tail of podoca lyx in wh ich prevents b inding. A l l of the early studies of the epithel ia l po lyanion and podoca lyx in in the kidney suggest a very important role for this mo lecu le in k idney deve lopment . Generat ion of p o d o c a l y x i n - n u l l mice demonstrated exactly how important this molecule is for glomerular development. 1.6.4 Lessons from the Podocalyxin Knockout P o d o c a l y x i n knockout mice were generated by homologous recombinat ion in w h i c h the major i t y o f exons f i v e , s i x , seven, and eight were de leted ; loss of express ion was conf i rmed by Northern blot (Doyonnas et a l . , 2001) . S ince C D 3 4 - n u l l mice do not have any major defects in tissues that normal ly co-express podoca lyx in and C D 3 4 (Cheng et a l . , 1996; S u z u k i et a l . , 1996) , abnormal i t ies were not expected in these t issues in p o d o c a l y x i n - n u l l mice either. Instead, cel ls that express on ly p o d o c a l y x i n , part icu lar ly podocytes, were expected to have more obvious defects (Doyonnas et a l . , 2001) . 1.6.4.1 Podocalyxin is Essential for Podocyte Morphogenesis T h e expected defects in podocytes lack ing podoca lyx in made the g lomeru l i an excel lent in i t ia l target fo r analysis in p o d o c a l y x i n - n u l l animals (Doyonnas et a l . , 2001) . For the most part, however , podocyte maturation is unaffected in knockout mice . Nephr in is a 56 component of the slit d iaphragm (discussed in section 1.6.5.1, be low) , and g lomeru lar epithelial protein ( G L E P P ) - l is a transmembrane tyrosine phosphatase o f podocytes; both are considered markers of podocyte di f ferent iat ion (Doyonnas 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 . , 2000) . Immunoh is tochemica l sta in ing for these two proteins is normal in mice lack ing p o d o c a l y x i n (Doyonnas et a l . , 2001) . H o w e v e r , ultrastructural analys is o f podocytes by t ransmiss ion e lectron m i c r o s c o p y ( T E M ) reveals d ramat ic morpho log ica l abnormal i t ies . There is a s ign i f icant reduct ion in the number of major processes and a complete absence o f foot processes and slit d iaphragms in p o d o c a l y x i n -nul l mice . Moreover , podocyte cel l bodies complete ly envelop the capi l lary loops, and there is a s t r ik ing presence o f junct iona l complexes between adjacent podocytes. These defects b lock normal filtrate product ion, as the bladders of knockout mice are complete ly empty just before bir th, in contrast to w i ld type l ittermates. Other observat ions inc lude vacuoles w i t h i n podocytes , t h i c k e n i n g o f the cap i l la ry endothel ia l ce l l layer , fewer endothe l ia l fenestrae, and an apparent reduct ion in the v o l u m e of ur inary spaces (Doyonnas et a l . , 2001) . The electron micrograph in F igure 1-9, inc luded for orientation purposes , depicts a n o r m a l k i d n e y podocy te e x t e n d i n g majo r processes and foot processes around a g l o m e r u l a r c a p i l l a r y . S i m i l a r l y , c r o s s - s e c t i o n s taken th rough podocytes and capi l lar ies of w i ld type and p o d o c a l y x i n - n u l l mice are depicted in F igure 1-10, where many of the differences described above are clear ly v is ib le . 57 Figure 1-9: Electron Micrograph of a Normal Glomerular Podocyte with Extended Major Processes and Foot Processes Surrounding a Blood Vessel. O r ig ina l magni f icat ion : 7 3 0 0 X . Reproduced and adapted w i th k ind permiss ion of Spr inger Science and Business M e d i a ( M u n d e l and K r i z , 1995). 5 8 Figure 1-10: T E M ' s of Kidneys from Wildtype and Podocalyxin Knockout Mice. Junct ional complexes (JC) are v is ib le between podocytes (Pod) in podoca l yx in -nu l l mice . These mice also lack major processes ( M P ) and foot processes (FP ) and have th ickened endothel ia l ce l ls ( 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 permiss ion o f T h e Rockefe l l e r Un ivers i ty Press (Doyonnas et a l . , 2001) . 59 T h e podocyte phenotypes observed in podoca lyx in knockout mice are consistent wi th a role for podoca lyx in in decreasing cel l adhesion and affect ing cel l morphology . A l t h o u g h podocyte maturat ion markers appear n o r m a l , the ce l ls have an immature m o r p h o l o g y overa l l . A s in 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 an imals retain tight junct ions instead of f o r m i n g slit diaphragms (Doyonnas et a l . , 2001) . Thus , it seems l ike l y that under normal condi t ions , either podocalyx in alters distr ibution of tight junct ion proteins by means o f its cy top lasmic interaction partners, or s i m p l y that express ion o f h igh leve ls o f this b u l k y , negat ive ly charged mo lecu le on the ap ica l surface may phys ica l l y d isp lace junct ions to a more basal locat ion. T h e lack of foot processes may i m p l y a more spec i f i c role fo r p o d o c a l y x i n in af fect ing ce l l m o r p h o l o g y through its extensive negat ively charged d o m a i n in addi t ion to its conserved cy top lasmic tai l and interactions w i th the actin cytoskeleton. Regard ing the increased presence of cy top lasmic vacuoles , this is also a characterist ic of human and rodent models of renal disease, and it may be an alternative pathway for fi ltrate product ion, since the presence of tight junct ions and the lack of sl it pores b lock the normal pathway (Toth and Takebayash i , 1992). T h e th icken ing of capi l la ry endothel ial cel ls in podocalyx in knockout mice may be related to the loss of endothel ial podoca lyx in or it may be due to an increase in capi l lary pressure as a result of the blockage in fi ltrate product ion (Doyonnas et a l . , 2001) . Thus , p o d o c a l y x i n -nu l l mice certainly demonstrate that podoca lyx in is a v i tal component of the deve lop ing k idney . 60 1.6.4.2 The Kidney Defect in Podocalyx in -Nul l Mice is the Apparent Cause of Perinatal Lethality in these Animals T h e st r ik ingly abnormal podocytes in podoca lyx in -de f i c ien t animals c lear ly represent a serious defect in these an imals and, in fact , it is the apparent cause o f their lethal i ty wi th in the first day of birth (Doyonnas et a l . , 2001) . A l l genotypes are present in expected frequencies throughout development, but this changes shortly after birth, such that there are no l ive podoca lyx in -nu l l mice one day later (Table 1-1). Unfortunately , death at this ear ly t ime point precludes a more thorough assessment o f potent ia l l y w idespread impl icat ions of the kidney defect. 61 N u m b e r of animals/genotype A g e of embryos +/+ +/-(post coi tum) -/- 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 A g e of newborns (post partum) 1 d 52 100 0 0 Table 1-1: Frequency of podxVl+, podxt'", and podxt1' Animals during Development. A l t h o u g h normal ratios of podoca lyx in -nu l l animals are present throughout development , they al l die wi th in 24 hours of birth. Adapted f r o m (Doyonnas et a l . , 2001) . 62 Other newborn mice wi th anuric renal fa i lure have also been descr ibed to die w i th in the f i rst day o f b i r th , m a k i n g it l i ke l y that this is the reason for the death of p o d o c a l y x i n knockout animals ( B u l l o c k et a l . , 1998; D a v i s et a l . , 1995). It is important to remember, however , that other mice with anuric renal fa i lure general ly have other s ignif icant defects or lack k idneys entirely. The podoca lyx in knockout was thus the most select ive, lethal anuric cond i t ion descr ibed at the t ime, but c o n f i r m a t i o n that the k idney defect is the def in i t ive cause of death is not easy to obtain (Doyonnas et a l . , 2001) . 1.6.5 Other Molecules Involved in Glomerular Development S o m e patients wi th g lomeru lar diseases have mutat ions in genes encod ing structural proteins invo lved in integrity of the podocyte cytoskeleton or sl it d iaphragms, such as a -act in in -4 , nephrin, podoc in , and CD2-assoc ia ted protein (Boute et a l . , 2 0 0 0 ; K a p l a n et a l . , 2 0 0 0 ; K e s t i l a et a l . , 1 9 9 8 ; K i m et a l . , 2 0 0 3 ) . H o w e v e r , most pat ients w i t h g lomeru losc leros is do not have mutations in these genes. Instead, express ion of these genes may be dysregulated by mutations in other genes, such as W T 1 (Gao et a l . , 2004) . In fact , it has been shown that mutat ions in W T 1 can be a factor in ear l y -onset g l o m e r u l o s c l e r o s i s . It is also poss ib le that a b n o r m a l podocyte f u n c t i o n caused by mutation of a single gene may initiate a series of responses that includes dysregulat ion of other podocyte genes. A l t h o u g h there are o b v i o u s l y numerous proteins required for g lomerular development, I have selected two molecu les to discuss that are part icular ly relevant fo r m y studies. N e p h r i n is in i t ia l l y expressed on the basolateral surface of deve lop ing podocytes at the S-shaped body stage of g lomerular development, not un l ike podoca lyx in ( K a w a c h i et a l . , 2 0 0 2 ; Schnabel et a l . , 1989)). I have taken advantage of this 63 fact when generating transgenic mice (described in chapter f ive) . In add i t ion , W T 1 is essential fo r g lomerular development , and it is k n o w n to be a regulator o f podoca l yx in expression (Gao et a l . , 2 0 0 4 ; G u o et a l . , 2 0 0 2 ; K re idberg et a l . , 1993; Palmer et a l . , 2 0 0 1 ; Stanhope-Baker et a l . , 2004) . 1.6.5.1 Nephrin N e p h r i n is a 180 k D a transmembrane protein be long ing to the i m m u n o g l o b u l i n (Ig) super fami ly ( rev iewed in (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 extracel lular Ig - l ike mot i fs , and in kidneys it is loca l i zed exc lus ive ly to the slit d iaphragm region. It was f irst ident i f ied in a genome-wide screen to locate the gene responsible for the autosomal recessive congenital nephrotic syndrome of the F inn ish type ( C N F ) . T h e f i l t ra t ion barr ier in C N F patients is d is rupted , resul t ing in symptoms o f nephrot ic syndrome. T h e effects are seen ear ly , w i th severe prote inur ia in utero, loss of foot processes, and death w i th in two years in the absence of a kidney transplant. C l o s e to 50 different mutations in the gene encoding nephrin (NPHS1) have been detected in these patients. In the d e v e l o p i n g g l o m e r u l u s , nephr in is expressed on the basolateral surface o f podocytes below junct ional complexes ( K a w a c h i et a l . , 2002) . It migrates in conjunct ion w i t h junc t iona l proteins and is eventual ly restr icted to the site of the sl it d iaphragm. N e p h r i n k n o c k o u t mice rap id ly deve lop severe prote inur ia , they exh ib i t part ial foot process effacement, loss of sl i t d iaphragms, and narrowing of f i l t rat ion sl i ts, and they die wi th in 2 4 hours of birth (Putaala et a l . , 2001) . A l t h o u g h nephrin expression is decreased 64 in P A N , other studies address ing the role o f nephr in in g l o m e r u l a r diseases are inconc lus ive (reviewed in (Salant and T o p h a m , 2003)) . The precise role of nephrin in mainta in ing slit d iaphragm 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 ( rev iewed in (T ryggvason , 1999)). A mode l fo r the invo lvement of nephrin in this type of structure relies on it interacting homoph i l i ca l l y w i th other nephrin molecules on neighbour ing foot processes, as expected for Ig - l ike adhesion molecu les . There are two Ig - l ike domains p r o x i m a l to the p lasma membrane and six more d is ta l , separated by a spacer sect ion. Based on the predicted 4 0 n m sl it pore w id th , it is poss ib le that the s ix distal Ig - l ike domains w o u l d interact w i th those on oppos ing nephr in molecules and that unpaired cysteine residues wou ld f o r m intermolecular disulphide bridges. Sl i t pores may then f o r m in between the non - in te rac t ing , p r o x i m a l I g - l i k e d o m a i n s of ne ighbour ing nephr in molecu les . T h i s model has yet to be proven, but the necessity for nephrin in g lomerular development and slit d iaphragm structure is clear. 1.6.5.2 WT1 A s descr ibed in section 1.3 .1 , W T 1 is a z inc f inger t ranscr ipt ion factor expressed in g lomeru la r podocytes (Pa lmer et a l . , 2 0 0 1 ; Rober ts , 2 0 0 5 ) , and there is considerable ev idence i m p l i c a t i n g W T 1 in normal podocyte func t ion ( G u o et a l . , 2002) . It is f i rst detectable at day nine of mouse development in the l i n i n g of the coe lomic cavity and in the urogenital r idge ( A r m s t r o n g et a l . , 1993). Express ion is maintained in mesothel ia l cel ls l i n ing major organs throughout development, but it is also strongly expressed in the 65 renal ves ic le and S -shaped body stages by cel ls that later become podocytes. It is then restr icted to podocytes in the adult k idney (Roberts , 2 0 0 5 ) . D e n y s - D r a s h syndrome ( D D S ) g e n e r a l l y i n v o l v e s d e v e l o p m e n t o f g l o m e r u l a r n e p h r o p a t h y w i t h g lomeru losc le ros i s ; 9 4 % of D D S patients have mutat ions in wtl ( G u o et a l . , 2002) . M o r e o v e r , one o f the c o m m o n wtl mutat ions seen in these patients can a lso cause g lomerulosclerosis in mice (Patek et a l . , 1999). In addi t ion , some patients wi th nephrotic syndrome and isolated cases of g lomerulosc leros is have wtl mutations (Ito et a l . , 1999; Y a n g et a l . , 1999). L o s s o f W T 1 has p ro found effects on g l o m e r u l a r deve lopment in m i c e . Targeted inact ivat ion of W T 1 by delet ion o f the f irst exon prevents mesenchymal induct ion and results in renal agenesis; nul l mice die between embryon ic day 13 and 14 (K re idberg et a l . , 1993). W T 1 ablat ion by smal l in ter fer ing R N A ( s i R N A ) in d e v e l o p i n g k idney explants conf i rms this result (Dav ies et a l . , 2004) . W h e n s i R N A is added to explants corresponding to embryonic day 9 (E9) , ureteric buds fa i l to grow. However , i f s i R N A is instead added at E l 1, ureteric buds remain intact, but nephrogenesis is severely impai red . W T 1 is therefore required at several stages of development , where it p lays numerous roles. W h e n W T l - n u l l m i c e are rescued us ing a human wtl t ransgene, nephrogenesis is recovered, but mice can sti l l develop crescentic g lomerulonephri t is or mesangial sclerosis after b i r th , depending on the level o f W I T expression (Guo et a l . , 2002) . W T l - d e f i c i e n t mice w i th a single copy of the human transgene surv ive at least unt i l birth. H o w e v e r , 66 only 2 6 % have two k idneys ; 14 % have one k idney , and 60 % lack k idneys complete ly and die w i t h i n 48 hours of b i r th . T h e s u r v i v i n g m i c e a l l su f fe r f r o m d e l a y e d nephrogenesis and congenital nephrotic syndrome with severe a lbuminur ia and die wi th in 20 days. Transgenic mice with two copies of human wtl al l have at least one k idney , and 7 6 % have two. They also have a less s t r ik ing delay in nephrogenesis , but they st i l l develop adult-onset nephrotic syndrome wi th a lbuminur ia , and 26 % die wi th in 150 days. S i m i l a r l y , mice with one copy of murine wtl develop both k idneys , but also exhibi t adult -onset nephrot ic syndrome, and 11 % die w i th in 150 days. W T 1 is therefore c lear l y important for nephrogenesis and normal kidney function throughout l i fe . 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 igh ly expressed in k idney , other s ign i f i cant areas o f expression include the hematopoietic and vascular systems. 1.7.1 Overview of Hematopoiesis D u r i n g development of the hematopoietic system, there are thought to be two main waves of hematopoies is , although there is st i l l some controversy regarding exact ly when and where each ce l l type is f irst generated. P r imi t i ve hematopoiesis begins at E 7 . 5 in mice and involves product ion, in the b lood islands of the yo lk sac, of the first c i rcu lat ing b lood cel ls ( rev iewed in (Dz ie rzak et a l . , 1998; K e l l e r et a l . , 1999)). These ce l ls are large, nucleated erythroid cel ls and are required for surv ival and rapid growth of the embryo . T h i s is f o l l o w e d by product ion o f y o l k sac -der ived hematopoiet ic progenitors , w h i c h 67 enter the vasculature and c i rcu late unt i l replaced by de f in i t i ve hematopoiet ic ce l l s . Def in i t i ve hematopoiesis begins in the aorta-gonad-mesonephros ( A G M ) region at E l 0 . 5 -11.5 and is characterized by product ion of enucleated erythrocytes, l y m p h o i d cel ls , 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 ver ( F T L ) , the main site o f fetal hematopoiesis . Hematopo ies is also occurs transiently in the fetal spleen ( S P L ) before becoming f i r m l y established in the bone marrow ( B M ) , where an elaborate set of envi ronmental signals regulate prol i ferat ion and differentiat ion (reviewed 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 al l hematopoiet ical ly active tissues throughout development (Doyonnas et a l . , 2005) . For example , most hematopoiet ic cel ls in E 1 0 - 1 2 murine y o l k sac and peripheral b lood express podoca l yx in (F igure 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 -pos i t ive ce l ls at this stage express markers of the erythroid l ineage, but a very smal l percentage instead express the stem cel l factor receptor c - K i t and the pan-hematopoiet ic marker C D 4 5 . In co lony f o r m i n g assays, these cel ls g ive rise to erythroid and m y e l o i d co lon ies , suggesting that podoca l yx in is expressed on pr imi t i ve erythrocytes as we l l as p r imi t i ve m y e l o i d or mult i l ineage progenitors. P o d o c a l y x i n expression in y o l k sac and peripheral b lood gradual ly decreases over t ime, both in terms of expression levels and frequency of podocalyx in -pos i t i ve cel ls . 68 100 1'1 1'2 1'3 14 l'5 1'6 iV 18 19 YS PB FL Spleen BM \ 100 75 V 50 V 25 n i—r 1—I I I I 18 NB 2 4 1 Wk 2wk 4wk Day PC Day PP F i g u r e 1-11: P o d o c a l y x i n is 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 ssues t h r o u g h o u t D e v e l o p m e n t . P C : post c o i t u m , P P : post partum. A d a p t e d f r o m (Doyonnas et a l . , 2005) . T h e n , as hematopo ies is shifts to F T L at E l 5 , 75 % of ce l l s in this t issue express podoca lyx in (F igure 1-11) (Doyonnas et a l . , 2005) . A t this stage, there are two dist inct popu lat ions o f p o d o c a l y x i n express ing ce l l s . T h e ce l l s express ing lower leve ls o f p o d o c a l y x i n are either p r i m i t i v e or de f in i t i ve ery thro id ce l l s , w h i l e the populat ion express ing the highest leve ls conta ins de f in i t i ve hematopoiet ic progeni tors . A g a i n , podoca lyx in expression in this tissue decl ines over the next few days. S i m i l a r l y , S P L and B M both conta in p o d o c a l y x i n - p o s i t i v e populat ions upon acquis i t ion o f hematopoiet ic act iv i ty . In perinatal m i c e , most cel ls expressing podoca lyx in are erythroblasts or early h e m a t o p o i e t i c p rogen i to rs . P o d o c a l y x i n e x p r e s s i o n then decreases to v i r t u a l l y undetectable levels by birth. However , there is a distinct burst of podoca lyx in expression in hematopoiet ic tissues immediate ly after birth, but this is again f o l l o w e d by a gradual dec l ine . Thus , the establ ishment o f each hematopoiet ical ly active tissue co inc ides with increased expression of podoca lyx in . P o d o c a l y x i n expression in the developing hematopoietic system has also been assessed in c h i c k e n ( M c N a g n y et a l . , 1997 ; S u o n p a a et a l . , 2005) . A s in m i c e , there are two populat ions of p o d o c a l y x i n express ing ce l ls in early av ian embryos ( M c N a g n y et a l . , 1997). E r y t h r o i d ce l ls express low leve ls 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 igh ly expressed on mult ipotent hematopoiet ic progenitors. S ign i f i cant l y , p o d o c a l y x i n -posit ive cel ls are found adhering to the ventral wal l of the dorsal aorta before E 4 ; this is the precise locat ion of the f i rst def in i t i ve H S C s ( M c N a g n y et a l . , 1997; Suonpaa et a l . , 2005) . P o d o c a l y x i n is also found on hematopoietic progenitor cel ls in bone marrow of 1-70 week o ld c h i c k s . T h u s , p o d o c a l y x i n ' s expression pattern is comparable in deve lop ing mammals and avians. In adult , p o d o c a l y x i n express ion is restricted to ce l l s of the p late let/megakaryocyt ic l ineage and a rare populat ion of cel ls wi th a stem cel l phenotype (Doyonnas et a l . , 2 0 0 5 ; M c N a g n y et a l . , 1997 ; M i e t t i n e n et a l . , 1999). T h e s e L i n S c a - 1 + c - K i t + ( L S K ) p o d o c a l y x i n + ce l ls give rise to m y e l o i d and l y m p h o i d l ineages in serial transplantation exper iments, suggesting that podoca lyx in may be a marker of L T R - H S C s (Doyonnas et a l . , 2005) . A l t h o u g h erythroid cel ls in adult do not normal l y express p o d o c a l y x i n , it is rapidly upregulated by erythroid progenitors in response to hemolyt ic anemia (Doyonnas et a l . , 2005) . T h i s suggests that podoca lyx in is expressed by early erythroid progenitors only when high rates of erythropoiesis are required, such as throughout development and under c o n d i t i o n s of e ry th ropo ie t i c stress. O n e study a lso reported express ion o f p o d o c a l y x i n m R N A in n u m e r o u s mature h e m a t o p o i e t i c l i n e a g e s , i n c l u d i n g B l ymphocy tes , T l ymphocy tes , and m y e l o i d ce l l s , but the cor responding protein is not expressed in these cel ls (Kerosuo et a l . , 2004) . Notab ly , however , p o d o c a l y x i n m R N A levels are much higher in k idney and in endothel ial cel ls than in any hematopoiet ic cel ls . Thus , podoca lyx in is expressed by hematopoietic stem cel ls and platelets, but not in other mature hematopoietic l ineages under steady state condit ions in adult. 1.7.3 Hematopoiesis in the Podocalyxin Knockout Since p o d o c a l y x i n is expressed by a subset o f hematopoiet ic ce l l s , i n c l u d i n g early hematopoiet ic progenitors, the hematopoiet ic system of podoca l yx in knockout animals 71 was assessed for defects (Doyonnas et a l . , 2 0 0 1 ; Doyonnas et a l . , 2005) . E 1 5 F T L (Figure 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 cy tometry w i th antibodies against hematopoiet ic progenitors (Sca-1 and c - K i t ) , erythroid ce l ls ( T e r l 19), megakaryocytes and platelets ( C D 4 1 ) , m y e l o i d ce l ls ( M a c l ) , B ce l l s ( B 2 2 0 ) , T ce l ls ( C D 3 ) , and granulocytes (Gr -1 ) (Figure 1-12 and (Doyonnas et a l . , 2001)) . There are no detectable dif ferences in the frequencies of any hematopoiet ic l ineages in p o d o c a l y x i n -def ic ient mice throughout development. T h e related protein, C D 3 4 , is also expressed by hematopoiet ic progenitors, thereby p rov id ing the poss ib i l i ty of funct ional compensat ion by this f a m i l y member ( A n d r e w s et a l . , 1989; Berenson et a l . , 1988; D o y o n n a s et a l . , 2001) . For this reason, c o m p o u n d knockout m i c e l a c k i n g both m o l e c u l e s were a lso generated and analyzed (Doyonnas et a l . , 2005) . Surpr is ing ly , however , hematopoiesis is also normal in mice lack ing both molecules (Figure 1-12). 7 2 100 90 80 70 60 50 40 § 30 ° 20 10 0 CU O CU > ro O) CD c I CT> r i • WT • Podo KO • CD34 KO DDKO 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 of al l hematopoietic l ineages are normal 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 mice , there is a funct ional defect in these hematopoiet ic ce l ls . Shor t - term homing assays demonstrate that injected ce l ls l a c k i n g either podoca lyx in or C D 3 4 migrate wi th 2 0 - 3 0 % lower e f f i c iency to the bone marrow than their w i ld type counterparts, and that loss o f both molecu les has an addi t ive effect (Doyonnas et a l . , 2005) . H o w e v e r , despite this defect, when cel ls f r o m any of these mice are injected in suff ic ient numbers, they can fu l l y reconstitute lethal ly irradiated recipients. T h i s result, a long w i th the expression pattern of podoca lyx in , in part icular , impl ies that wh i le these molecu les are not essential , they may faci l i tate the c ross ing of endothel ial barriers for entry into or exit f r o m , hematopoiet ic m i c r o e n v i r o n m e n t s (Doyonnas et a l . , 2 0 0 5 ) . Hematopoiet ic cel ls of the yo lk sac become less adherent in order to leave b lood is lands, precursors in F T L must cross into the vasculature to migrate to S P L and B M , and severe anemia leads to an e f f lux of erythroid progenitors f r o m the B M in order to establ ish addit ional sites of erythropoiesis. Each of these situations corresponds to an increase in podoca lyx in expression. 1.7.4 Podocalyxin in the Vasculature In addit ion to its expression in the hematopoietic system, podoca lyx in is also a universal marker of vascu lar endothe l ia l ce l l s ( D o y o n n a s et a l . , 2 0 0 5 ; Horva t et a l . , 1986 ; Mie t t inen et a l . , 1990). It is expressed in b lood vessels of k idney , lung, heart, brain, smal l intestine, and other tissues (Horvat et a l . , 1986). P o d o c a l y x i n is found on endothelial cel ls l i n i n g a w ide range of vessels, f r o m the coronary artery to the s inusoids of l i ver and spleen and the special ized postcapi l lary venules in l y m p h nodes termed H E V (Horvat et 74 a l . , 1986; M i e t t i n e n et a l . , 1990; Sassetti et a l . , 1998). B y i m m u n o T E M , it has been shown that podoca l yx in is loca l i zed in a patchy pattern on the lumina l face of vascular endothelial cel ls (Horvat et a l . , 1986). Despi te the widespread express ion of podoca l yx in on b lood vessels , the vasculature appears normal in deve lop ing p o d o c a l y x i n knockout animals (Doyonnas et a l . , 2001) . There are no detectable di f ferences in the staining pattern o f the endothel ia l marker , P E C A M - 1 in E 1 6 bra in , k i d n e y , lung , or gut. Fur thermore , e lectron m i c r o s c o p y o f newborn lungs shows the format ion of wel l -developed pulmonary capi l lar ies. The related protein, C D 3 4 , is also ubiqui tous ly expressed in vasculature, however , so it is possible that it cou ld be funct ional ly compensat ing for loss of podocalyx in (Doyonnas et a l . , 2 0 0 1 ; F i n a et a l . , 1990). In fact , upregulat ion of C D 3 4 in lung o f E 1 8 podoca lyx in -de f i c ien t animals has been detected by real t ime R T - P C R and immunohistochemistry (Doyonnas et a l . , 2001) . A l t h o u g h there are no obv ious defects in the vasculature, approximately 25 % of podoca lyx in -def i c ient embryos exhibit m i l d to severe edema (Doyonnas et a l . , 2001) . Th i s may be a result of leaky vessels or, alternatively, the kidney defect wou ld l ike ly lead to an increase in b lood pressure, w h i c h may be rel ieved by loss of f l u i d f r o m the vessels. F u n c t i o n a l l y , of course, it w o u l d be easier to assess defects in the vascular system in adult an imals , but perinatal lethality precludes this analysis. 1.7.5 Podocalyxin Expression in Hemangioblasts Express ion of podoca lyx in by hematopoietic and vascular endothel ial cel ls impl ies that it cou ld also be expressed by the c o m m o n precursor of both ce 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 ce l ls in the A G M region at E l 1.5 (Hara et a l . , 1999). P o d o c a l y x i n + C D 4 5 ce l l s f r o m the A G M dif ferent iate into both angioblasts and hematopoiet ic ce l ls in vitro, depending on the growth cond i t ions , and they are also capable of long - term l ympho id and m y e l o i d reconstitution in transplantation exper iments , suggest ing the presence on L T R - H S C s in this popu lat ion , a l though this cannot be conc lus ive ly demonstrated without per forming single ce l l transplants (Hara et a l . , 1999). T h u s , a l though not c o n f i r m e d , this data is suggest ive o f p o d o c a l y x i n expression on hemangioblasts. 1.8 Podocalyxin in the Brain Another interesting site of podoca lyx in expression is the brain, where it is detected in the marginal zone of the cerebral cortex, the cerebe l lum, and the superior co l l i cu lus 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 ce l l s in the d e v e l o p i n g cerebel lum hints that it may be invo lved in enabl ing neurons to migrate and detach f r o m radial g l i a . In situ h y b r i d i z a t i o n demonstrates that p o d o c a l y x i n m R N A is , in fact , expressed in many regions throughout development and in adult brain , wi th the highest expression levels in cerebral cortex and cerebel lum postnatally (V i ture i ra et a l . , 2005) . In the f o r e b r a i n , p o d o c a l y x i n is expressed in the o l f a c t o r y b u l b , neocor tex , and h ippocampus. It is expressed in deve lop ing neurons and a long speci f ic axonal pathways. T h e funct ional s ign i f i cance of p o d o c a l y x i n express ion in the brain and any potential defects in podoca lyx in knockout animals are st i l l under invest igat ion, but its pattern of expression hints at a role in pro l i ferat ion , migra t ion , or neuronal d i f ferent iat ion in the central nervous system (V i tu re i ra et a l . , 2005) . A n t i - a d h e s i v e forces may aid in the 76 detachment and migrat ion o f neuronal cel ls and leading processes, or podoca l yx in may be act ive ly invo lved in axonal path f i n d i n g through its interact ions wi th P D Z d o m a i n -contain ing proteins (Or lando et a l . , 2 0 0 1 ; V i tu re i ra et a l . , 2005) . 1.9 Additional Phenotypes in Podocalyxin-Null Animals P o d o c a l y x i n is expressed by podocytes , hematopoiet ic progenitors , platelets, vascular endothel ia , a subset of neurons, and mesothel ia l cel ls l i n i n g many organs; defects are therefore expected in these tissues in mice l a c k i n g podoca lyx in (Doyonnas et a l . , 2 0 0 1 ; Horvat et a l . , 1986; Ker jaschki et a l . , 1984; Ke rshaw 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 . , 1999; Sassetti et a l . , 1998). C D 3 4 may func t iona l l y compensate for p o d o c a l y x i n loss in hematopoiet ic progenitors and b lood vessels , potential neuronal defects are being act ively investigated, and podocytes are c lear ly abnormal (Doyonnas et a l . , 2001) . T h e f ina l area of interest is therefore the l i n i n g o f body cavi t ies . S t r ik ing l y , approx imate ly 30 % of 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 herniat ion of the gut, k n o w n 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 n o r m a l p h y s i o l o g i c a l process that occurs at E 1 2 as the rap id ly g r o w i n g organs exceed the l i m i t i n g space of the peritoneal cav i t y ; however , in normal m i c e it is resolved as the peritoneal cav i ty expands, general ly by E 1 6 ( K a u f m a n , 1998). U s i n g t imed mat ings , it was determined that resolut ion of the ompha loce le is de layed in a l l p o d o c a l y x i n - n u l l mice , but 7 0 % do retract the gut before birth (Doyonnas et a l . , 2001) . T h e delay may be a result of increased adhesion upon loss of podoca lyx in f r o m exposed surfaces. Thus , in another tissue where C D 3 4 cannot funct ional ly compensate for loss o f podoca lyx in , there is an apparent increase in cel l adhesion. 77 1.10 Podocalyxin in Cancer U p to this point I have concentrated on the normal funct ion and expression pattern of p o d o c a l y x i n , but I w i l l now f o c u s on cases where p o d o c a l y x i n e x p r e s s i o n is dys regu la ted . P o d o c a l y x i n has been i m p l i c a t e d in numerous mal ignant s i tuat ions , i n c l u d i n g breast cancer, test icular cancer, prostate cancer, and leukemia (Casey 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 ; Somasir i et a l . , 2004). 1.10.1 Podocalyxin in Breast Cancer A c c o r d i n g to analys is o f a tissue microarray of 272 invas ive breast ca rc inomas and corresponding long - term outcome data, we have shown that podoca lyx in upregulation is correlated w i th poor outcome in a dist inct subset of tumours (Somasi r i et a l . , 2004) . Immunohistochemist ry was used to compare podoca lyx in expression in tumour samples. A score of " 0 " corresponds to a lack of podoca lyx in staining in tumour ce l l s , and was found in 60 % of cases. Less than 10 % of tumour cel ls were stained (group 1) in 23 % of samples, 12 % of cases exhibi ted intense staining in less than half of the cel ls or diffuse staining in more than 10 % (group 2). The f inal group (group 3) made up 6 % of the array and had intense podoca lyx in staining in the majority of tumour cel ls . Surv iva l rates were compared between patients f r o m each group, and a statistically s ignif icant difference was noted between group 3 and the rest of the patients, combined (Figure 1-13). T h e mean surv iva l t ime was 9.0±1.8 years for patients in group 3, wh i le those patients wi th tumours expressing low or no podoca lyx in l i ved on average 15±0.5 years. A l t h o u g h there are no 78 s ign i f icant di f ferences in h is to log ica l subtype, tumour s ize , or l y m p h node metastasis between groups, there are p ropor t iona l l y more h igh grade, estrogen receptor ( E R ) -negat i ve t u m o u r s in the h i g h p o d o c a l y x i n g r o u p . F u r t h e r m o r e , p o d o c a l y x i n overexpression is a statistically s igni f icant independent predictor of poor outcome with more than an eight - fo ld relative risk compared to podocalyx in low or negative samples. 79 Group 0 Group 1 Group 2 1 — Group 3 0 5 10 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 express ion and group 3: h igh p o d o c a l y x i n expression (Somasi r i et a l . , 2004) . 80 The mechan ism leading to increased podoca l yx in expression has not yet been determined (Somasi r i et a l . , 2004) . H o w e v e r , the human podxl gene is located on chromosome 7 q 3 2 -q 3 3 , w h i c h is in between two regions prev ious ly ident i f ied as ch romosomal gain sites in ductal breast ca rc inoma and breast tumour ce l l l ines ( 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 protein , N H E R F 1 is often expressed at lower leve ls in E R - n e g a t i v e breast tumours in c o m p a r i s o n to E R -posi t ive tumours , and many o f the tumours express ing high levels 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 may be a cont r ibu t ing factor ( 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 though the events l e a d i n g up to it can o n l y be hypothesized at this stage, p o d o c a l y x i n is upregulated in a subset o f breast tumours in patients w i t h poor outcomes . There is cur rent ly a larger screen o f breast tumours underway to conf i rm these results. 1.10.2 Podocalyxin in Prostate Cancer G e n e t i c approaches were used to ident i f y p o d o c a l y x i n dys regu la t ion as a factor in prostate cancer (Casey et a l . , 2006) . S ince prostate cancer aggressiveness, w h i c h varies w ide ly among patients, is in f luenced by f a m i l y history , a genetic component is suspected ( K l e i n et a l . , 1998). A g e n o m e - w i d e screen o f over 5 0 0 af fected s ib l ings st rongly impl icated chromosome 7 q 3 2 - q 3 3 in tumour aggressiveness (Wit te et a l . , 2000) , and this result has been conf i rmed in subsequent studies (Paiss et a l . , 2 0 0 3 ; Wi t te et a l . , 2003) . In prostate tumours, this region also exhibi ts a h igh f requency of a l le l i c imbalance , w i th the podxl gene contained wi th in the smallest region of imbalance (Nev i l l e et a l . , 2002) . 81 M u t a t i o n a l analys is was performed on genomic podxl f r o m the probands of 17 f a m i l i e s ident i f ied p rev ious l y (Casey et a l . , 2 0 0 6 ) . T h e re lat ionship between prostate cancer , t u m o u r a g g r e s s i v e n e s s , and podxl var iants was then assessed i n a f a m i l y - b a s e d assoc iat ion study. Several c o m m o n mutat ions were ident i f ied , i n c l u d i n g a var iab le i n -f rame delet ion in the f i rst exon , four missense mutat ions, and two si lent var iants. T h e presence o f one or two copies of the in - f rame delet ion variant, w h i c h results in loss o f serine and pro l ine residues in the extracel lu lar d o m a i n , increases the relat ive r isk , w i t h two copies doub l ing the r isk of deve lop ing more aggressive prostate cancer. T h e presence of missense mutat ions in exon two increases the r isk of deve lop ing prostate cancer by approx imate ly 50 % , but has no effect on aggressiveness. T h e other mutations were not r i sk fac to rs , and add i t iona l var iants outs ide o f the podxl locus d i d not show any associat ion w i t h prostate cancer either, i m p l y i n g that the mutations descr ibed above are genuine r isk factors . W h i l e the func t iona l i m p l i c a t i o n s of these mutat ions are not yet k n o w n , it may be that they result in decreased negative charge on p o d o c a l y x i n , w h i c h may disrupt the associat ion of p o d o c a l y x i n w i th the actin cy toske leton , or ce l l mot i l i t y and invasiveness may be increased as a result of perturbation of downstream targets. 1.10.3 Podocalyxin in Testicular Cancer P o d o c a l y x i n express ion in test icular cancer , the most c o m m o n type o f so l id tumour in young adult males , was the f irst report of podoca l yx in in mal ignant cel ls (Schopper le et a l . , 2 0 0 3 ) . It is impor tant to f i n d markers to d i s t ingu ish between the t w o types o f test icular ge rm ce l l tumours , seminomatous and nonseminomatous ( N S G C T ) , because they require different treatments. P o d o c a l y x i n is preferential ly expressed by N S G C T , and 82 not in protein lysates f r o m normal tissue. P o d o c a l y x i n is also found in the supernatants of cultured embryonal ca rc inoma ce l l l ines, and it may therefore be a useful serum marker for detect ion o f N S G C T (Schopper le et a l . , 1992). Interest ingly , the N S G C T f o r m of podoca l yx in is considerably larger than the f o r m found in k idney , suggesting that it may undergo addi t ional post - t ranslat ional mod i f i ca t ions in these tumours (Schopper le et a l . , 2003) . 1.10.4 Podocalyxin in Leukemia Potent ia l express ion o f p o d o c a l y x i n in l e u k e m i a was assessed for several reasons: the related protein , C D 3 4 is expressed by many , but not a l l , l eukemic blasts, p o d o c a l y x i n is expressed by normal hematopo ie t ic progeni tors , and the p o d o c a l y x i n t ranscr ipt ional regulator, W T 1 is expressed by the major i ty of blasts in acute m y e l o i d l e u k e m i a ( A M L ) and acute l ymphob las t i c l e u k e m i a ( A L L ) (Doyonnas 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 . , 1995 ; P a l m e r et a l . , 2001) . A M L and A L L tissue microarrays and biopsy spec imens were assayed for p o d o c a l y x i n express ion ( K e l l e y et a l . , 2005) . P o d o c a l y x i n was detected in blasts in 77 % of 39 A M L cases, w i th strong expression in 41 % of samples. It was also detected in 81 % of 27 A L L cases, w i th strong expression in 22 % . A l t h o u g h C D 3 4 is also expressed in many l e u k e m i c blasts, there is no s ign i f i can t cor re la t ion between C D 3 4 and p o d o c a l y x i n express ion . P o d o c a l y x i n expression in m y e l o i d sarcomas was also assessed in order to address a proposed role in fac i l i ta t ing tissue inf i l t rat ion ; it was expressed in 87 % of 15 cases w i th strong expression in 53 % . S ince podoca lyx in expression is regulated by W T 1 in podocytes , expression of W T 1 was assessed in l e u k e m i c blasts: 4 4 % o f A M L cases and 7 8 % of A L L cases 83 exhibi ted nuclear W T 1 , but there was no correlat ion w i t h podoca l yx in express ion. T h i s suggests that p o d o c a l y x i n express ion is somehow dysregulated in l e u k e m i a or , s ince podoca l yx in is normal l y expressed by hematopoiet ic progenitors, perhaps express ion by leukemic blasts just f o l l o w s the normal expression pattern, w h i c h may be independent o f W T 1 in hematopoiesis . A g a i n , the funct ional relevance of podoca lyx in expression in this type of cancer is u n k n o w n , but its presence c o u l d be used as a marker to increase the sensit iv i ty of assays designed to detect leukemia . 1.10.5 Podocalyxin in Hepatocellular Carcinoma H e p a t o c e l l u l a r c a r c i n o m a ( H C C ) is one o f the f i v e l e a d i n g causes o f cancer death w o r l d w i d e (P isani et a l . , 1999). Unfor tunate ly , this type of cancer is often detected too late, thereby l i m i t i n g treatment opt ions, and m a k i n g the f i ve - year surv iva l rate on ly f i ve percent ( E l - S e r a g et a l . , 2001) . F i n d i n g addit ional markers of H C C may faci l i tate earl ier detection and better outcome (Chen et a l . , 2004) . One major difference between H C C and normal l i ver tissue can be in the type o f b lood vessels they contain. N o r m a l vessels l i n i n g the hepatic s inusoid a l low free d i f fus ion of macromolecu les , but not larger part ic les, v i a smal l fenestrations (Chen et a l . , 2004) . In contrast, vasculature w i th in tumours is often a b n o r m a l l y permeable , a l l o w i n g passage o f larger m o l e c u l e s , and even metastasis o f cancerous ce l l s , through w idened c e l l - c e l l j unc t ions , larger fenestrat ions, t ranscel lu lar holes, and an irregular basement membrane (Hash izume et a l . , 2000) . S t r ik ing l y , C D 3 4 is h igh l y upregulated in endothe l ia l ce l l s o f H C C in c o m p a r i s o n to normal l i ve r t issue ( R u c k et a l . , 1995) . N o t a b l y , C D 3 4 is general ly not detectable in most s o l i d tumours , such as breast cancer, l y m p h o m a , m y e l o m a , or neuroblastoma ( rev iewed in (S i lvestr i et 84 a l . , 1992)) , whi le podoca lyx in has been found in several , such as those already d iscussed, W i l m s ' tumours (descr ibed b e l o w ) , and h i g h grade o v a r i a n tumours ( u n p u b l i s h e d observat ions, 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 ted in (Somas i r i et a l . , 2004)) . In a search for other markers of H C C using c D N A microar ray analys is o f normal tissue and tumours f r o m 58 patients, podoca lyx in and C D 3 4 were both f o u n d to be upregulated in H C C (Chen et a l . , 2004) . T h i s result was c o n f i r m e d by immunoh is tochemis t r y : 11 o f 12 H C C tissue sections were posit ive for p o d o c a l y x i n , w h i l e al l 5 normal tissue sections l a c k e d p o d o c a l y x i n . T h e r e was a lso a h i g h l y s ta t i s t i ca l l y s i g n i f i c a n t increase i n podoca lyx in and C D 3 4 expression in H C C on a tissue microarray conta in ing 3 5 0 samples (Chen et a l . , 2004) . It is thought that perhaps this dramatic upregulat ion contributes to the leakiness of vasculature in H C C . Regardless of the funct iona l imp l i ca t ions o f C D 3 4 and podoca l yx in express ion, they may be useful markers fo r earl ier detect ion o f this type o f cancer. 1.10.6 Podocalyxin in Wilms' Tumours W i l m s ' tumour is the most f requent ly occur r ing pediatr ic k idney cancer and the fourth m o s t c o m m o n c h i l d h o o d m a l i g n a n c y ( M i l l e r et a l . , 1 9 9 5 ) . T h e p o d o c a l y x i n t ranscr ipt ional regulator, W T 1 is mutated in 10-15 % of W i l m s ' tumours , and there is also ev idence to impl icate p53 in some cases; however , the major i ty of W i l m s ' tumours have no k n o w n cause. In contrast to the other cancers descr ibed above , p o d o c a l y x i n express ion is s ign i f i cant l y reduced in W i l m ' s tumours relat ive to no rmal fetal k i d n e y , a c c o r d i n g to c D N A microar ray analys is of 6 4 tumour samples ( S t a n h o p e - B a k e r et a l . , 2 0 0 4 ) . S u r p r i s i n g l y , h o w e v e r , p o d o c a l y x i n and W T 1 e x p r e s s i o n leve ls 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. This 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 C o n d i t i o n a l 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 T r a n s g e n i c C o n s t r u c t Murine podxl cDNA was excised from pBluescript using BamHI and Xhol restriction enzymes, and the transgenic pCCALL-2 vector (generously provided by Dr. Corrinne Lobe) was digested with Bgll l 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 pCCALL-2 derivative Z/EG and ligated into the pCCALL-2-/?o<ix/ construct, such that podocalyxin and GFP were expressed concurrently. 2.1.2 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 C o n s t r u c t The podocyte-specific transgenic construct was generated using the murine NPHSI promoter provided by Dr. Sue Quaggin. Two oligonucleotides (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 Pad 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 ) us ing the X h o l and E c o R l sites of the m u l t i p l e c l o n i n g site. M u r i n e podxl was then 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 vector v ia the SacII and B g l l l sites and l igated into p I R E S 2 -E G F P between SacII and B a m H I . F i n a l l y , the murine NPHS1 promoter was c loned into this p l a s m i d between X h o l and the new P a d site. A f t e r test ing C M V promoter -d r i ven p o d o c a l y x i n express ion f r o m this construct in vitro, X h o l and S f i l restr ict ion enzymes were used to isolate the NPHSl promoter and murine podxl for generation of transgenic mice . 2.1.3 Murine Podocalyxin Expression Vector The mur ine podxl expression vector used for in vitro studies was created as descr ibed in section 2 . 1 . 2 , but the ubiquitous C M V promoter was retained and the podocy te - spec i f i c NPHSI promoter was not inc luded. 2.1.4 Chicken Podocalyxin Expression Vectors A const ruct had p r e v i o u s l y been generated f o r express ion of a f u s i o n prote in that i n c l u d e d the ex t race l lu la r ( N - t e r m i n a l ) por t ion o f c h i c k e n podxl in the p c D N A 3 . 1 e x p r e s s i o n v e c t o r ( p c D N A 3 . l - c h M E P 2 1 F c ) . P C R , u s i n g 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'-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 ' , was used 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 construct (c lone 4 D 1 ( M c N a g n y et a l . , 1997)) and to add an 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 was l igated 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 was then exc ised w i t h B a m H I and X b a l . Th i s f ragment was l igated 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 expression construct. P o d o c a l y x i n express ion leve ls were l o w i n cel ls transfected w i t h the pcDNA3.1-/?0<sW construct, so n e w constructs were generated us ing the p I R E S 2 - E G F P express ion vector . C h i c k e n podxl c D N A was a m p l i f i e d f r o m the pcDNA3.1 -/?o f lW construct us ing pr imers 5 ' - C C A C T G C T T A C T G G C T T A T C G - 3 ' and 5 ' - A C A A C A G A T G G C T G G C A A C - 3' . The P C R product was l igated into the p C R 2 . 1 - T O P O c l o n i n g vector , exc ised w i t h E c o R l , and l igated into p I R E S 2 - E G F P . T h i s was then sequenced after f i rst c h e c k i n g the inser t ' s or ientation. 2.1.5 Chicken Podocalyxin Mutants A l a , A s p , a n d A D T H L c h i c k e n podxl mutants were generated by s i t e - d i r e c t e d mutagenesis us ing the T rans fo rmer S i te -D i rec ted Mutagenes is K i t (C lon tech , M o u n t a i n V i e w C A ) , accord ing to manufacturer 's instruct ions. D r . R e g i s D o y o n n a s generated the a lan ine mutants by s i te -d i rected mutagenesis 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 construct . I generated the other mutants as descr ibed b e l o w . T h e p c D N A 3 . 1 - c h i c k e n podxl vector was i n i t i a l l y used as a template fo r creat ion o f the aspart ic ac id mutants us ing pr imers 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 ' (putative P K C site) and 5 ' -A G G T G A T G G A A G A C G G C T C T G A A A T - 3 ' (putat ive C K I I s i te) , as w e l l as 5 ' -C C T G A A A C A C T A G T G G G C C C G T - 3 ' f o r m u t a t i o n o f the X b a l site to S p e l f o r 9 0 select ion purposes. R e d lettering denotes codons o f interest. H o w e v e r , convers ion o f the th reon ine res idue i n the putat ive C K I I site to an aspart ic a c i d res idue i n v o l v e d mutagenesis o f a l l three residues i n the c o d o n , w h i c h p roved d i f f i c u l t . Instead, the prev ious ly 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 was eventual ly used as a template because c o n v e r s i o n o f the a lan ine res idue to aspart ic a c i d requ i red mutagenesis of just two residues. S i m i l a r l y , 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 ' was used to insert a premature 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 the C - t e r m i n a l D T H L sequence by mutat ing the last g lu tamic ac id residue to a stop c o d o n , thereby creat ing the A D T H L mutant. The Ata i l mutant was created by us ing 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 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 ' 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 ' to insert a premature stop c o d o n after the jux tamembrane C C H Q R F sequence. T h e P C R product was then l igated into p C R 2 . 1 - T O P O , exc ised w i th B a m H I and X b a l , and l igated into the p c D N A 3 . 1 - N -terminal podxl construct. The al ternat ively sp l i ced f o r m o f c h i c k e n podxl was generated by a m p l i f i c a t i o n o f the C ' t e r m i n a l por t ion o f podxl f r o m a pB luesc r ip t c l o n i n g vector conta in ing podxl's sp l ice var iant ( 4 B 3 ( M c N a g n y et a l . , 1997)) . T h e P C R product created w i t h the pr imers 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 pCR2.1 -TOPO, excised with BamHI and X b a l , and inserted into the pcDNA3.1-N-terminal podxl construct. In addition, the above variants were all later cloned into the p I R E S 2 - E G F P expression vec to r . The p r imers 5 ' - C C A C T G C T T A C T G G C T T A T C G - 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 pcDNA3.1 constructs. The P C R products were inserted into pCR2.1-T O P O , excised with E c o R l , and ligated into the multiple cloning site of p IRES2-EGFP. 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 IRES2-EGFP 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 Nhel 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 pCR2.1 -TOPO using the Nhel 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 conta in ing podxl's t ransmembrane region and cy top lasmic ta i l . A l l constructs were sequenced after complet ion . 2.2 Cell Culture 2.2.1 Culture Conditions M C F - 7 and M D C K cel ls were rout inely mainta ined in A d v a n c e d D - M E M / F 1 2 m e d i u m ( Inv i t rogen # 1 2 6 3 4 - 0 1 0 , B u r l i n g t o n O N ) supplemented w i t h 5 % fetal b o v i n e se rum ( F B S ) , H E P E S , g lutamine, pen ic i l l i n , and streptomycin. N S O mouse m y e l o m a ce l ls (generously p rov ided by D r . G a r y M c L e a n and D r . J o h n Schrader) were rout inely mainta ined in D - M E M supplemented wi th 10 % F B S , s o d i u m pyruvate, non-essent ia l amino acids , g lutamine, p e n i c i l l i n , and st reptomycin . C H O cel ls were cultured in D - M E M supplemented wi th 10 % F B S , g lutamine, (3-mercaptoethanol, p e n i c i l l i n , and st reptomycin . F D C P - 1 cel ls were maintained in R P M I supplemented w i th 10 % F B S , 2 % W E H I - 3 B cond i t ioned 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 , pen ic i l l i n , and streptomycin. R l e m b r y o n i c stem ce l ls ( E S C s ) were rout inely mainta ined in D - M E M supplemented wi th 15 % F B S , 10 ng/ml L I F , sod ium pyruvate, non-essential amino acids , g lutamine, (3-mercaptoethanol , p e n i c i l l i n , and streptomycin. E S C s were cultured on mouse e m b r y o n i c f ibroblast feeder layers or gelatin coated plates. 93 2.2.2 Transfection Techniques M C F - 7 and M D C K ce l l s were t ransfected w i t h 3 0 p g D N A u s i n g the D M R I E - C t ransfect ion reagent ( Invitrogen) a c c o r d i n g to manufac tu re r ' s instruct ions. D N A was d i luted in 3 0 0 p i serum-f ree D M E M / F 1 2 and s l o w l y added to 15 u l D M R I E - C , also p rev ious l y d i lu ted in 3 0 0 p i D M E M / F 1 2 . T h i s was m i x e d gent ly , incubated for 15 minutes, and di luted w i th 2.4 m l D M E M / F 1 2 . T h e transfection mixture was transferred to 10 c m dishes conta in ing 3 0 - 5 0 % conf luent cel ls and incubated for 6 -8 hours. M e d i a was replaced w i th D M E M / F 1 2 conta in ing 5 % F B S and g lu tamine , and stable c lones were selected based on resistance to 4 0 0 pg/ml G 4 1 8 . 15 x 10 6 N S 0 cel ls were electroporated in 5 0 0 u l c o l d P B S w i t h 2 0 p g D N A in 0 .4 c m cuvettes (200 V , 9 5 0 p F , t ime constant ~ 30 msec) . C e l l s were then washed wi th culture media , and replated as bu lk cultures and in 9 6 we l l plates for c lona l select ion. Cu l ture in 1.5 mg/ml G 4 1 8 was used to generate stable transfectants. C H O ce l l s were transfected in 6 0 m m dishes w i t h the L i p o f e c t a m i n e P l u s reagent ( Invitrogen) accord ing to manufacturer 's instructions. P lus reagent (8 p i ) was added to 2 p g D N A , prev iously di luted in 2 5 0 p i med ia l a c k i n g serum and ant ibiot ics . T h i s mixture was incubated for 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 di luted in 2 5 0 p i serum-free media . These components were c o m b i n e d and incubated for 15 minutes before they were transferred to plates o f ce l ls conta in ing 2 m l of f resh serum-free media . A f t e r 3 -hour incubat ion at 37°C, 3 ml o f m e d i a conta in ing serum was added, and expression was assessed 2 days later. 9 4 5.6 x IO 6 E S C s were electroporated in 0.8 m l P B S conta in ing 20 p g D N A (240 V , 5 0 0 p F , t ime constant ~ 7 msec) . C e l l s were then a l l o w e d to recover on ice for 20 minutes , washed w i t h cu l ture m e d i a , and transferred to 2 1 0 - c m plates. Stable c lones were obtained by cu l tur ing in 150 pg/ml G 4 1 8 . 2.3 Expression Analysis 2.3.1 Antibodies T h e antibodies used in this thesis are shown in (Table 2-1 and Tab le 2-2) . 95 Antibody Type Concentration Procedure Source a-ezrin ms IgG! 25 pg/ml Confocal 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, F C M 2 : Sigma-Aldrich a - G F P rb IgG (pAb) 10 pg/ml IF Invitrogen a - N H E R F l rb (pAb) 3 u,g/ml Confocal Abeam a - P E C A M - 1 rat IgG 2 a 5 pg/ml IHC B D Biosciences a-ch podocalyxin ms IgG[ neat Confocal, F C (McNagny et al., 1992) a-ch podocalyxin ms IgGi 1:5 Western (McNagny et al., 1992) a-ms podocalyxin rat IgG! 5 pg/ml Confocal, F C M B L (Woburn M A ) a-ms podocalyxin rat IgG, 5 pg/ml IHC (Hara et al., 1999) ms IgG; Isotype control 25 pg/ml Confocal D A K O (Mississauga ON) 96 Antibody Type Concentration Procedure Source ms I g G , (biotin) Isotype control 25 u.g/ml C o n f o c a l R & D Systems (Minneapo l i s M N ) rat I g G , Isotype control 5 p,g/ml F C , I H C Cedar lane (Hornby O N ) rb I g G Isotype control 3 pg/ml C o n f o c a l Jackson ImmunoResearch Laborator ies (Westgrove P A ) pha l lo id in A l e x a F l u o r - 5 6 8 25 units/ml C o n f o c a l Invitrogen Table 2 - 1 : Primary Antibodies used in this Thesis. p A b : po lyc lona l antibody ch : ch i cken , rb: rabbit, ms: mouse F C : f l o w cytometry , IF : immunof luorescence , I H C : immunohistochemist ry . 97 Antibody Label Concentration Procedure Source a - m s Ig A P C 2 pg/ml F C B D B iosc iences a - m s Ig H R P 0.1 pg/ml Western D A K O a - m s I g G , A l e x a F luor 488 8 - 20 pg/ml C o n f o c a l Invi trogen a - m s IgG[ A l e x a F l u o r 568 20 pg/ml C o n f o c a l Invitrogen a - ra t I g G biot in 1 u,g/ml F C Southern B io tech ( B i r m i n g h a m A L ) a - ra t I g G biot in 5 pg/ml I H C V e c t o r Laborator ies (Bur l ington O N ) a - r b IgG A l e x a F l u o r 488 20 u.g/ml C o n f o c a l Invitrogen a - r b IgG A l e x a F luor 488 4 pg/ml I F Invitrogen streptavidin A l e x a F l u o r 568 5 pg/ml C o n f o c a l Invitrogen streptavidin A P C 0.5 pg/ml F C B D B iosc iences streptavidin P E 1.25 p,g/ml F C B D B iosc iences Table 2-2: Secondary Antibodies used in this Thesis. A P C : a l lophycocyan in , H R P : horse radish peroxidase, P E : phycoerythr in rb: rabbit, ms: mouse F C : f l o w cytometry , IF: immunof luorescence , I H C : immunohis tochemist ry . 98 2.3.2 Flow Cytometry and Cell Sorting C e l l s were washed w i t h F A C S buf fer (10 % F B S and 0.05 % s o d i u m az ide in P B S ) , labe l led w i t h p r imary ant ibodies (Table 2 -1) fo r 2 0 - 3 0 minutes on i ce , washed w i t h F A C S buffer , incubated w i th secondary ant ibodies (Table 2 -2) fo r 2 0 minutes on i ce , washed again , and stained wi th the v iab i l i t y marker 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 iosc iences , M i s s i s s a u g a O N ) . In most exper iments , 10 0 0 0 v iable cel ls were co l lected w i th a F A C S C a l i b u r f l o w cy tometer (Bec ton D i c k i n s o n ) and ana lyzed w i t h F l o w J o software (Tree Star Inc, A s h l a n d O R ) . C e l l sort ing was performed by A n d y Johnson w i th a B D F A C S Vantage cel l sorter in the B i o m e d i c a l Research Centre 's F A C S fac i l i t y . M C F - 7 c e l l s t ransfected w i t h m u r i n e podxl were assessed 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 io t iny la ted ant i - rat I g G , , and p h y c o e r y t h r i n ( P E ) - or a l l o p h y c o c y a n i n (APC) - con jugated streptavidin ( S A ) . M C F - 7 cel ls transfected w i th ch icken podxl mutants were assessed us ing mouse ant i - ch icken podoca l yx in or b iot iny lated mouse ant i - f lag and A P C - c o n j u g a t e d ant i -mouse 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 th the empty vector were used as negative controls fo r podoca lyx in staining. Transfected E S C s were assessed fo r mur ine p o d o c a l y x i n expression us ing rat ant i -mouse p o d o c a l y x i n , b iot iny lated anti - rat IgG[ , and phycoery thr in (PE) - con jugated streptavidin ( S A ) . Rat IgG[ was used as a negative control for podoca lyx in staining. 99 2.3.3 Western blotting C e l l s w e r e l y s e d w i t h R I P A b u f f e r p l u s p r o t e a s e i n h i b i t o r s , i n c l u d i n g phenylmethanesul fony l f luor ide ( P M S F ) and a protease inhib i tor cockta i l ( S i g m a - A l d r i c h #P8340). R I P A buffer contained 10 m M phosphate buffer , 150 m M sod ium chlor ide , 1 % N P - 4 0 , 1 % s o d i u m d e o x y c h o l a t e , 0.1 % s o d i u m d o d e c y l su l fate ( S D S ) , 2 m M ethy lenediaminetet raacet ic ac id ( E D T A ) , and 5 0 m M s o d i u m f luo r ide . Prote ins were separated by S D S - p o l y a c r y l a m i d e gel e lec t rophores is ( P A G E ) and t ransfer red to n i t rocel lu lose membranes. M e m b r a n e s were b locked for 30 minutes wi th 5 % s k i m m i l k in T r i s buf fered sal ine ( T B S ) p lus 0 .05 % 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 incubat ion in p r imary ant ibody (mouse an t i - f l ag or mouse a n t i - c h i c k e n p o d o c a l y x i n ) d i luted in T B S - T and 3 5 -minute washes wi th T B S - T (Table 2 -1) . M e m b r a n e s were then probed w i t h secondary ant ibody (horse radish 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 I g G ) d i luted i n 2 - 3 % B S A in T B S - T for 45 minutes and washed 3 x 5 minutes w i th T B S - T (Table 2 -2 ) . Western blots were developed after expos ing f i l m ( K o d a k ) to membranes that were prev ious ly incubated w i t h enhanced chemi luminescence ( E C L ) reagent (Perk in E l m e r , W o o d b r i d g e O N ) for 1 minute. 2.3.4 RT-PCR m R N A was isolated us ing p M A C S m R N A isolat ion kits ( M i l t e n y i B i o t e c , A u b u r n C A ) accord ing to manufacturers instruct ions. c D N A was generated us ing Thermosc r ip t R T -P C R kits ( Invitrogen) accord ing to manufacturer 's instructions. M u r i n e podxl c D N A was detected by P C R w i t h pr imers 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 ' and 5 ' -100 T A C T C G A G T G G G T T G T C A T G G T A A C C - 3 ' . G F P was detected w i t h 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 ' a n d 5 ' - T C C T T G A A G A A G A T G G T G C G - 3 ' . Hypoxanth ine phosphor ibosy l transferase ( H P R T ) was used as a posit ive contro l , w i th 5 ' -C T C G A A G T G T T G G A T A C A G G - 3 ' a n d 5 ' - T G G C C T A T A G G C T C A T A G T G 3 ' pr imers. 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 for (3-glactosidase expression, they were washed w i th P B S , and f i x e d for 5 minutes at room temperature in 0 .2 % glutaraldehyde, 0.01 % s o d i u m d e o x y c h o l a t e , 0 . 0 2 % N P - 4 0 , 100 m m m a g n e s i u m c h l o r i d e , and 5 m m e thy lene -bis(oxyethylenenitr i lo)tetraacetic ac id ( E G T A ) in P B S . C e l l s were then washed 3 t imes and stained overn igh t at 37 ° C u s i n g 1 mg/ml X - g a l , 5 m M K 3 F e ( C N ) 6 , 5 m M K 4 F e ( C N ) 6 , and 2 m M magnes ium chlor ide in P B S . 2.4.2 PCR to Detect Transgene G e n o m i c t r a n s g e n i c m u r i n e podxl w a s de tec ted 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 ' -T A C T C G A G T G G G T T G T C A T G G T A A C C - 3 ' . G F P was detected w i t h p r imers 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 ' . 101 2.5 Adhesion Assays M C F - 7 cel ls were plated in tr ipl icate in 6 we l l plates (5 x 10 5 cel ls per wel l ) and cultured for 2 4 hours. Suspens ion cel ls inc luded those in culture med ia as w e l l as those obtained after wash ing w i th P B S for 1 minute. Adherent cel ls were removed w i th t ryps in , d i luted wi th med ia , and counted as w e l l . 2.6 Confocal Microscopy C e l l s were cultured on glass cover s l ips and f i xed for 20 minutes w i th 37 °C preheated 4 % P F A . A l l subsequent steps were per formed at r o o m temperature. C e l l s were then rehydrated w i th P B S , permeabi l i zed with 0.1 % T r i ton X - 1 0 0 for 10 -15 minutes (or 0.5 % T r i ton X - 1 0 0 for 10 minutes for actin label l ing) , and washed 2 x 1 0 minutes w i th P B S before i m m u n o l a b e l l i n g . C o v e r sl ips were b locked for 4 0 minutes w i t h 10 % goat serum and 1 % B S A in P B S , and then r insed br ie f l y w i th P B S . Samples were incubated w i t h pr imary ant ibodies (Table 2-1) in 1 % B S A for 1 hour, f o l l o w e d by 4 10-minute washes wi th P B S or 1 % B S A in P B S . Secondary antibodies (Table 2-2) were also d i luted in 1 % B S A in P B S and incubated wi th samples for 1 hour, f o l l o w e d by a single 10-minute wash wi th P B S or 1 % B S A in P B S . For actin labe l l ing , A l e x a F l u o r 568-conjugated pha l lo id in (in 1 % B S A ) was added for 15 minutes at this stage, f o l l o w e d by a 10-minute wash w i t h P B S . N u c l e i were label led w i th 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 ) fo r 2 minutes , and ce l ls were washed w i th P B S or 1 % B S A in P B S 4 x 10 minutes . C o v e r s l ips were mounted w i t h f l u o r o m o u n t - G (Southern B io tech ) . C e l l s transfected w i th the 102 empty vector were used as negative controls fo r p o d o c a l y x i n sta in ing. R a b b i t I g G was used as a negative control for N H E R F - 1 staining. B i o t i n y l a t e d ezr in and p o d o c a l y x i n dual l a b e l l i n g requi red add i t iona l steps to prevent cross- react iv i ty and background f r o m endogenous b iot in . Streptavidin was added dur ing the b l o c k i n g step at 1 pg/ml to b l o c k ce l lu la r b i o t i n , and this was f o l l o w e d by 3 10 -minute washes wi th P B S to remove excess unbound streptavidin. In contrast to other dual labe l l ing exper iments , in this case antigens had to be label led sequent ial ly . P o d o c a l y x i n was label led f i rst , w i t h the pr imary ant ibody 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 luor -488-con jugated a n t i - m l g G , . C o v e r sl ips were then b locked w i th 10 % mouse serum and 1 % B S A in P B S for 4 0 minutes f o l l o w e d by two add i t iona l P B S washes. E z r i n was then labe l led w i t h b io t iny la ted 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 labe l l ing , as descr ibed above. C e l l s transfected w i th the empty vector were used as negative contro ls fo r p o d o c a l y x i n sta in ing. M o u s e IgG[ was used as a negative control fo r ezr in staining. S l ides were examined using an O l y m p u s F l u o v i e w F V 1 0 0 0 confoca l mic roscope ( 1 0 0 X o i l immers ion object ive, 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 object ive, N A : 1.40). T r i p l e - l a b e l l e d images were co l lected sequent ia l ly , and confoca l sections were acquired in 0 . 1 8 0 [xm or 0 . 2 3 0 p m steps. M i c r o g r a p h s were generated w i t h several merged confocal planes or vert ical sections of confoca l stacks us ing O l y m p u s F l u o v i e w F Y 1000 sof tware ( ve rs ion 1.3b). Photos were ar ranged w i t h A d o b e P h o t o s h o p and A d o b e Il lustrator software. 103 2.7 Scanning Electron Microscopy (SEM) C e l l s were g r o w n on glass cover s l ips and f i x e d us ing 2.5 % glutaraldehyde w i t h 1 % tannic ac id in 0.1 M cacodylate buffer. S E M processing was performed by Der r i ck H o m e in the B i o l m a g i n g Fac i l i t y at the Un ivers i ty o f B r i t i sh C o l u m b i a : samples were post - f i xed w i t h buf fered 1 % o s m i u m tetrox ide, dehydrated in a graded series of ethanols , and c r i t i ca l po int d r ied . Images were then co l lec ted us ing a H i t a c h i S 4 7 0 0 F E S E M , and photos were arranged using A d o b e Photoshop and A d o b e Il lustrator software. 2.8 Transmission Electron Microscopy (TEM) 2.8.1 TEM of Cell Lines C e l l s were g rown on f i l ters (1 urn pore size: Bec ton D i c k i n s o n ) and then f i xed for 1 hour in 1.5 % glutaraldehyde and 1.5 % paraformaldehyde in 0.1 M s o d i u m cacodylate buffer ( p H 7.3) . T h e f i l ters were washed w i th 0.1 M s o d i u m cacodylate buffer , and then post -f i x e d for 30 minutes on ice in buffered 1 % o s m i u m tetroxide. T h e y were washed w i t h d is t i l led water and stained en bloc fo r 30 minutes w i th 1 % uranyl acetate. Samples were then dehydrated through a graded series of ethanols and inf i l t rated wi th propylene ox ide and P o l y b e d . A f t e r embedd ing in P o l y b e d , the samples were po lymer i zed for 2 4 hours at 6 0 °C. T h i n sect ions were prepared, stained w i th uranyl acetate and lead citrate, and v i e w e d and photographed on a P h i l i p s 3 0 0 electron microscope operated at 60 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 sh C o l u m b i a . Negat ives were scanned into d ig i ta l 104 format and contrast adjusted us ing the Image Ad jus tments too l . F igures were arranged using A d o b e Photoshop and A d o b e Il lustrator software. 2.8.2 TEM of Kidneys K i d n e y s were i so la ted f r o m E 1 8 m i c e , cut into 4 - 6 p ieces , and f i x e d in 2 .5 % glutaraldehyde in 0.1 M cacodylate buffer overnight at 4°C. Der r i ck H o m e completed the process ing and i m a g i n g at the Un i ve rs i t y of 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 w i t h cacody late buffer , pos t - f i xed w i th 1 % o s m i u m tetrox ide, washed w i t h d is t i l l ed water, stained en bloc w i th 2 % uranyl acetate, and r insed w i t h d i s t i l l ed water. Samples were then dehydrated us ing a graded series o f ethanols and embedded in resin. T h i n sections were imaged using a H i tach i H 7 6 0 0 T E M , and f igures were arranged using A d o b e Photoshop and A d o b e Il lustrator software. 2.9 Immunofluorescence of Tissue Sections 2.9.1 Sample Preparation M i c e were anesthetized w i th 25 mg/ml avert in intraper i toneal ly and perfused w i th 4 % paraformaldehyde ( P F A ) . T issues were removed and f i x e d in 2 % P F A for 2 hours at 4 °C , f o l l o w e d by incubat ion in 20 % sucrose overn ight at 4 °C . E x c e s s l i q u i d was removed , samples were p laced in T issue T e k embedd ing m e d i u m (Sakura F inetek U S A Inc, T o r r a n c e C A ) , f r o z e n in l i q u i d n i t rogen , and stored at - 8 0 °C . T i s s u e s were 105 sectioned us ing a cryostat set at 6 - 1 2 p m , dr ied overnight , and rehydrated in P B S for 1 hour at 4 °C. 2.9.2 Tissue Staining T issue sections were b locked overnight w i th 25 % goat serum and 10 % B S A in 0.3 % T r i ton X - 1 0 0 in P B S . Sect ions were then incubated w i th a n t i - G F P (Table 2-1) in 10 % goat serum, 10 % B S A , and 0.3 % Tr i ton X - 1 0 0 in P B S for 1.5-2 hours at 4 °C. Samples were washed 5 x 12 minutes w i th 0.3 % T r i ton X - 1 0 0 in P B S at 4 °C. Sect ions were incubated w i th secondary ant ibody ( A l e x a F l u o r - 4 8 8 - c o n j u g a t e d goat ant i - rabbi t I gG ) (Table 2-2) in 10 % goat se rum, 10 % B S A , and 0.3 % T r i ton X - 1 0 0 in P B S fo r 1.5-2 hours at 4 °C and washed as above. C o v e r s l ips were mounted w i th f l u o r o m o u n t - G . T i s s u e sect ions f r o m C 5 7 B L / 6 ( B 6 ) m i c e were used as negat ive cont ro ls fo r G F P staining. S l ides were examined using a Ze iss microscope w i th a 4 0 X object ive or a 6 3 X o i l immers ion objective. 2.10 Immunohistochemistry T issues were embedded in T issue T e k , f rozen in l i q u i d n i t rogen, and stored at - 8 0 °C . F rozen samples were sect ioned, f i x e d in acetone, and rehydrated in P B S . Samples were stained us ing the Vectasta in A B C K i t , accord ing to manufacturer 's instructions (Vector Laborator ies ) . S l ides were b l o c k e d w i t h 10 % F B S in P B S , l abe l led w i t h p r i m a r y antibodies (ant i -mouse podoca lyx in or P E C A M - 1 ) fo r 3 0 - 4 5 minutes (Table 2 -1 ) , washed with P B S , label led wi th secondary antibody (biot inylated anti-rat IgG) (Table 2-2) for 3 0 -106 4 5 minutes, and washed again. Rat I g G , was used as a negative control for p o d o c a l y x i n and P E C A M - 1 s ta in ing . S l ides were then incubated in 0 .3 % hydrogen perox ide in methanol fo r 1 5 - 3 0 minutes to quench endogenous peroxidase. T h i s was f o l l o w e d by 2 3 -minute P B S washes, incubat ion in the A B C reagent fo r 3 0 - 4 5 minutes, and another P B S wash. S l ides were developed w i th the d iaminobenz id ine ( D A B ) substrate, and washed in P B S . N u c l e i were label led wi th 1 % methyl green fo r 2 -3 minutes and sl ides were washed wi th P B S and di lute a m m o n i a . Samples were then dehydrated through a graded series of ethanols, f o l l o w e d by methanol and xy lene , and cover s l ips were sealed w i th Permount . S l i d e s were e x a m i n e d us ing a Z e i s s mic roscope w i t h a 2 0 X object ive or a 6 3 X o i l immers ion objective. 2.11 Mice A l l m i c e were mainta ined on a B 6 background in the B i o m e d i c a l Research Cent re ' s mouse fac i l i t y . M i c e were genotyped for the wi ld type or knockout al lele of podxl by P C R u s i n g 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 ' , 5 ' -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 ' - A G T G A G A G A C A C A T T G G G T A A C T -3 ' . T h e first pr imer is a c o m m o n pr imer , w i th a sequence found in the f i f th exon o f podxl, the second p r imer ' s sequence is found in the neomycin resistance cassette of knockout mice , and the sequence o f the third pr imer is found in the f i f th intron o f wi ld type animals . T h e expected P C R products are 5 5 0 bp (knockout ) and 7 6 0 bp (wi ld type) in length. 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 ransgenic m i c e were genotyped f o r presence o f the transgene using the G F P - s p e c i f i c pr imers 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 bp P C R product. 107 CHAPTER 3 : OVEREXPRESSION OF WILDTYPE AND MUTANT PODOCALYXIN IN EPITHELIAL CELLS 3.1 Rationale A t the outset of this study, there were on ly two previous publ icat ions based on in vitro analys is of 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 rst descr ibed p o d o c a l y x i n as a l i gand for L -select in in H E V (Sassetti et a l . , 1998) , w h i l e the second suggested that p o d o c a l y x i n inhibi ted c e l l - c e l l adhesion and disrupted ce l l junct ions (Takeda et a l . , 2000) . It was clear that further analys is was required in order to dec ipher p o d o c a l y x i n ' s func t ion and to understand its roles m e c h a n i s t i c a l l y . La te r papers p rov ided b i o c h e m i c a l ev idence 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 act in cy toske leton through ezr in and members o f the N H E R F f a m i l y of adapter proteins ( L i et a l . , 2 0 0 2 ; Or lando 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 func t iona 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 was therefore expressed in numerous ce l l l ines in order to c lar i fy its funct ion and mechan ism of action.* • Some data presented in this chapter can be found in the f o l l o w i n g art icles: 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 . , and 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 inducer of mic rov i l l us fo rmat ion . 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 . , Prent ice, L. , G i l k s , C . B . , C h i a , S . K . , G e l m o n , K . A . , K e r s h a w , D . B . , H u n t s m a n , 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 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 an independent predictor o f breast cancer progression. Cancer Res, 64, 5 0 6 8 - 5 0 7 3 . 108 In add i t ion , a part icular ly interesting feature of p o d o c a l y x i n ' s normal expression pattern is its tendency to be expressed in c e l l s w i t h un ique and in t r icate c e l l su r face morpho log ies . F o r example , podoca lyx in is expressed by megakaryocytes , w h i c h extend long processes when generating platelets ( M c N a g n y et a l . , 1997; M ie t t inen et a l . , 1999). A s d iscussed in sect ion 1.6, it is a lso essent ia l fo r f o r m a t i o n o f the elaborate foo t processes assoc ia ted w i t h mature k i d n e y podocy tes . A s s e s s i n g the p o s s i b i l i t y o f p o d o c a l y x i n h a v i n g a more g loba l role in regu la t ing c e l l sur face m o r p h o l o g y was therefore another major goal of this study. K i d n e y and breast epithel ial ce l l l ines were the m a i n model systems used for our in vitro studies since in addi t ion to p o d o c a l y x i n ' s essential role in normal k idney deve lopment ( D o y o n n a s et a l . , 2 0 0 1 ) , our early studies a lso descr ibed p o d o c a l y x i n as a pred ic t ive marker of poor outcome in human breast cancer ( rev iewed in section 1.10.1) (Somasi r i et a l . , 2004) . S p e c i f i c a l l y , two ce l l l ines were used for the major i ty of exper iments : M D C K and M C F - 7 cel ls . M D C K cel ls were chosen because they are a wel l - character i zed k idney ce l l l ine that had been used in other podoca lyx in - re la ted publ icat ions ( L i et a l . , 2 0 0 2 ; O r lando et a l . , 2001) . M C F - 7 breast ca rc inoma cel ls were used since they are a w e a k l y invas i ve ep i the l ia l ce l l l ine that expresses l o w leve ls o f endogenous p o d o c a l y x i n , in compar ison to the more invas ive , h igh ly podoca lyx in -pos i t i ve M D A - 2 3 1 cel l l ine (F igure 3-1) (Somasi r i et a l . , 2004) . O f note, podoca l yx in is absent f r o m the non - invas i ve T 4 7 D breast ce l l l ine . T h u s , M D C K and M C F - 7 ce l l l ines were appropriate for overexpress ion studies to address the funct ion of podoca lyx in . 109 U o k D — | ••—Podocalyxin 42kDH $=ERK 1/2 F igure 3-1: Endogenous H u m a n Podoca lyx in Express ion i n Breast C a r c i n o m a Lines. Western blot analysis demonstrates that metastatic MDA-231 cells express considerably higher levels of podocalyxin than the less invasive T47D 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 suitable assay system fo r assessing p o d o c a l y x i n ' s f u n c t i o n , va r ious podoca lyx in mutants were generated and tested us ing this system. These studies prov ided important mechanist ic insights into podoca l yx in ' s funct ions. 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 was c loned into the p I R E S 2 - E G F P express ion vector to enable ectopic express ion in vitro. T h i s vector fac i l i tates express ion of any gene of 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 ) sequence. In a d d i t i o n , a n e o m y c i n resistant cassette a l lows fo r selection of stably expressing transfectants. M C F - 7 and M D C K cel ls were transfected w i th 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 , or the empty vector as a control . Stably transfected clones were isolated based on expression o f podoca lyx in or G F P in combinat ion wi th the neomycin- res istance gene (F igure 3 -2) . I l l 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 in i t ia l examinat ion of transfected ce l l s , the most obv ious di f ference between those t ransfected w i t h p o d o c a l y x i n or empty vector was that ce l l s e c t o p i c a l l y express ing p o d o c a l y x i n f o r m e d large clusters o f suspension cel ls rather than conf luent monolayers of adherent ce l l s , as shown in F igure 3 - 3 . Quant i tat ive exper iments revealed that there were c lose to fou r t imes as many ce l l s in suspension in the podoca lyx in - t rans fec ted populat ion (F igure 3 -4) . These cel ls maintained v iab i l i t y , as assessed by f l o w cytometry and rep lat ing o f disaggregated ce l l s . These exper iments d id not e x p l a i n , however , the m e c h a n i s m lead ing to decreased cel l -substrate adhesion. It is somewhat counter intui t ive that a protein expressed on the apical surface of epithel ia l ce l ls cou ld lead to decreased adhesion at the basolateral surface. Further experiments (described below) prov ided some insights into this phenomenon. M o r e o v e r , in support of prev iously publ ished work (Takeda et a l . , 2000) , there were also apparent alterat ions in c e l l - c e l l j unc t ions in M C F - 7 ce l ls overexpress ing p o d o c a l y x i n (F igure 3 -5) (Somas i r i et a l . , 2004) : instead o f a we l l a l igned apical bar of s ta in ing, as in the vector cont ro l , p o d o c a l y x i n - p o s i t i v e populat ions d isp layed d isorganized loca l i za t ion of the tight j unc t ion protein, Z O - l . T h u s , the data con f i rmed that p o d o c a l y x i n funct ions 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 , and ce l l - subst ra te interactions. 113 Empty vector Podocalyxin Figure 3-3: Transfected MCF -7 Cells Ectopically Expressing Murine Podocalyxin Exhibited Decreased Cell-Substratum Interactions. Scale bar: 50 u m . 114 c 100 g '55 c 80 0) a in 60 3 </> C Isi 40 a> o 20 o 0 T 1 Empty vector Podocalyx in Figure 3-4: Podocalyxin Decreases Cell Substrate Adhesion. A f t e r M C F - 7 cel ls transfected w i th empty vector or vector encoding murine podoca lyx in were sorted based on podoca l yx in express ion, 5 x 10 5 cel ls were plated per we l l in 6 - w e l l plates. One day later, suspens ion and adherent ce l l s were counted in t r ip l icate we l l s . Representative of 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 ce l l s transfected w i th empty vector or vector encod ing mur ine p o d o c a l y x i n and label led w i t h antibodies against Z O - 1 (green) and podoca l yx in (red). C e l l junct ions were abnormal and monolayers were disrupted upon ectopic expression of podoc a l y x i n . Scale bar: 15 UMTI (Somasi r i et a l . , 2004) . 116 3.2.3 Podocalyxin Recruits NHERF1 to the Apical Surface of Cells T h e prev ious exper iments c lear ly supported the role o f p o d o c a l y x i n in d is rupt ing ce l l adhes ion , but h o w it a c c o m p l i s h e d this was not i m m e d i a t e l y obv ious . S ince the act in cytoskeleton plays an important role in cel l adhesion, molecules that have been suggested to l i n k p o d o c a l y x i n to act in were examined . A l t h o u g h b i o c h e m i c a l ev idence suggested that p o d o c a l y x i n c o u l d interact w i th 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 on ly been shown 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 . , 2001) . T a k i n g advantage o f the fact that M C F - 7 cel ls express N H E R F 1 , its loca l i zat ion wi th respect to podoca lyx in was assessed by confoca l microscopy . In ce l l s express ing both m o l e c u l e s , p o d o c a l y x i n and N H E R F 1 were c lear ly a p i c a l l y co loca l i zed (F igure 3 -6) . In cel ls transfected w i th empty vector, however , N H E R F 1 was cons is tent ly expressed throughout the c y t o p l a s m ( F i g u r e 3 - 6 C , left panel ) . T h u s , p o d o c a l y x i n express ion led to a dramatic increase in ap ica l l y l oca l i zed N H E R F 1 wi th a concomitant decrease in cy top lasmic stain ing (F igure 3 - 6 C , r ight panel) . S ince N H E R F f a m i l y proteins f u n c t i o n as s c a f f o l d i n g proteins and interact w i t h ac t in , it had been suggested that they cou ld play a role in proper loca l i zat ion of podoca l yx in (Cheng 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 . , 2004) . It was therefore very int r igu ing that p o d o c a l y x i n , not N H E R F 1 , was responsible for apical loca l i zat ion of both molecu les (F igure 3 -6) . Regard less , the interact ion of p o d o c a l y x i n w i th this scaf fo ld ing protein i m p l i e d a l i n k to the cytoskeleton and a potential c lue to p o d o c a l y x i n ' s role in b l o c k i n g ce l l adhesion. 117 Empty vector Podocalyxin B NHERF1 Podocalyxin Podocalyxin NHERF1 DAPI Figure 3-6: Confocal Images Demonstrated Apical Recruitment of NHERF1 by Podocalyxin. M C F - 7 ce l ls transfected w i th empty vector or vector encod ing mur ine podoca lyx in were l a b e l l e d w i t h D A P I (blue) and ant ibod ies 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 and N H E R F 1 . Imag ing by M a r c i a L. M c C o y , Univers i ty o f B r i t i sh 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 ga in ing some insights into p o d o c a l y x i n ' s funct ion as an ant i -adhesion molecu le , it was possible to concentrate on its potential role in in f luenc ing cel l morpho logy . E lect ron m i c r o s c o p y was used to assess the ce l l surface o f ep i the l ia l ce l l s transfected w i t h p o d o c a l y x i n or empty vector. In part icular , the fo rmat ion of m i c r o v i l l i was assessed by electron m i c r o s c o p y because there is some ev idence to suggest that the p o d o c a l y x i n b i n d i n g prote in , N H E R F 1 may be i n v o l v e d in the generat ion o f such structures. F o r example , the intestinal brush border m i c r o v i l l i of one strain of N H E R F l - n u l l mice are s t r ik ing ly d isorganized in c o m p a r i s o n to those o f w i ld t ype l i ttermates ( M o r a l e s et a l . , 2 0 0 4 ) . Fu r th e rm ore , when M C F - 7 ce l l s are treated w i t h es t rad io l , they upregulate N H E R F 1 m R N A , and the number and length o f m i c r o v i l l i on the cel l surface increases (Ed iger et a l . , 1999; V i c et a l . , 1982). W h i l e this certainly does not prove that N H E R F is responsible for m i c r o v i l l u s fo rmat ion , in combinat ion wi th the above data it is suggestive enough to warrant further invest igat ion . 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 in podocalyx in - t ransfected M C F - 7 cel ls . T h e dif ference between the two ce l l populat ions was st r ik ing : a l though control cel ls had some 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 ce l l s were covered w i t h these ce l l surface prot rus ions , as s h o w n by scann ing electron m i c r o s c o p y ( S E M ) (F igure 3 -7 ) . A very s imi la r result was observed in transfected M D C K cel ls (F igure 3 -8 ) , demonstrat ing that this dramatic phenotype was not an artifact of one particular ce l l l ine. B o t h ce l l l ines were 119 also e x a m i n e d by t ransmiss ion e lectron m i c r o s c o p y ( T E M ) , w i t h consistent results (Figure 3-9) . 120 Empty vector Podocalyxin Figure 3-7: Podocalyxin Induced Microvillus Formation in M C F - 7 Cells. S E M ' s o f M C F - 7 ce l l s t ransfected w i t h empty vector or vector e n c o d i n g m u r i n e podoca lyx in . Scale bar: 2 p m . Representative o f two independent experiments. 121 Empty vector Podocalyxin Figure 3-8: Podocalyxin Also Induced Microvillus Formation in MDCK Epithelial Cells. S E M ' s o f M D C K ce l l s t ransfected w i t h empty vector or vector e n c o d i n g m u r i n e podoca l yx in . Scale bar: 2 u m . 122 Empty vector Podocalyxin MCF-7 MDCK I n 1 f a • Q M wmmn Figure 3 - 9 : TEM's of Epithelial Cells Transfected with Empty Vector or Vector Encoding Murine Podocalyxin. Scale bar: 1 p m . Representat ive o f two independent exper iments. Imaging by A . W a y n e V o g l , Un ivers i t y o f B r i t i s h C o l u m b i a . 123 3.3 Generation and Analysis of Podocalyxin Mutants In order to gain further ins ights into the mechan isms 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 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 fo rmat ion , a panel o f ch icken p o d o c a l y x i n mutants was generated, as depicted in F igure 3 -10. M o s t mutations were in the cy top lasmic tai l as the h i g h leve l o f conserva t ion in this reg ion suggested f u n c t i o n a l i m p o r t a n c e , and mutat ions were expected to b l o c k or promote interact ion w i th k n o w n and u n k n o w n b ind ing partners. T h e mutants ranged f r o m those l a c k i n g entire domains to those w i th s ing le point mutat ions , as f o l l o w s : 1) serine and threonine residues o f t w o potent ial phosphory lat ion sites were mutated, alone or in combinat ion , to alanine residues to b lock phosphory lat ion in the A l a mutants; 2) the same residues were converted to aspartic ac id residues to m i m i c const i tut ive phosphory lat ion in the A s p constructs ; 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 fo r interact ion w i t h N H E R F p r o t e i n s ; 4) A t a i l l a c k e d the ent i re c y t o p l a s m i c ta i l w i t h the e x c e p t i o n o f the j u x t a m e m b r a n e sequence C C H Q R F , w h i c h was retained as a membrane anchor (this mutant lacked al l potential phosphory lat ion 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 ez r in -b ind ing site); 5) A E C lacked the major i ty of the extracel lular domain ( i n c l u d i n g the m u c i n d o m a i n and the cys te ine -bonded g lobu lar d o m a i n ) and instead encoded an extracel lu lar f lag - tag , as we l l as podoca l yx in ' s t ransmembrane region and its f u l l - l e n g t h c y t o p l a s m i c t a i l ; and 6) the f i n a l cons t ruc t , A l t . s p l i c e , c o n t a i n e d p o d o c a l y x i n ' s natural ly occur r ing spice var iant , w h i c h lacks m u c h o f the c y t o p l a s m i c tai l . 124 Wildtype Ala Asp ADTHL Atail Alt. splice AEC Figure 3-10: Schematic of Podocalyxin Mutants. L i g h t b lue : ext racel lu lar d o m a i n , green: f l a g - t a g , red: t ransmembrane d o m a i n , purple: cy top lasmic ta i l ( inc lud ing C - t e r m i n a l D T H L in w i l d t y p e , A l a , A s p , and A E C ) , y e l l o w : c y t o p l a s m i c ta i l o f spl ice var iant , hor izonta l l ines : g l ycosy la t ions , A : point mutat ion to a lanine, and D: point mutat ion to aspartic ac id . 125 A v i a n p o d o c a l y x i n was chosen as the basis fo r these experiments fo r three reasons: 1) it is 8 5 % ident ica l to m a m m a l i a n p o d o c a l y x i n in its intracel lu lar d o m a i n , 2) as w i th the murine protein used in section 3 . 2 , w i ld type av ian podoca lyx in also induced m i c r o v i l l u s fo rmat ion i n M C F - 7 ce l ls , and 3) I cou ld select ively detect its ectopic expression us ing a spec ies -spec i f ic m o n o c l o n a l antibody that reacts w i th native, f i x e d , and denatured f o r m s of the molecu le ( 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 descr ibed above were c l o n e d into the p c D N A 3 . 1 express ion vector and transfected into M C F - 7 ce l l s . Unfo r tunate ly , there was a consistent p r o b l e m wi th a l l 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 studies: i n b u l k popu la t ions o f t ransfected c e l l s , ce l l s express ing p o d o c a l y x i n were cont inuous ly lost f r o m cultures. A l t h o u g h this was partly due to loss o f suspens ion ce 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 cu l ture techniques to retain suspension cel ls d id not complete ly solve the prob lem. In i t ia l ly , this led to rec lon ing of constructs into an alternative express ion vector ( p I R E S 2 - E G F P ) . A s this d id not so lve the p r o b l e m either , F A C S was used to ser ia l l y sort cul tures fo r p o d o c a l y x i n express ion . A l t h o u g h this enabled generat ion o f populat ions c o n t a i n i n g higher proport ions of p o d o c a l y x i n - p o s i t i v e ce l l s , expression was st i l l not stable. F igure 3-11 shows F A C S prof i les of transfected cel ls before sort ing, and after one, two , or three rounds o f sor t ing . 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 ce l l s d id increase 126 after each round, the cel ls that were cultured after each sort are shown in the white boxes, and this level of podoca lyx in expression was clear ly not maintained. A l t h o u g h not perfect ei ther , the most successfu l strategy for generat ing stably express ing cultures was to isolate and expand single podoca lyx in -pos i t i ve c lones. U s i n g this strategy, at least three c lones were iso lated for each mutant. M o s t exper iments were per fo rmed w i t h these c lones, and key results were conf i rmed wi th bulk populat ions. 127 > GFP Figure 3 - 1 1 : Podocalyxin Expression After Multiple Rounds of Cell Sorting. Transfected M C F - 7 cel ls were sorted based on p o d o c a l y x i n and G F P expression. C e l l s in white boxes were expanded and ser ia l ly sorted. Thus , ce l ls i n the f irst white box were sorted and expanded. These cel ls gave rise to the cel ls depicted in the second F A C S plot. S i m i l a r l y , on ly the cel ls i n the second white box were retained; these gave rise to a l l of the cel ls depicted in the third F A C S blot , and so on . 128 3.3.1.2 Podocalyxin Mutants were All Expressed and were of Expected Molecular Weights Western blott ing was used to assess the expression o f each mutant in c lona l populat ions (F igure 3 -12) . Th i s also enabled conf i rmat ion that each f o r m of p o d o c a l y x i n was o f the appropriate molecu lar weight . The lower band was non -spec i f i c , as it was also observed in empty vector transfected cel ls (Vector 6). Fu l l - l eng th p o d o c a l y x i n was approx imate ly 160 k D a , as expected ( W i l d t y p e 7 and W i l d t y p e 13) , as were mutants 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 (Atai l 2 and Ata i l 8) and the alternatively sp l iced f o r m of p o d o c a l y x i n (A l t . spl ice 5) , w h i c h al l lack most of podoca l yx in ' s cy top lasmic ta i l , were not iceably shorter, as up to 71 amino acids had been deleted. Expected ly , t runcat ion of the four C - t e r m i n a l amino acids made l itt le di f ference to the size of 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 most of the ext racel lu lar d o m a i n was detected us ing an a n t i - f l a g ant ibody , w h i c h recogn i zed the f l a g - t a g inserted in p lace o f the deleted por t ion o f p o d o c a l y x i n . A s p red ic ted , this mutant was c o n s i d e r a b l y shorter than f u l l - l e n g t h podoca lyx in due to the absence of the heavi ly g lycosy lated m u c i n domain ( A E C 8). 129 Figure 3-12: Western Blots Demonstrated Expression of Podocalyxin Mutants of the Expected Molecular Weights in Clonal Populations. Note the s l ight ly smal ler size o f podoca lyx in mutants w i th shorter cy top lasmic tai ls . C e l l s transfected w i t h the empty vector were used as negat ive contro ls . S i z e markers were measured in 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 many exper iments were performed wi th al l mutants, there was no detectable di f ference between wi ld type podoca lyx in and the phosphory lat ion mutants ( A s p and A l a ) or between podoca lyx in ' s spl ice variant (A l t . spl ice) and the f u l l cy top lasmic tail delet ion (Atai l ) . F o r this reason, repetit ive in format ion has been omitted by i n c l u d i n g analys is o f just f i ve constructs in each f igure : empty vector , w i ld type p o d o c a l y x i n , A D T H L , A t a i l , and A E C . Western blott ing was used to detect protein expression of the mutants described in the f o l l o w i n g exper iments (F igure 3 -13) , and surface express ion was c o n f i r m e d by f l o w cy tometry , as shown in F igure 3 - 1 4 . Express ion levels were comparab le , w i t h the except ion o f the extracel lular delet ion, A E C , w h i c h could not be accurately compared due to the use of an independent antibody for its detection. 131 chPodocalyxin Figure 3-13: Western Blots Demonstrated Expression of Podocalyxin in Clonal Populations Used for Subsequent Experiments. Note the sl ight decrease in size when the cy top lasmic tai l was deleted. S ize markers were measured in 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. Transfected ce l ls were c l o n a l l y sorted, expanded, and labe l led w i th ant ibodies 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) . Dot ted l ines represent background staining measured on empty vector-transfected cel ls . Representative of 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 Prev ious exper iments 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 cel ls (Figure 3 -6) , and it is k n o w n that p o d o c a l y x i n ' s four C - te rmina l amino acids are the P D Z recogni t ion site for 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 . , 2006) . If p o d o c a l y x i n and N H E R F 1 were truly interact ing in M C F - 7 ce l l s , then co loca l i za t ion w o u l d be lost in ce l ls expressing podoca l yx in 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 mic roscopy was therefore used to assess this interact ion. A s s h o w n in merged images taken at the a p i c a l surface o f t ransfected c e l l s , w i l d t y p e p o d o c a l y x i n s h o w e d st rong 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 C - t e r m i n a l truncation mutants (Atai l and A D T H L ) d i d not (F igure 3 -15) . In contrast, the A E C mutant l a c k i n g the extracel lular domain but retain ing the entire cy top lasmic tai l o f podoca l yx in was f o u n d m a i n l y a p i c a l l y l o c a l i z e d and c o l o c a l i z e d w i t h N H E R F 1 . T h i s fur ther demonstrates that p o d o c a l y x i n recruits N H E R F 1 to the ap ica l d o m a i n through its C -terminal D T H L motif . 134 NHERF1 Podocalyxin Vector Wildtype ADTHL H Atail AEC Isotype Figure 3-15: Confocal Analysis of N H E R F 1 and Podocalyxin at the Apica l Surface of Transfected M C F - 7 Cells. C e l l s were l a b e l l e d w i t h D A P I (blue) and ant ibod ies 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 . T h e isotype contro l sample was labe l led 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), and an isotype control for N H E R F 1 (green) to demonstrate the speci f ic i ty o f N H E R F 1 labe l l ing . C e l l s transfected w i t h the empty vector were used as negative controls fo r p o d o c a l y x i n staining. Scale bar: 5 u m . Representative of two independent experiments. 135 A p i c a l recruitment of N H E R F 1 cou ld be c lear ly v isua l i zed in vert ical s l ices o f confoca l stacks (Figure 3 -16) . C y t o p l a s m i c N H E R F 1 staining was v is ib le in cel ls transfected w i t h C - t e r m i n a l delet ions of p o d o c a l y x i n , w h i l e an increase in apical N H E R F 1 stain ing was accompan ied by a decrease i n c y t o p l a s m i c sta in ing in the w i ld type and A E C samples. These exper iments 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 interact ion partners in vivo. Importantly , the podoca lyx in -dependent recruitment of N H E R F 1 to the apica l ce l l surface may have i m p l i c a t i o n s fo r funct iona l regulat ion o f N H E R F f a m i l y members. 136 N H E R F 1 Podocalyxin Wildtype A D T H L Vector Isotype A E C Atail Figure 3-16: Analysis of NHERF1 and Podocalyxin in Vertical Slices of Confocal Stacks. T r a n s f e c t e d M C F - 7 c e l l s were l a b e l l e d w i t h D A P I (b lue) and ant ibod ies 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 co loca l i za t ion o f N H E R F 1 and p o d o c a l y x i n . T h e isotype cont ro l sample was l a b e l l e d w i t h D A P I (b lue) , a n t i -p o d o c a l y x i n (red), and an i so type cont ro l fo r N H E R F 1 (green) to demonstrate the spec i f i c i t y o f N H E R F 1 l abe l l i ng . C e l l s transfected w i t h the empty vector were used as negat ive cont ro ls fo r p o d o c a l y x i n s ta in ing . Sca le bar: 5 u.m. Representat ive o f t w o independent experiments. 137 3.3.4 Analysis of Essential Sequences Required for Morphological Changes T h e effect of loss o f the p o d o c a l y x i n / N H E R F l interaction on m i c r o v i l l u s fo rmat ion was then assessed by electron m i c r o s c o p y us ing c l o n a l l y iso lated M C F - 7 ce l ls t ransfected wi th podoca lyx in mutant constructs (F igure 3 - 1 7 A and F igure 3 - 1 8 A ) . 3.3.4.1 Interaction with NHERF1 is Not Required for Formation of Microvilli T E M demonstrated that, as expec ted , cont ro l ce l l s had f e w m i c r o v i l l i w h i l e c e l l s transfected wi th f u l l - l e n g t h podoca l yx in d isp layed increased m i c r o v i l l i (F igure 3 - 1 7 B ) . S t r i k i n g l y , ce l l s express ing 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 numbers to f u l l - l e n g t h p o d o c a l y x i n t ransfectants . S E M a n a l y s i s c o n f i r m e d these observat ions (F igure 3 - 1 7 C ) . T h i s demonstrated that d i rect interact ion o f p o d o c a l y x i n w i th N H E R F proteins was surpr is ingly not required for fo rmat ion 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 ) Schemat ic o f p o d o c a l y x i n and mutants, as descr ibed in F igure 3 - 1 0 . (B ) T E M ' s o f transfected M C F - 7 ce l ls . Scale bar: 1 p m . (C ) S E M ' s of transfected M C F - 7 ce l ls . Scale bar: 2 p m . Representat ive o f two independent exper iments. T E M i m a g i n g by A . W a y n e V o g l , Un ivers i t y o f B r i t i sh C o l u m b i a . 139 3.3.4.2 Podocalyxin's Extracellular Domain is Required for Formation of Microvilli Converse l y , T E M revealed that the extracel lular domain o f podoca l yx in was essential for m i c r o v i l l u s fo rmat ion : there was no increase in m i c r o v i l l u s number in ce l ls express ing the A E C mutant bear ing only the f lag - tagged transmembrane and cy top lasmic d o m a i n o f p o d o c a l y x i n (F igure 3 - 1 8 B ) , even though this mutant was able to a p i c a l l y recru i t N H E R F 1 (see F igure 3 - 1 5 , above) . T h i s result was conf i rmed v i a S E M analys is (Figure 3 - 1 8 C ) . 1 4 0 Wildtype Vector AEC Figure 3-18: Deletion of the Major i ty of Podocalyxin's Extracel lular 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 and A E C mutant. ( B ) T E M ' s o f t ransfected M C F - 7 cel ls . Scale bar: 1 u m . (C) S E M ' s o f transfected M C F - 7 ce l l s . Sca le bar: 2 p m . Representat ive o f t w o independent exper iments . T E M i m a g i n g by A . W a y n e V o g l , Un ivers i t y o f B r i t i sh C o l u m b i a . 141 In order to quantitate this effect , a l l m i c r o v i l l i observed in s ix r a n d o m 15 0 0 0 X S E M f ie lds were enumerated for each mutant (F igure 3 -19). F u l l - l e n g t h , A D T H L , and Ata i l t ransfectants a l l had at least t w i c e as many m i c r o v i l l i as v e c t o r - c o n t r o l and A E C t ransfectants . It is therefore 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 d o m a i n , t ransmembrane reg ion , and s i x a m i n o acids 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 ta i l were suff ic ient to induce N H E R F 1 - i n d e p e n d e n t format ion of m i c r o v i l l i . 142 Microvilli / 50 pm2 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 ix random 5 0 u m 2 f ie lds were enumerated for M C F - 7 cel ls transfected w i t h each p o d o c a l y x i n construct. E r ro r bars represent standard dev ia t ion . Representat ive o f two independent experiments. 143 It has been s h o w n that maintenance o f podocyte foot process integr i ty is c r i t i c a l l y d e p e n d e n t on the n e g a t i v e l y - c h a r g e d g l y c o s y l a t i o n s d e c o r a t i n g p o d o c a l y x i n ' s extracel lu lar d o m a i n ( A n d r e w s , 1979; Ker jaschk i et a l . , 1984; Se i le r et a l . , 1975) ; it was therefore not par t i cu la r l y s u r p r i s i n g that de let ion o f the entire m u c i n d o m a i n a lso prevented 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 ce l l s . H o w e v e r , the fact that the h i g h l y c o n s e r v e d , N H E R F - b i n d i n g , c y t o p l a s m i c ta i l o f p o d o c a l y x i n was d i spensab le f o r m i c r o v i l l u s f o r m a t i o n was quite unexpected. It is k n o w n that the integrity of the act in cytoskeleton is essential fo r main ta in ing ce l l shape, and that l inkage o f act in to integral membrane proteins is important for generating and support ing ce l l surface protrusions, i n c l u d i n g m i c r o v i l l i (Revenu et a l . , 2004) . S ince N H E R F 1 and N H E R F 2 can connect p o d o c a l y x i n to the actin cytoskeleton through ezr in (Mora les 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 . , 2006) , it was in i t ia l l y assumed that p o d o c a l y x i n ' s invo l vement in 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 interact ion w i t h N H E R F proteins. M o r e o v e r , N H E R F 2 interact ions w i th p o d o c a l y x i n have recently been s h o w n to co inc ide c lose l y w i t h the f o r m a t i o n of a " p r e - a p i c a l d o m a i n " in M D C K ce l ls (as d iscussed in sect ion 1.5.4), and it was postulated that the associat ion was a prerequisite for fo rmat ion of this d o m a i n ( M e d e r et a l . , 2005) . H o w e v e r , the current data suggested that, a l though the C -terminal tai l o f p o d o c a l y x i n was suff ic ient to act ive ly recruit N H E R F 1 to apica l p lasma 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 and , indeed , the ap ica l target ing o f p o d o c a l y x i n to the p lasma membrane occurred in the absence of any direct interact ion w i th N H E R F . 144 3.3.5 Interaction with NHERF is Unnecessary for Colocalization of Podocalyxin with Ezrin S i n c e m i c r o v i l l i f o r m a t i o n c o i n c i d e s w i t h d r a m a t i c reo rgan i za t ion o f the a p i c a l membrane d o m a i n , po lymer i zat ion of f -ac t in at the core o f m i c r o v i l l i , and recruitment o f members o f the E R M f a m i l y of. proteins to the ap ica l d o m a i n , p resumably to act as l inkers between the cy toske le ton and t ransmembrane proteins ( rev iewed in ( L o u v e t -V a l l e e , 2000) ) , the loca l i za t ion o f ezr in and f - a c t i n were assessed in transfected ce l l s . In terest ing ly , ce l l s t ransfected w i t h a l l f o r m s o f 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 , regardless of whether or not they contained a N H E R F - b i n d i n g sequence, d isp layed increased apica l recruitment of ezr in and strong co loca l i za t ion of p o d o c a l y x i n w i t h this protein (F igure 3 -20) . Increased ap ica l recrui tment o f ez r in was espec ia l l y evident in vert ical sections of confoca l stacks, where strong ezr in staining corresponded to cel ls expressing ectopic podoca l yx in (F igure 3 -21) . T h u s , recruitment of ezr in to the apica l membrane occurred in the absence of any direct interact ion of p o d o c a l y x i n w i t h N H E R F . In contrast, recruitment of ezr in appeared to be somewhat less pronounced in the A E C mutant, again demonst rat ing the impor tance o f the ext race l lu lar d o m a i n in podoca lyx in funct ion. 145 Ezrin Podocalyxin Vector Wildtype ADTHL Atail AEC Isotype Figure 3-20: Confocal Analysis of Podocalyxin and Ezrin at the Apical Surface of Transfected MCF - 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 DAPI (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 Ezr in P o d o c a l y x i n Merge Figure 3-21: Vertical Sections of Confocal Stacks Demonstrated Increased Apical Recruitment of Ezr in 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 Occurs in the Absence of NHERF Binding Furthermore, apica l recruitment of f -ac t in was also independent of N H E R F - b i n d i n g . A l l f o r m s o f p o d o c a l y x i n demonst rated 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 recrui tment was notably less robust in the absence of the extracel lu lar d o m a i n (F igure 3 - 2 2 and F i g u r e 3 -23) . Cons is tent w i t h e lectron m i c r o s c o p y results, ce l l s express ing extracel lu lar d o m a i n - c o n t a i n i n g podoca l yx in mutants exhib i ted a clear punctate stain ing pattern on the apica l surface o f ce l ls in protruding structures indicat ive of 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 ha l lmarks typ ica l o f m i c r o v i l l i , they were e x a m i n e d at h igh 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 , ce l l s express ing A D T H L and A t a i l mutants each c lear ly demonstrated the presence o f act in f i laments in the m i c r o v i l l a r core , as shown both in long i tud ina l and cross -sect ions of ind i v idua l m i c r o v i l l i (F igure 3 -24) . In summary , podoca l yx in was able to recruit actin to m i c r o v i l l i in 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 ext race l lu lar domain was essential fo r m i c r o v i l l u s format ion . 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 Figure 3-23: Ver t ica l Sections of Confocal Stacks Demonstrated A p i c a l Recruitment of f -Ac t in in Podocalyxin-Transfected Cells. M C F - 7 cel ls transfected w i th w i ld type and mutant podoca l yx in were assessed for f -ac t in (red), p o d o c a l y x i n (green), and nuclear (blue) labe l l ing in vert ical sections o f confoca l s tacks . C e l l s t ransfected w i t h the empty vector were used as negat ive cont ro ls fo r podoca lyx in staining. Scale bar: 5 u m . Representative of two independent experiments. 150 Figure 3-24: Actin Filaments were Visible in Individual Microvi l l i . H i g h magn i f i ca t ion T E M ' s demonstrated the presence o f act in f i laments i n m i c r o v i l l i of ce l ls transfected w i t h w i ld type p o d o c a l y x i n as w e l l as A D T H L and Ata i l mutants. Sca le bars: 0.1 p m . ( A ) L o n g i t u d i n a l sections. (B ) C ross sections. Imaging by A . W a y n e V o g l , Un ivers i t y of B r i t i s h 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 in two epithel ial ce l l l ines in order to assess its effects on ce l l adhes ion and m o r p h o l o g y . In add i t ion to decreas ing ce l l -substrate adhesion and caus ing alterations in c e l l - c e l l junct ions , podoca l yx in was found to recruit N H E R F 1 and induce 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 in ep i the l ia l ce l l s p rov ided some ins ights into p o d o c a l y x i n ' s m e c h a n i s m of ac t ion . T h e p o d o c a l y x i n -dependent apical recruitment of N H E R F 1 was unexpected, but the requirement for the C -terminal D T H L sequence was not surpr is ing. In contrast, the we l l - conserved cy top lasmic tail was found to be dispensable for m i c r o v i l l u s fo rmat ion , wh i le the extracel lular domain was essential. 3.4.2 Podocalyxin's Role in Determining Cell Morphology Exper imenta l ev idence f r o m the 1970's suggested that h igh l y -g l ycosy la ted and heav i l y -s ia ly lated glycoproteins play a key role in mainta in ing the integrity o f the foot processes of k idney podocytes , as d iscussed in sect ion 1.6.3 ( A n d r e w s , 1979 ; Se i le r et a l . , 1977; Se i le r et a l . , 1975) . W i t h the subsequent ident i f i ca t ion of p o d o c a l y x i n as the major component of the podocyte g l y c o c a l y x and the tight corre lat ion between its express ion and podocyte foot process morphogenes i s in vivo, this m o l e c u l e became the p r ime candidate as a regulator of foot process fo rmat ion (Dekan et a l . , 1 9 9 1 ; K e r j a s c h k i et a l . , 1984; Sawada et a l . , 1986; Schnabel et a l . , 1989). G e n e targeting studies a l l owed us to 152 c o n f i r m that this m o l e c u l e is indeed requi red f o r the generat ion o f foo t processes : a l t h o u g h 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 recursors , these f a i l to u n d e r g o morphogenesis (described in section 1.6.4) (Doyonnas et a l . , 2001) . M o r e o v e r , this defect is rescued by k idney - spec i f i c ectopic expression o f podoca lyx in (see chapter 5). W i t h the current exper iments, these observations can be general ized: rather than be ing a podocyte -spec i f i c p h e n o m e n o n , express ion o f p o d o c a l y x i n was also suf f ic ient fo r induc t ion o f morphogenes is , as measured through m i c r o v i l l u s f o r m a t i o n , in two dif ferent ep i the l ia l ce l l l ines , 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 expressed and ap ica l l y targeted in normal m a m m a r y ep i the l ium in vivo (Somas i r i et a l . , 2004) and may be required fo r the extensive remode l ing that occurs in 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 induces m i c r o v i l l u s fo rmat ion is unclear , m y experiments suggest that an essential component of podoca l yx in ' s act iv i ty as a c e l l m o r p h o g e n is its ex t race l lu la r d o m a i n : mutants l a c k i n g this d o m a i n , though a p i c a l l y targeted, were unab le to induce m i c r o v i l l u s f o r m a t i o n . S e v e r a l p rev ious exper iments in vivo, though indirect , support this not ion : treatment of k idney podocytes to neutral ize the negatively charged s ia l ic ac id residues on the podocyte surface leads to a dramat ic loss o f podocyte interdig i tat ing foot processes ( A n d r e w s , 1979; G e l b e r g et a l . , 1996 ; Se i le r et a l . , 1977; Se i le r et a l . , 1975). T h i s is bel ieved to be due to a direct effect o n p o d o c a l y x i n f o r severa l reasons : p o d o c a l y x i n is the m o s t h i g h l y e x p r e s s e d s ia log lycoprote in on these ce l ls , its expression dur ing embryogenesis perfect ly co inc ides w i t h f o r m a t i o n o f these structures, and d isrupt ion o f the podxl gene leads to fa i lure to 153 produce foot processes dur ing embryogenesis (Doyonnas et a l . , 2 0 0 1 ; K e r j a s c h k i et a l . , 1984; Schnabel et a l . , 1989). It was therefore not surpr is ing that delet ion of the entire m u c i n domain also prevented m i c r o v i l l u s fo rmat ion . A l t h o u g h the precise mechan ism by w h i c h this effect occurs is e lus ive , we propose two models . T h e f irst model prov ides a b iophys ica l explanation for the induct ion of mic rov i l l us fo rmat ion by podoca lyx in . In this m o d e l , generation of addit ional p lasma membrane may s i m p l y serve to evenly disperse the b u l k y , negat ively charged m u c i n domains (F igure 3 -25) . T h i s model is consistent w i t h the prev ious reports of a dose-dependent w e a k e n i n g of ce l l junct ions induced by e c t o p i c express ion o f p o d o c a l y x i n and the t ight co r re la t ion between p o d o c a l y x i n overexpression and breast cancer metastatic index (Somas i r i et a l . , 2 0 0 4 ; T a k e d a et a l . , 2000) . It is also consistent w i th recent reports of podoca l yx in 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 (Meder et a l . , 2005) . 154 Low / no podocalyxin l l III | f-actin cell junctions integrins microvilli podocalyxin protein X F i g u r e 3 - 2 5 : T w o M o d e l s o f 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 . 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 hand , a surpr is ing result o f m y studies is the apparent d ispensabi 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 potent ia l phosphory la t ion sites and mot i fs w i th the capacity fo r d i rect ly b i n d i n g ezr in 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 ; Serrador et a l . , 2 0 0 2 ; T a k e d a et a l . , 2001) . T h e format ion of m i c r o v i l l i is t ightly l inked to the recruitment of f -ac t in and E R M proteins to the apical membrane d o m a i n . W i t h the d iscovery that podoca lyx in binds the N H E R F f a m i l y of 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 of adaptors is required for p o d o c a l y x i n func t ion ( L i et a l . , 2 0 0 2 ; M e d e r et a l . , 2 0 0 5 ; Schmieder 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 , 2001) . Unexpected ly , however , m y results demonstrated that fo rmat ion o f m i c r o v i l l i does not require direct interaction of p o d o c a l y x i n w i th N H E R F proteins. In support of this f i n d i n g , a l though one strain o f N H E R F l - n u l l mice impl icated this adapter protein in intestinal m i c r o v i l l u s fo rmat ion or organizat ion ( M o r a l e s et a l . , 2 0 0 4 ) , a second strain f a i l e d to support the observat ion (Sheno l ikar et a l . , 2002) . N o t a b l y , p o d o c a l y x i n is not expressed in the intestinal brush 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 fo rmat ion in this t issue, it w o u l d have to be independent o f p o d o c a l y x i n ( M c N a g n y et a l . , 1997) . Regard less , m y data suggest that p o d o c a l y x i n may be able to recruit ez r in and f - a c t i n by interact ing w i th another mo lecu le v ia its ext racel lu lar d o m a i n . T h i s interact ion m a y then transduce the s ignal to in i t iate m i c r o v i l l u s f o r m a t i o n (F igure 3 - 2 5 ) . T h i s p rov ides an a l ternat ive hypothesis to the b iophys ica l mode l proposed above and depends upon loss of a spec i f ic b ind ing site in podoca lyx in ' s extracel lular domain . 156 3.4.3 Significance of Recruitment of NHERF by Podocalyxin A l t h o u g h m y resul ts p r e c l u d e a d i rec t f u n c t i o n a l ro le f o r N H E R F / p o d o c a l y x i n interact ions in f o r m a t i o n o f m i c r o v i l l i and estab l ish ing the ap ica l ce l l d o m a i n , ap ica l recruitment o f N H E R F 1 by podoca lyx in is l i ke l y to be very important for other aspects of podoca lyx in and N H E R F funct ion . N H E R F proteins have been shown to be invo l ved in a var iety o f processes i n c l u d i n g i o n transport , s ignal t ransduct ion , g rowth c o n t r o l , and receptor internal izat ion, and they have also been shown to b ind a w ide variety of l igands and undergo o l i gomer i za t ion ( rev iewed in section 1.4.2) (Shenol ikar 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 . , 2005) . T h e fact that p o d o c a l y x i n is a potent inducer o f N H E R F recruitment to m i c r o v i l l i may suggest a new l i n k between 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 numerous b i o l o g i c a l processes (F igure 3 -26) . Regardless of m i c r o v i l l u s fo rmat ion , the podocalyx in -dependent loca l i zat ion of N H E R F proteins cou ld be quite important in their regulat ion. 157 NHERF + no Podocalyxin NHERF + high Podocalyxin signal receptor transduction internalization I I KEY: cell junctions integrins III f-actin microvilli NHERF-1 Figure 3-26: Model of Podocalyxin's Role in the Function of NHERF 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 shown to act as a pro - or ant i -adhesin depending upon its ce l lu lar context, as d iscussed in sections 1.5.2 and 1.5.3. F o r example , when expressed in H E V podoca l yx in acts as a l igand for L - se lec t in on l ymphocytes , but it is l i k e l y that this pro -adhesive funct ion is a special except ion rather than the general rule. T o act as a select in l igand podoca l yx in must be decorated w i th 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 found on most other cel ls ( M i c h i e et a l . , 1993 ; Sassetti et a l . , 1998; Segawa et a l . , 1997). In contrast, when overexpressed in M D C K ce l l s , w h i c h lack the appropriate enzymes fo r m o d i f y i n g p o d o c a l y x i n for s e l e c t i n - b i n d i n g , the mo lecu le was f o u n d to b lock c e l l - c e l l aggregation and mod i f y cel l junct ions (Takeda et a l . , 2000) . L i k e w i s e , we have p rev ious 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 var ious deve lopmenta l defects (omphaloce le and anuria) that are consistent w i th excessive ce l l adhesion in its absence ( rev iewed in sections 1.6.4.1 and 1.9) (Doyonnas et a l . , 2001) . F i n a l l y , we have f o u n d that p o d o c a l y x i n express ion is upregulated in metastatic breast cancer ce l l s and decreases cel l -substrate adhesion when overexpressed in vitro ( (Somasir i et a l . , 2004) and F igure 3 -3) . O u r current results suggest that the ab i l i t y to act as either a p r o - or ant i -a d h e s i n m a y 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 express ion on 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 adhes ion m o l e c u l e s at their t ips , c o u l d fac i l i ta te the reported L - se lec t in -dependent l eukocy te r o l l i n g and t ransendothel ia l m i g r a t i o n (G i ra rd et a l . , 1999 ; 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 . , 1995) . C o n v e r s e l y , in most 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 , ap ica l d o m a i n may protect ce l l s f r o m n o n - s p e c i f i c adhes ion . 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 in breast may promote tumour c e l l d i s s e m i n a t i o n by 159 in i t iat ing a general d isrupt ion of cel l adhesion, part icular ly under condi t ions where apica l membrane domains are expanded due to breakdown in polar i ty . T h i s w o u l d help init iate metastatic spread by a different, although not necessari ly mutua l l y exc lus i ve , m e c h a n i s m f r o m the w e l l d e s c r i b e d e p i t h e l i a l - m e s e n c h y m a l t r a n s i t i o n w h i c h s p e c i f i c a l l y downregulates ce l l - ce l l junct ions ( K a n g and Massague , 2004) . It is also tempting to speculate that the recruitment of f - a c t i n to the apica l membrane of ce l l s to generate m i c r o v i l l i might deplete act in f r o m the basal surface o f these ce l l s (Schmieder et a l . , 2 0 0 4 ) , thereby prevent ing stable interact ions o f integr ins w i t h the extracel lular matr ix . T h i s hypothesis is supported by the observat ion that cel ls transfected w i th 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 tai l generate abundant m i c r o v i l l i and exhib i t decreased ce l l - subst ratum adhes ion , w h i l e ce 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 ext race l lu lar d o m a i n do not generate m i c r o v i l l i and remain adherent ( (Somasi r i et a l . , 2 0 0 4 ) , F igure 3 - 2 5 , and unpub l i shed observat ions) . Further studies del ineat ing the mechanisms by w h i c h apical podoca l yx in and basolateral integrins compete for actin may c lar i fy this process. 160 CHAPTER 4 : GENERATION OF TRANSGENIC MICE CONDITIONALLY OVEREXPRESSING PODOCALYXIN 4.1 Rationale There were fou r major reasons f o r at tempt ing to generate a mouse overexpress ing podoca lyx in : a) L o s s - o f - f u n c t i o n and g a i n - o f - f u n c t i o n e x p e r i m e n t s are t w o very p o w e r f u l approaches fo r e luc idat ing the no rmal func t ion o f a poor ly understood protein. The podoca l yx in knockout mouse has a dramatic phenotype in that it dies w i th in 2 4 hours o f b i r th , but this prevents us f r o m f u l l y understanding p o d o c a l y x i n ' s funct ions, especia l ly in adult an imals ( rev iewed in section 1.6.4) (Doyonnas et a l . , 2 0 0 1 ) . M u c h i n f o r m a t i o n has been o b t a i n e d f r o m in vitro 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 e x p e r i m e n t s (see chapter 3 ) , but there are m a n y quest ions remain ing , w h i c h may be answered us ing 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 m a i n l y f u n c t i o n s to b l o c k inappropr iate c e l l adhes ion . B y overexpress ing p o d o c a l y x i n in t issues where adhes ion is impor tant , numerous b i o l o g i c a l processes c o u l d be altered in an attempt to c la r i f y their importance and gain mechanis t i c insights. F o r e x a m p l e , b l o c k i n g adhesion o f leukocytes m a y m i m i c diseases such as leukocyte adhesion def ic iency syndromes, by prevent ing leukocyte ro l l i ng and f i r m arrest (Bunt ing et a l . , 2 0 0 2 ) . 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 vascu lar endothe l ia may m i m i c 161 some character is t ics o f other d isorders , l i k e ar thr i t is , by inc reas ing vascu la r permeabi l i ty (Dor ia et a l . , 2006) . c) P o d o c a l y x i n knockout mice die wi th in one day of bir th, presumably as a result o f a major k idney defect. T h i s precludes analysis of defects in other tissues in adult mice . Therefore , by c ross ing an induc ib le p o d o c a l y x i n overexpressor mouse to a podocy te - spec i f i c C re mouse, it w o u l d be possible to generate mice spec i f i ca l l y expressing p o d o c a l y x i n in the area where it is thought to be absolutely essential . T h i s may enable the m i c e to overcome the lethal phenotype, thereby a l l o w i n g analysis of other podoca lyx in -def ic ient tissues in adult. d) W e have recent ly s h o w n that p o d o c a l y x i n is an exce l len t predictor of poor outcome in human breast cancer ( rev iewed in sect ion 1.10.1) ( S o m a s i r i et a l . , 2004) . It is poss ib le that the ant i -adhesive func t ion o f p o d o c a l y x i n a l l o w s fo r more rap id d i s s e m i n a t i o n o f tumour ce 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 fo r podoca lyx in in breast cancer progression cou ld be addressed by spec i f i ca l l y overexpress ing p o d o c a l y x i n in m a m m a r y t issue. A f t e r i n d u c i n g the f o r m a t i o n o f t u m o u r s 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 , d isease progression cou ld be f o l l o w e d in these mice and compared to non-transgenics. There are therefore a n u m b e r o f interest ing quest ions that c o u l d be addressed us ing condi t ional podoca l yx in overexpressing mice . 4.2 The Cre-loxP System T h e C r e - l o x P system is a versat i le tool a l l o w i n g induc ib le express ion , or de le t ion , o f genes o f interest ( r e v i e w e d in ( L o b e and N a g y , 1998) ) . C r e is a bac te r iophage 162 recombinase that se lect ive ly induces s i te - spec i f i c recombinat ion between 3 4 bp l o x P consensus sequences (F igure 4 - 1 ) . T h u s , after inser t ing a gene in between two l o x P sequences, C re can be used to delete the intervening sequences, l eav ing behind a s ingle l o x P site. It is poss ib le to make numerous t issue-spec i f ic knockouts by generating just a single mouse l ine wi th a transgene consist ing of the gene of interest f lanked by l o x P sites and then cross ing it w i t h any o f the many m i c e ava i lab le that express C r e in a t issue-spec i f i c or tempora l l y regulated manner . L i k e w i s e , this system can also be used to condi t ional ly overexpress a gene. In this case, the l o x P sites f lank a spacer that separates the promoter f r o m the gene o f interest and inc ludes a stop codon and po ly -adeny la t ion ( p o l y A ) sequences. T h i s element can subsequently be deleted, thereby fac i l i ta t ing gene expression f r o m a c o m m o n promoter. Thus , the C r e - l o x P system was deemed suitable for inducib le expression of podoca lyx in in mice . 163 1> D>— + Cre recombinase V Figure 4-1: Schematic of Cre-Mediated Recombination. C r e recombinase (hexagon) mediates h o m o l o g o u s r e c o m b i n a t i o n between l o x P sites (triangles), thereby exc i s ing the intervening (black) sequence and connect ing the f l a n k i n g (purple) sequences. 164 4.3 Transgenic Construct There are several basic l o x P - c o n t a i n i n g constructs ava i lab le for generat ing transgenic m i c e ; the one that I used 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 o v e r e x p r e s s i n g transgenic is ca l led Z / E G , and it encodes several useful features (F igure 4 - 2 and (Lobe et a l . , 1999; N o v a k et a l . , 2000)) . In i t ia l l y , p o d o c a l y x i n and G F P expression were b locked by a /acZ/neomycin resistance (Pgeo) fus ion spacer cassette, w h i c h inc luded a stop codon 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, and was therefore exc isab le by Cre -mediated s i te -speci f ic recombinat ion . T h e C M V enhancer/(3-actin promoter was used to dr ive express ion of the gene o f interest (podxl, in this case) , w h i c h was inserted upstream of an I R E S sequence and GFP, enab l ing expression 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 romote r i n i t i a l l y d rove ub iqu i tous express ion o f (3-galactosidase ((3-gal), but C r e - e x c i s i o n led to express ion , instead, 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 )\ Podocalyxin | IRES-GFP loxP loxP i + Cre CMV-p actin promoter Podocalyxin IRES-GFP loxP Figure 4-2: Schematic of Transgenic Construct Before and After Cre-Mediated Recombination. The or ig ina l construct encoded the Pgeo fus ion 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 and G F P were encoded d o w n s t r e a m and were therefore not expressed. C r e - m e d i a t e d r e c o m b i n a t i o n deleted the spacer e lement and induced expression o f podoca l yx in and G F P . 166 4.4 In Vitro Validation of Transgenic Construct P r i o r to us ing the construct to generate t ransgenic m i c e , a s i m i l a r precursor p l a s m i d l a c k i n g G F P was f irst tested in vitro by transfect ing the mur ine m y e l o m a ce l l l ine , N S O , w h i c h was chosen for ease o f t ransfect ion (see sect ion 2.1.1) . A f t e r p roduc ing stable n e o m y c i n resistant l ines conta in ing the construct , the ce l ls were transiently transfected w i t h the p C I - C r e express ion p l a s m i d (generously p rov ided by Dr . F a b i o Ross i ) . F l o w cytometry was then used to assess podoca lyx in expression (Figure 4-3). A s expected, due to the transient nature o f the C r e t ransfect ion, some cel ls expressed p o d o c a l y x i n , w h i l e many d id not; the e f f i c iency w o u l d be expected to increase w i th stable C r e transfect ion. Thus , this construct was found to funct ion appropriately and the Z/EG-podxl p lasmid was used to generate stable embryon ic stem cel l ( E S C ) l ines. 167 Empty vector X _>, o o T3 O CL CSI o I'' ><• I" " 111 • • 1 1 1 1 1 I 0 200 400 600 800 1000 Podocalyxin feC# • i i I i i i i I i i i i | i i i i | i i i i | 0 200 <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 inear ized construct was introduced into mur ine R l E S C s by e lectroporat ion. C e l l s were then a l l o w e d to recover , after w h i c h t i m e they were selected f o r n e o m y c i n resistance. A p p r o x i m a t e l y 4 0 0 drug-resistant E S C co lon ies were p i cked and transferred to 9 6 we l l plates. Those colonies that cont inued to g row were rep l ica plated onto feeder layers for cont inued expansion and preparation of stocks and onto gelat in -coated plates fo r ana lys is (F igure 4 -4 ) . A s s e s s m e n t o f c lones 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 igure 4 -5) and screening ce l ls , before and after d i f ferent iat ion, fo r P-galactosidase express ion. T h i s ensured that the transgene had integrated into a site that w o u l d fac i l i tate express ion in many ce l l types. F i v e c lones met these cr i ter ia and were maintained for 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 cel ls that had incorporated the construct (green circles) were selected based on resistance to n e o m y c i n ( G 4 1 8 ) . These c o l o n i e s were then screened for f3-galactosidase express ion, and posit ive c lones (blue) were further assessed after generating f rozen 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 of E S C s . Podocalyxin c D N A , encoded by the transgenic construct, was detected in P-glactosidase-positive clones, but not in parental R l ESCs. 171 4.6 Transgene Expression Analysis After Cre-Mediated Recombination in ESCs T h e next step in the select ion process required test ing the f i ve c lones fo r proper C r e -mediated exc is ion of the spacer and subsequent expression of podoca lyx in . R T - P C R was used to c o n f i r m the presence o f podoca lyx in m R N A after Cre- t ransfect ion (Figure 4 -6) . It was also noted that parental R l E S C s expressed a l o w level o f endogenous podoca l yx in . T h i s was not unexpected because it has been s h o w n that human E S C s a lso 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 exp ress ion o f exogenous podoca lyx in (above background levels) in al l f i ve c lones (F igure 4 -7 ) . A g a i n , C r e was transfected transiently, and the ef f ic iency w o u l d be improved upon sustained C r e expression in vivo. Furthermore, G F P expression was detected in al l c lones, both by R T -P C R (F igure 4 - 8 ) , and by f luorescence m i c r o s c o p y . Based on this express ion data, as we l l as co lony m o r p h o l o g y , two c lones were chosen for generat ion of c h i m e r i c m i c e ( 2 G 1 2 and 2 B 6 ) . 172 « Podocalyxin F i g u r e 4-6: E x p r e s s i o n o f P o d o c a l y x i n m R N A i n E S C s . R T - P C R was used to detect podoca l yx in m R N A i n E S C s after transient transfection w i t h C r e , and in posit ive control ( F D C P - 1 ) cel ls . 173 2 B 6 ( 2 2 % ) C «i 080803 ESC.007 4 ifs x CD O o "D O CL 080803 ESC .008 • 21 5 Sfo-T,... 2 G 1 2 ( 7 % ) 080803 E S C J 13 1) 080803 ESC.012 11 2 A 7 (14%) FSC 080803 ESC 016 3 E 3 ( 2 2 % ) 080803 ESC .017 ^ 090 803ESC.021 = 1 if2' SV ! « ' • r . J i ^ 080803 ESC 020 si • r " " j a i o -• 3 H 7 (45%) 3 ESC .025 • 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 in parental R l ESCs . 175 4.7 Production of Chimeric Mice C h i m e r i c mice were generated w i th the 2 B 6 c lone by Cor r inne L o b e ' s laboratory us ing the technique o f E S C - m o r u l a aggregation ( L o b e et a l . , 1999). A l t h o u g h aggregations were performed repeatedly wi th several c lones, only one exper iment led to product ion of c h i m e r i c m i c e , and unfor tunate ly none o f the e x p e r i m e n t s resul ted in g e r m l i n e transmission of the transgene. 4.8 Explanation for Lack of Germline Transmission in Transgenic Mice T h e E S C clones were then sent to J i l l L a h t i ' s laboratory fo r an addi t ional attempt at generating ch imer ic mice . K a r y o t y p i n g is often completed before generating transgenic m i c e w i t h cu l tured E S C s , so t w o c lones ( 2 G 1 2 and 3 E 3 ) were ka ryo typed before proceeding. U s i n g this technique, it was determined that both c lones were t r i somic fo r c h r o m o s o m e eight , through creat ion of what appeared to be i sochromosomes (F igure 4 -9 ) . T r i s o m y eight is a fa i r l y c o m m o n c h r o m o s o m a l aberrat ion not iced in cu l tured E S C s , as it gives cel ls a growth advantage ( L i u et a l . , 1997). Unfortunately , although this genetic defect does not inh ib i t p roduct ion o f c h i m e r i c m i c e , it does prevent germl ine t ransmiss ion o f the transgene because the abnormal ce l ls cannot f o r m an entire v iab le a n i m a l . S ince both tested c lones had the same structural c h r o m o s o m a l aberrat ion, it is l i ke l y that the or ig inal R l E S C s were also t r i somic for chromosome eight. Th i s precluded further use of these cel ls for generating transgenic animals . 176 .1 I II )L << >< >< d <« IMJ II »l M M • 7 V « » 10 11 « C i t l I I i f f t II * 14 15 16 17 ttt Figure 4-9: Karyotyping Uncovered the Presence of Trisomy Eight in Tested Clones. 177 4.9 Summary and Conclusions W e were interested in generating transgenic m i c e cond i t iona l l y expressing p o d o c a l y x i n as a tool for assessing podoca lyx in ' s role in breast cancer, fo r invest igat ing the effect of a general loss o f adhesion in numerous ce l l types, and in order to rescue p o d o c a l y x i n knockout mice by speci f ica l ly expressing podoca lyx in in k idneys , where it is presumed to be essential fo r v iab i l i t y o f mice . A transgenic construct was generated w h i c h enabled expression of podoca l yx in f r o m a ubiquitous promoter , on ly after C re -med ia ted exc is ion of a spacer e lement. T h i s construct was used to generate E S C c lones that exh ib i ted excel lent express ion o f the transgene before and after d i f ferent iat ion , as assayed by (3-galactosidase express ion. Several c lones were f o u n d to be acceptable for generation of t ransgenic m i c e based on m o r p h o l o g y as w e l l as e f f i c ien t C r e - m e d i a t e d i n d u c i b l e express ion of p o d o c a l y x i n and G F P . H o w e v e r , in i t ia l attempts at generat ing c h i m e r i c mice that propagated the transgene in the germl ine f a i l e d , and the E S C s were therefore examined for ch romosomal aberrations. T w o c lones were tested, and both were found to possess an addi t ional copy o f c h r o m o s o m e eight. T h i s exp la ins the lack o f ge rml ine t ransmiss ion , and necessitates the generation o f new E S C c lones , beg inn ing w i t h new, untransfected E S C s . In the meant ime, a more direct strategy was used to address one goal of this study: the repair of p o d o c a l y x i n - k n o c k o u t k idneys was attempted us ing a k i d n e y -speci f ic podoca lyx in transgenic mouse (chapter 5). 178 CHAPTER 5 : REPAIR OF KIDNEY DEFECT IN PODOCALYXIN-NULL MICE 5.1 Rationale L o s s - o f - f u n c t i o n exper iments can prov ide considerable insights into protein funct ion , so p o d o c a l y x i n - n u l l m i c e had been generated prev ious ly (Doyonnas et a l . , 2001) . Defects were expected in t issues where p o d o c a l y x i n is expressed: podocytes of the k i d n e y , hematopoiet ic progenitor ce l l s , megakaryocy tes , vascular endothel ia , mesothel ia l ce l l s l i n i n g organs, and a subset o f neuronal ce l l s (Doyonnas 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 . , 1999; Horvat et a l . , 1986; Ker jaschk i et a l . , 1984; M c N a g n y et a l . , 1997; M i e t t i n e n et a l . , 1990 ; M i e t t i n e n et a l . , 1999 ; V i t u r e i r a et a l . , 2005) . H o w e v e r , delet ion of the podxl gene resulted in perinatal lethal i ty , prevent ing analys is of defects in adult m i c e . Further analys is suggested that loss of p o d o c a l y x i n in podocytes of the k idney was the cause of this dramatic phenotype. P o d o c a l y x i n - n u l l mice were a n u r i c , e x h i b i t e d inc reased b l o o d pressure , and were f o u n d to have drast ic m o r p h o l o g i c a l abnormal i t ies in k idney g lomeru l i upon analysis by electron mic roscopy (Doyonnas et a l . , 2001) . In order to study defects in adult mice l a c k i n g p o d o c a l y x i n , we attempted to rescue p o d o c a l y x i n - n u l l m i c e by e c t o p i c a l l y exp ress ing p o d o c a l y x i n s p e c i f i c a l l y in p o d o c y t e s . T h e f i r s t st rategy i n v o l v e d g e n e r a t i n g p o d o c a l y x i n 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 sys tem descr ibed in the prev ious chapter . H o w e v e r , due to delays in that strategy, it was dec ided that a more direct strategy was requ i red . T h e new strategy i n v o l v e d generat ing a 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 transgene. 179 5.2 Transgenic Construct The promoter fo r the NPHS1 gene, w h i c h encodes nephr in (discussed in sect ion 1.6.5.1, and rev iewed in (Salant and T o p h a m , 2 0 0 3 ; T r y g g v a s o n , 1999)) , was chosen to dr ive ectopic express ion of p o d o c a l y x i n in podocytes . In the g l o m e r u l u s , nephr in is f o u n d exc lus i ve l y in the sl it d iaphragm region o f podocytes , and nephr in and p o d o c a l y x i n are in i t ia l l y expressed at a s imi la r t ime point in development ( K a w a c h i et a l . , 2 0 0 2 ; Schnabel et a l . , 1989). T h e p I R E S 2 - E G F P expression vector was chosen as the backbone fo r the transgenic construct. T h i s vector inc luded a n e o m y c i n resistance cassette, as w e l l as a mul t ip le c l o n i n g site downstream of a ubiqui tous promoter . M u r i n e p o d o c a l y x i n c D N A was c loned into the mul t ip le c l o n i n g site, upstream of GFP, w h i c h was expressed us ing an I R E S sequence. Before generating transgenic mice , the NPHS1 promoter was inserted upstream of podxl, and the ubiqui tous C M V promoter was deleted (F igure 5 -1) . T h i s enabled podoca l yx in and G F P expression spec i f ica l l y in 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 de let ion o f the ub iqu i t ious promoter , the t ransgenic construct was tested by t ransfect ion into C H O ce l l s . T rans ient t ransfect ion resul ted in e x p r e s s i o n o f both podoca lyx in and G F P , as detected by f l o w cytometry (F igure 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 transgenic construct was l inear ized after successfu l l y test ing it in vitro; the f ina l construct contained on ly the NPHS1 promoter upstream of podxl and GFP. T h i s D N A fragment was injected into the pronucle i o f fer t i l i zed eggs in the B i o m e d i c a l Research Cent re 's transgenic fac i l i t y . A p p r o x i m a t e l y 4 0 0 inject ions were per fo rmed, resul t ing in 51 pups. G e n o t y p i n g results suggested that f i ve m i c e contained the transgene, but on ly one founder produced consistently strong signals for the transgene by P C R . 5.5 Transgene Expression Analysis Generat ion o f t ransgenic m i c e by oocyte m i c r o i n j e c t i o n often results in inser t ion o f mul t ip le copies of the transgene into a s ingle site. T rad i t iona l l y , transgene copy number has therefore been assessed in order to a p p r o x i m a t e express ion leve ls . H o w e v e r , express ion can also be affected by many other factors. F o r example , the transgene may insert into a region o f heterochromatin that cou ld prevent its express ion. A l te rnat i ve l y , it may be located next to a strong promoter , w h i c h c o u l d alter express ion . In contrast, insert ional mutagenesis c o u l d lead to d is rupt ion o f another gene's normal express ion pattern or even creation o f a fus ion protein. Therefore , when generating transgenic m i c e , several founders shou ld be assessed to ensure that any phenotypes are the result o f the transgene itself , and not the result o f the transgene's insert ion site. A l l f i ve founders were therefore in i t ia l l y assessed fo r transgene expression. 184 5.5.1 Transgene Expression in Glomeruli of Founders' Kidneys In that podocytes were the transgene's target ce l ls , the founders ' k idneys were assessed for express ion, after f i rst insur ing that the transgene had been transmitted to subsequent pups. It is important to remember that the founders were otherwise normal an imals w i th addi t ional , ectopic expression o f podoca l yx in (as we l l as G F P ) f r o m the transgene. Thus , podoca l yx in expression cou ld not be assessed in these a n i m a l s ; the fore ign G F P protein was therefore used as a marker . G F P express ion was very fa int , 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 us ing an a n t i - G F P ant ibody. T h e founder w i th the strongest transgene signal by P C R also had the brightest G F P staining in k idney g lomeru l i (F igure 5 -3) . T h i s founder was k n o w n as 3 -3 as it was the third pup f r o m the third set of inject ions. T w o other founders ( 1 - 9 and 3 -10) also had reasonable levels o f G F P express ion , w h i l e the last two ( 1 - 6 and 1-8) were ext remely weak. A n a l y s i s at higher magn i f i ca t ion showed a d ist inct ive sta in ing pattern, as shown in the image of a s ingle g lomerulus in F igure 5 - 4 . T h i s pattern is typ ica l o f ap ica l membrane labe l l ing of g lomerular podocytes. 185 B6(wt) 1-9 3-3 Figure 5-3: GFP Expression in Glomeruli of Transgenic Founders. A f t e r pe r fus ion w i t h para fo rmaldehyde ( P F A ) , k i d n e y s were f r o z e n , sect ioned , 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 ant ibod ies , as desc r ibed i n chapter 2 . B r i g h t green s t a i n i n g represents G F P in g l o m e r u l i , w h i l e y e l l o w s t a i n i n g is b a c k g r o u n d immunof luorescence . T issues f r o m wi ld type (B6 ) mice were used as negative controls for G F P staining. Scale bar: 50 p m . 186 Figure 5-4: Glomerulus from Founder with Highest GFP Expression Levels. A f t e r P F A p e r f u s i o n , k i d n e y s were f r o z e n , s e c t i o n e d , and i m m u n o l a b e l l e d w i t h ant ibodies against G F P . Pos i t i ve sta in ing can be seen i n the g lomeru lus . T issues f r o m wi ld type (B6) mice were used as negative controls for G F P staining. Scale bar: 20 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 expression in k idney , it was also necessary to ident i fy sites o f inappropriate express ion. Other areas where p o d o c a l y x i n is no rmal l y expressed, such as hematopo ie t ic ce l l s and vascu lar t issues, were important to examine because ectop ic express ion in these areas w o u l d prevent analysis of defects in ce l ls l a c k i n g p o d o c a l y x i n when crossed w i th p o d o c a l y x i n - n u l l animals . N o expression was detected in the l i ver or lung , where vascular expression w o u l d be not iceable (F igure 5 -5 ) , or in the per ipheral b lood (F igure 5-6) . A s podoca l yx in and nephrin can both be found in a subset of ce l ls in the brain ( 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 ; Putaala 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 examined , but G F P expression was not detected (F igure 5-7). In s u m m a r y , a l though a l l f i v e founders demonst ra ted some degree o f G F P express ion in k idney , on ly three had reasonable levels . Inappropriate expression of the transgene was not detected (Table 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 perfus ion, tissues were f rozen , sect ioned, and immuno labe l led w i th antibodies against G F P . Transgene expression was not detected in vascular endothel ia. T issues f r o m w i ld t ype (B6 ) m i c e were used as negative controls fo r G F P stain ing. K i d n e y sect ions f r o m founder 3 -3 were used as posit ive controls for G F P staining. Scale bar: 50 u.m. 189 IUU 10° 101 102 103 104 • GFP Figure 5-6: Transgene Expression was Undetectable in Peripheral Blood. Per iphera l b l o o d was iso lated f r o m progeny o f o r ig ina l founders and assessed for G F P expression by f l o w cytometry . G F P expression was compared to expression in peripheral b lood isolated f r o m wi ld type ( B 6 ) and 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 p e r f u s i o n , brains were f r o z e n , sec t ioned , and i m m u n o l a b e l l e d fo r G F P . A l t h o u g h there appeared to be some b a c k g r o u n d f luorescence , there was no spec i f i c expression in brain . T issues f r o m wi ld type (B6) mice were used as negative controls for G F P staining. K i d n e y sections f r o m founder 3 -3 were used as posit ive controls fo r G F P staining. Scale bar: 5 0 p m . 191 Founder K i d n e y Express ion Pattern B r a i n B l o o d L i v e r L u n g 3 -3 ++ - - -1-9 ++ - nd nd 3 - 1 0 + - nd nd 1-8 weak - nd nd 1-6 weak - nd nd T a b l e 5 - 1 : S u m m a r y o f T r a n s g e n e E x p r e s s i o n . (nd: not determined) 192 5.6 Breeding Scheme and Expected Numbers of Rescued Mice Ind iv idual colonies were expanded f r o m the three most suitable founders ( 3 - 3 , 1-9, and 3 -10) in order to obtain m i c e for further breeding experiments. Podxl*1*transgene*1' progeny were then crossed w i th podxl*''(transgene'') m ice to obtain m i c e heterozygous fo r both the podxl knockout al le le and the new, randomly integrated podxl transgene (Figure 5 -8) . O b v i o u s l y , it was necessary to use m i c e heterozygous fo r the podxl knockout a l le le because o f the demonst ra ted le tha l i t y o f h o m o z y g o t e s . A f t e r o b t a i n i n g podxl*1' transgene*1' a n i m a l s , these were in te rb red to o b t a i n t r a n s g e n e - c o n t a i n i n g m i c e h o m o z y g o u s fo r the podxl knockout a l le le . T h e expected f requency o f each potential genotype f r o m these breedings is shown in T a b l e 5 - 2 . M i c e o f interest were those express ing no endogenous p o d o c a l y x i n w h i l e also harbour ing at least one copy of the new transgene. The expected frequency of these rescued mice was 3/16, as shown in blue in Tab le 5 - 2 . 193 podxr' ^ transgene^' x podxl'' transgene ' i / + / " + / " ; / + / " +1-podxl transgene 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 u s e d 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 K i d n e y . podxl + / + podxl + / podxl +/+ transgene +/-transgene -/-transgene 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 C r o s s e s . Blue represents the frequency of potentially rescued mice. 1 9 4 5.7 Transgenic Mice Still Die within 24 Hours of Birth A f t e r intercrossing podxt'~transgene+'~ m i c e , a l l resul t ing pups that surv i ved beyond the first 2 4 hours were genotyped. A l t h o u g h normal M e n d e l i a n ratios were seen fo r podxl+l+ and podxt1' t ransgene pos i t i ve a n i m a l s , there was not a s ing le s u r v i v i n g podxt' transgene" mouse (Table 5-3) . A l t h o u g h only the first f i ve litters are shown in Tab le 5 - 3 , al l subsequent litters were also devoid of rescued pups. Notab ly , there was no evidence o f insert ional mutagenesis , as m i c e w i th the transgene were f o u n d in s i m i l a r numbers to those l a c k i n g it when m i c e heterozygous fo r endogenous podxl and the transgene were crossed wi th podxt1' an imals (Table 5-4) . 195 Lit ter podxl + , + Genotype podxl + l 1 0 6 0 2 3 3 0 3 1 2 0 4 3 4 0 5 2 4 0 T O T A L 9 19 0 Table 5-3: Actual Numbers of Transgene-Positive Mice of Each Genotype in the First Five Litters Resulting from Podxt'Transgene*1' Crosses Demonstrated a Lack of Rescue of Podxt1' Mice. 1 9 6 L i t ter transgene +/ transgene 1 podxl + / + podxl + / podxl + / + podxl + l podxl 1 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 Table 5-4: Actual Numbers of Transgene-Positive and Transgene-Negative Mice in Eight Litters Resulting from Breeding of Podxt'Transgene*1' Mice with Podxl*1' Transgene'1' Mice Demonstrated that Transgene-Integration was not Detrimental. 197 5.8 Potential Reasons for Lack of Rescue T h e l a c k o f s u r v i v i n g pups was c lear l y d i s a p p o i n t i n g , and it was also s o m e w h a t surpr is ing. There were, however , several possible explanations fo r this outcome: a) P o d o c a l y x i n may not have been expressed f r o m the transgene. A l t h o u g h un l ike l y , it was possible that the G F P expression observed i n k idneys of transgenic animals d id not correlate wi th ectopic podoca lyx in expression. b) P o d o c a l y x i n may have been expressed at the incorrect t ime dur ing development , thereby preventing proper maturation of podocytes. c) P o d o c a l y x i n may have been expressed at inadequate leve ls . T h i s was also not part icular ly l i ke l y because podxl+l~ m ice have w e l l - d e v e l o p e d , funct ional k idneys (Doyonnas et a l . , 2001) . d) E v e n i f p o d o c a l y x i n was expressed in the correct c e l l s , it may have been mis loca l i zed . e) There may be other lethal podocalyx in - re lated defects. 5.9 Transgenic Podocalyxin Expression Analysis In order to address the poss ib i l i t ies out l ined above, the express ion of p o d o c a l y x i n was first assessed. Th i s was accompl i shed by iso lat ing k idneys f r o m m i c e just pr ior to bir th. A t this t ime point , a l l genotypes were present in expected ratios. Fur thermore, ectopic podoca lyx in expression cou ld be analyzed by compar ing podxl'1'transgene* k idneys w i th podxl'1'transgene k idneys. R T - P C R was used to demonstrate the presence of podoca l yx in m R N A expressed f r o m the transgene (F igure 5 -9) . K i d n e y s isolated f r o m wi ld type and 198 p o d o c a l y x i n - n u l l mice l a c k i n g the transgene were used as posit ive and negative contro ls , respect i ve ly . 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 were express ing p o d o c a l y x i n m R N A , as shown in red in F igure 5 - 9 . Immunohis tochemist ry was used to c o n f i r m protein express ion , as shown in F igure 5 - 1 0 . A l t h o u g h there was o n l y a th in b r o w n layer of p o d o c a l y x i n stain ing in the podxl'transgene* g lomeru l i ( left panel) in compar ison to the widespread expression observed in wi ld type animals (middle panel) , it is important to remember that p o d o c a l y x i n is a lso normal l y expressed on a l l vascu lar endothel ia l ce l ls . T y p i c a l vascular staining is shown in the right panel , where P E C A M - 1 sta in ing has been used to indicate vasculature. In contrast to the p o d o c a l y x i n s ta in ing seen in the transgenic k idney , apical s ta in ing was most notably absent f r o m the outer layer o f ce l ls in the compact g lomerulus label led wi th antibodies against P E C A M - 1 . A t h igh m a g n i f i c a t i o n , it was also more evident that p o d o c a l y x i n was not on ly expressed, but was cor rect l y l o c a l i z e d to the ap ica l surface o f podocytes in t ransgenic a n i m a l s (F igure 5 -11) . Thus , a lack o f appropriate podoca lyx in expression was not the reason for the lack of rescue observed in breeding experiments. 199 E n d o g e n o u s + + T r a n s g e n i c + + - + P o d o c a l y x i n Figure 5-9: Podocalyxin mRNA was Expressed from the Transgene during Development in Kidneys of Transgenic Mice. A f t e r i so la t ion o f m R N A f r o m k i d n e y s o f E l 8 m i c e , R T - P C R was pe r fo rmed u s i n g /oafrZ-speci f ic pr imers . 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 contro ls , and k idneys f r o m E l 8 w i ld t ype ( B 6 ) m i c e were used as pos i t ive contro ls fo r expression o f podoca l yx in m R N A . 2 0 0 Podocalyxin staining PECAM-1 staining Podo KO + transgene Podo WT Figure 5-10: Immunohistochemistry was used to Detect Podocalyxin Expression in Kidney Glomeruli of Transgenic Mice during Development. K i d n e y s were isolated f r o m E l 8 m i c e , f rozen , sect ioned, f i x e d , and i m m u n o l a b e l l e d fo r p o d o c a l y x i n or P E C A M - 1 , as a vascu la r endothe l ia l c e l l - s p e c i f i c marker . N o t e the wi ld type sample d isp layed p o d o c a l y x i n stain ing on b lood vessels and podocytes , w h i l e g l o m e r u l i f r o m podx/1 m i c e express ing the transgene d i s p l a y e d on ly a th in layer o f p o d o c a l y x i n s ta in ing , ind icat ive of ap ica l l a b e l l i n g o f podocytes. 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 mice were used as negative controls fo r p o d o c a l y x i n sta in ing, and rat I g G , was used as a negative cont ro l fo r P E C A M - 1 sta in ing. Representat ive o f t w o independent experiments. Scale 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 isolated f r o m E l 8 m i c e , f r o z e n , sect ioned, f i x e d , and i m m u n o l a b e l l e d . A th in , dark layer o f p o d o c a l y x i n sta in ing was most obv ious around the outer edge of the g lomerulus , wh i le staining was absent in the isotype control sample. Scale bar: 10 p m . 202 5.10Morphological Analysis of Kidneys A l t h o u g h l ight m i c r o s c o p y was used to demonstrate that p o d o c a l y x i n was expressed appropriately in g lomerular podocytes, these images cou ld not be used to assess ce l lu lar morpho logy . T o address this issue, k idneys were again isolated f r o m animals just before birth and analyzed by T E M . P o d o c a l y x i n - n u l l m i c e exh ib i t t w o major m o r p h o l o g i c a l abnormal i t ies in podocytes ( rev iewed in sect ion 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 interdig i tat ing foot processes that no rmal l y surround capi l lar ies in the g lomeru lus , and ne ighbour ing podocytes mainta in tight junc t ions between ce l l bodies. D u r i n g no rmal maturation of podocytes, tight junct ions are lost, and replaced by slit d iaphragms between interd ig i tat ing foot processes ( rev iewed in sect ion 1.6.1). These character ist ics were therefore assessed by electron mic roscopy in k idneys o f podxl'transgene*1' an imals . T h e m o r p h o l o g i e s p r e v i o u s l y observed fo r w i l d t y p e and p o d o c a l y x i n - n u l l an ima ls were c o n f i r m e d (F igure 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 an ima ls express ing the k idney -spec i f i c transgene had not iceably improved podocyte morphologies . There was an absence o f t ight junct ions between podocytes , and interd ig i tat ing foot processes were very evident (Figure 5 -12) . H igher magni f icat ion images c lear ly showed the intricate foot processes (F igure 5 -13) . S l i t d iaphragms between foot processes were also detectable under c lose 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 is absolute ly required fo r morphogenesis of podocytes , and that ectopic express ion o f p o d o c a l y x i n in these ce l ls was suff ic ient for repair ing the defect. 203 Podo WT Absence of FP's RBC 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 iso lated f r o m E l 8 m i c e , f i x e d , sect ioned, and processed for T E M . E a c h image shows a cross -sect ion o f a cap i l la ry , and most inc lude c i rcu la t ing red b lood cel ls ( R B C ) . Podocy tes were situated outs ide the c a p i l l a r y , ex tend ing in terd ig i ta t ing foot processes ( F P ' s ) , w h i c h were v i s ib le in 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 samples . Scale bar: 2 u m . Imag ing 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 , Un ive rs i t y o f B r i t i s h C o l u m b i a . 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 int r icate foot processes i n w i l d t y p e and p o d o c a l y x i n knockout/t ransgene-posi t ive samples and the presence o f ce l l junct ions in the p o d o c a l y x i n knockout sample. Scale bar: 3 p m . Imag ing 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 , Un ive rs i t y o f B r i t i s h C o l u m b i a . 205 5.11 Summary and Conclusions P o d o c a l y x i n - n u l l an imals exhib i t perinatal lethality. T h i s phenotype was presumed to be due to a podocyte-related k idney defect since drastic morpho log ica l changes are observed in these cel ls in the absence of podoca lyx in (Doyonnas et a l . , 2001) . In an effort to rescue podoca l yx in knockout mice to faci l i tate analysis of defects in adult an imals , podoca l yx in was e c t o p i c a l l y expressed us ing the p o d o c y t e - s p e c i f i c NPHS1 p romoter . E x p r e s s i o n analysis demonstrated appropriate expression o f the transgene in several founder an imals , w i th substantial expression in one founder. E c t o p i c k i d n e y - s p e c i f i c expression o f podoca lyx in d id not prevent the perinatal lethality observed in k n o c k o u t a n i m a l s , but it d i d repair the m o r p h o l o g i c a l defects in the podocytes. It should be noted, however , that al though there was a dramatic d i f ference in podocyte m o r p h o l o g y between 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 and wi thout the transgene, it is poss ib le that the k i d n e y defect was not adequately repaired due to somewhat aberrant express ion . C a r e f u l examinat ion suggests that foot processes may have been s l ight ly flatter and slit pores marg ina l l y narrower in rescued mice compared to w i ld t ype contro ls . It may be that ectopic express ion levels were s l ight ly lower , or that express ion was init iated a l itt le later in g lomerular development than w o u l d be expected f r o m the endogenous podxl promoter . H o w e v e r , nephrin and p o d o c a l y x i n are expressed at a lmost ident ical t imes dur ing deve lopment (Putaala et a l . , 2 0 0 0 ; Putaala et a l . , 2 0 0 1 ; S c h n a b e l et a l . , 1989). Fu r th e rmore , 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 fe rence between podoca l yx in knockout mice expressing the transgene and those l a c k i n g it, in combinat ion w i t h the consistent lethal i ty w i t h i n 2 4 hours o f b i r th , suggests that p o d o c a l y x i n - n u l l 206 animals may actual ly die as a result of other podocalyx in - re lated defects. T h i s hypothesis is supported by the fact that some transgenic mice lack kidneys entirely but die w i th in 48 hours o f b i r th , rather than 2 4 hours ( G u o et a l . , 2002) . T h u s , a l though k idney fa i lu re w o u l d eventua l l y lead to death , p o d o c a l y x i n - n u l l an ima ls may actua l l y d ie ear l ie r because of an addit ional defect. T h i s is a very exc i t ing possib i l i ty and w i l l require further invest igation. 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 lose l y related to C D 3 4 and endog l ycan ( N i e l s e n et a l . , 2002) . C D 3 4 f a m i l y members have been proposed to funct ion in enhancing pro l i ferat ion , b l o c k i n g di f ferent iat ion, establ ishing polar i ty , and regulat ing cel l adhesion pos i t ive ly and negatively (Baumhueter et a l . , 1993; C h e n g et a l . , 1996; F a c k l e r et a l . , 1995 ; F ieger et a l . , 2 0 0 3 ; M e d e r et a l . , 2 0 0 5 ; Sassetti et a l . , 1998 ; T a k e d a et a l . , 2000) . 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 mot i fs that, in the case o f p o d o c a l y x i n and endog lycan , faci l i tate interaction wi th N H E R F adapter proteins (He et a l . , 1992; 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 . , 1997; 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 . , 2006) . T h i s associat ion is k n o w n to connect p o d o c a l y x i n w i t h the act in cy toskeleton v i a e z r i n , a l inkage that is disrupted in several disease models (Or lando et a l . , 2 0 0 1 ; Takeda et a l . , 2001) . P o d o c a l y x i n is w i d e l y d ist r ibuted on vascular endothel ia l ce l l s (Horvat et a l . , 1986 ; K e r s h a w et a l . , 1997a). It is also expressed by hematopoiet ic progenitors , platelets and megakaryocy tes , a subset o f neuronal ce l l s , and mesothel ia l ce l l s l i n i n g many organs (Doyonnas 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 . , 1997 ; M i e t t i n e n et a l . , 1999 ; V i t u r e i r a et a l . , 2005) . S t r i k i n g l y , it is most h igh l y expressed on the elaborate g lomeru lar podocytes o f k idney , where it is v i tal for proper fo rmat ion of elaborate foot process extens ions ( 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) . Impor tan t l y , 208 abnormal podoca lyx in expression is associated w i th a number o f pathological condi t ions. 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 in h u m a n , m i c e l a c k i n g podoca lyx in have poor ly developed podocytes, exhib i t anur ia , and die w i th in 2 4 hours of b i r th ( D o y o n n a s et a l . , 2 0 0 1 ) . In h u m a n , increased p o d o c a l y x i n exp ress ion is an independent p red ic to r o f poor o u t c o m e in breast cancer , w h i l e mutat ions in the p o d o c a l y x i n gene, podxl, are f o u n d in patients w i t h h igh l y aggressive prostate cancer (Casey et a l . , 2 0 0 6 ; Somas i r i et a l . , 2004) . P o d o c a l y x i n is also l i nked w i th several other types of mal ignancies . 6.1.1 Podocalyxin's Role in Cell Morphogenesis In order to gain insights into podoca l yx in ' s role in disease and in normal development , it was overexpressed in M C F - 7 breast ca rc inoma cel ls (chapter 3). T h i s b locked format ion of c o n f l u e n t m o n o l a y e r s , and c e l l s ins tead e x h i b i t e d decreased c e l l - s u b s t r a t u m in teract ions . E c t o p i c e x p r e s s i o n a lso i n d u c e d a d r a m a t i c increase in m i c r o v i l l u s f o r m a t i o n . P o d o c a l y x i n express ion on ce l ls w i t h c o m p l e x membrane extensions is a c o m m o n theme under 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 essent ial fo r podocyte foot process format ion (Doyonnas et a l . , 2 0 0 1 ; Schnabel et a l . , 1989). H o w e v e r , it is a lso expressed on a subset of neurons , and f o r m a t i o n o f ce l lu la r extensions by podocytes and neurons d isplay many s imi lar i t ies ( rev iewed in ( K o b a y a s h i et a l . , 2004)) . A l t h o u g h these two cel l types are func t iona l l y very d i f ferent , the act iv i t ies of both are c r i t i ca l l y dependent on intr icate architectures. These h igh l y branched ce l ls require an extensive network o f cytoskeletal constituents. M i c r o t u b u l e s and intermediate f i laments support major processes o f podocytes and neuronal axons and dendr i tes , w h i l e act in 2 0 9 f i laments f o r m the backbone o f foot processes and dendr i t ic spines. Fur thermore, many o f the same cytoske leta l proteins are f o u n d in podocytes and neurons, i m p l y i n g that s imi la r mechanisms underl ie the format ion of ce l lu lar extensions in both ce l l types. M o r e o v e r , p o d o c a l y x i n is a lso 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 d i f fe rent ia t ing into 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 . , 2005) . O v e r a per iod o f 4 - 1 0 hours, the entire megakaryocy t i c cy top lasm is converted into m i c r o t u b u l e - b a s e d proplate let extens ions ( rev iewed in (Patel et a l . , , 2 0 0 5 ) ) . In addit ion to fo rmat ion of these long extensions, the ends of proplatelets bend and bifurcate in an act in -dependent process termed end a m p l i f i c a t i o n ; this fac i l i tates generat ion o f thousands o f platelets f r o m each megakaryocy te . T h e exact mechan isms i n v o l v e d in fo rmat ion o f podocyte foot processes and dendr i t ic spines, as we l l as the b i furcat ion of proplatelets are u n k n o w n , but they are al l act in-based processes and they al l occur in cel ls e x p r e s s i n g the ac t in -assoc ia ted p ro te in , p o d o c a l y x i n . T h u s , I be l ieve that a g loba l f u n c t i o n o f p o d o c a l y x i n is to fac i l i ta te the generat ion o f m o r p h o l o g i c a l l y d i s t inc t structures. In support of this hypothesis , C h e n g et al recently publ ished that podoca lyx in is required fo r tubulogenes is in vitro ( C h e n g et a l . , 2005) . M D C K ce l ls are der ived f r o m k i d n e y tubular ce l l s and can be i n d u c e d to f o r m tubules in vitro. D e p l e t i o n o f endogenous podoca l yx in by s i R N A b locks tubulogenesis. A l t h o u g h this system is somewhat ar t i f ic ia l s ince k idney tubules do not normal l y express p o d o c a l y x i n , the results may be relevant to other tissues where podoca l yx in is typ ica l l y expressed (Dekan et a l . , 1990; T a k e d a et a l . , 2 1 0 2001) . F o r example , podoca lyx in in m a m m a r y epithel ial ce l ls (Somasi r i et a l . , 2004) and vascular endothel ia (Horvat et a l . , 1986) may faci l i tate r e m o d e l l i n g and angiogenesis , respectively . P o d o c a l y x i n is compr i sed of an extensive m u c i n - l i k e ext racel lu lar region 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 th the act in cy toske le ton through N H E R F proteins and ezr in . 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 in order to study p o d o c a l y x i n ' s m e c h a n i s m of ac t ion . S u r p r i s i n g l y , a l though interact ion w i t h the act in cytoskeleton is l i k e l y i n v o l v e d in fo rmat ion o f p o d o c a l y x i n -induced ce l lu lar extensions, deletion of the major i ty of podoca l yx in ' s cy top lasmic tai l d id not af fect m i c r o v i l l u s f o r m a t i o n . In contrast , and more expected ly , tubulogenes is in M D C K cel ls does require p o d o c a l y x i n ' s int racel lu lar d o m a i n ( C h e n g et a l . , 2005) . T h e apparently contradictory ev idence suggests that alternative mechan isms may be ut i l i zed under certain cond i t ions , or that the two types of morphogenet ic alterations occur v i a somewhat different pathways. The requirement for the m u c i n - l i k e d o m a i n , however , is supported by al l avai lable data. Podoca lyx in -dependent m i c r o v i l l u s fo rmat ion was comple te l y ablated upon delet ion of the extracel lular d o m a i n , foot process architecture is drast ical ly affected by neutral izat ion o f p o d o c a l y x i n ' s negative charge, and tubulogenesis is m u c h less extensive when the m u c i n d o m a i n is r e m o v e d , whether genet ica l ly or by treatment w i th inh ib i tors o f O -l i n k e d g l ycosy la t ion (see chapter 3) ( A n d r e w s , 1979 ; C h e n g et a l . , 2 0 0 5 ; Se i le r et a l . , 1977). P o d o c a l y x i n may st imulate the fo rmat ion o f ce l lu la 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 th its role in ce l l morpho logy , there is c lear ly m o u n t i n g ev idence to impl i ca te p o d o c a l y x i n , a long wi th C D 3 4 , in decreasing ce l lu lar adhesion. Interestingly, it appears that C D 3 4 and p o d o c a l y x i n may e m p l o y several m e c h a n i s m s to act as g loba l a n t i -adhesins. They have been shown to decrease cel l -substratum adhesion, as we l l as c e l l - c e l l adhesion, both by interfering wi th c e l l - c e l l junct ions and by b l o c k i n g aggregation of cel ls in suspension (chapter 3) (Doyonnas 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 . , 2000) . Decreased c e l l - c e l l adhesion between mul t ip le podoca l yx in or C D 3 4 expressing cel ls in suspension can easi ly be exp la ined by steric hindrance or charge repuls ion between s ia lomuc ins on nearby cel ls . Express ion of podoca l yx in at the tips of m i c r o v i l l i , also generated in a podoca lyx in -dependent manner, w o u l d further increase the ant i -adhes ion phenotype, as m i c r o v i l l i are the f i rst point o f contact between cel ls . S i m i l a r l y , c e l l - c e l l junct ions may s i m p l y be affected by re loca l i zat ion o f p o d o c a l y x i n or C D 3 4 toward the junct ions , thereby f o r c i n g ne ighbour ing ce l ls 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 junct ions between adjacent k idney podocytes d u r i n g deve lopment c o i n c i d e s prec ise ly w i t h p o d o c a l y x i n express ion and m i g r a t i o n toward the basal ce l l surface (Schnabel et a l . , 1989). A g a i n , m i c r o v i l l u s fo rmat ion near the ap ica l sur face, between these ce l ls w o u l d fur ther serve to increase the spac ing between cel ls . 213 Decreased ce l l - subst ratum interact ions are more d i f f i cu l t to exp la in because C D 3 4 and p o d o c a l y x i n are genera l l y a p i c a l m e m b r a n e proteins . In the unusual H G E C culture sys tem, where some p o d o c a l y x i n is aberrant ly l o c a l i z e d to the basolateral sur face, decreased ce l l - subst ra tum interact ions can be exp la ined by steric h indrance or charge repuls ion , where the negat ively charged m u c i n - l i k e d o m a i n of podoca lyx in is expressed in the v i c i n i t y o f the s i m i l a r l y charged l a m i n i n or c o l l a g e n ex t race l lu la r m a t r i x components . In most ce l l s , though, this is not the case. H o w , then, does an apical protein affect adhesion at the basal .surface? A n int r iguing poss ib i l i ty is that podoca lyx in - induced act in recruitment for generat ion o f apica l ce l l surface structures may deplete actin f r o m the basal sur face , thereby d e c r e a s i n g the s tab i l i t y o f i n t e g r i n - m e d i a t e d adhes ion complexes at the basal surface (F igure 6 -1) . In support of this idea, cel ls transfected w i th p o d o c a l y x i n mutants that induced m i c r o v i l l u s f o r m a t i o n also d isp layed decreased ce l l adhesion, whereas the extracel lu lar delet ion mutant d i d not induce m i c r o v i l l u s fo rmat ion or decrease c e l l adhes ion . S i m i l a r l y , p o d o c a l y x i n may w e a k e n c e l l - c e l l j unc t ions by t itrating actin away f r o m these junct ional proteins as w e l l . M o r e o v e r , since podocyte foot processes conta in an extens ive network o f act in f i l a m e n t s , p o d o c a l y x i n m a y affect mul t ip le aspects of podocyte architecture by i n d u c i n g act in reorganizat ion. Thus , loss of p o d o c a l y x i n m a y ind i rec t l y af fect cy toske le ta l interact ions w i t h proteins o f the sl i t d iaphragm c o m p l e x in addit ion to those on the basal ce l l surface. Th i s w i l l require further inves t iga t ion o f the in te rp lay between act in rec ru i tment by ap ica l and basolatera l proteins. 2 1 4 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 I I III * t T : f-actin cell junctions integrins microvilli Figure 6-1: Model Demonstrating a Possible Mechanism for Decreased Cell-Substrate Adhesion Induced by Apical Podocalyxin Expression. A p i c a l recruitment of f -ac t in by p o d o c a l y x i n may titrate act in away f r o m basal integrins and c e l l - c e l l junct ional proteins, thereby weaken ing these interactions. 215 6.1.3 Podocalyxin-Dependent NHERF Localization P o d o c a l y x i n ' s role in apical recruitment of N H E R F 1 is another s igni f icant result of this work . 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 recogni t ion 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 . , 2006) . S ince N H E R F f a m i l y members can l ink proteins indirect ly to the act in cy toske le ton through E R M d o m a i n - d e p e n d e n t in teract ions w i t h cy toske le ta l l i n k e r m o l e c u l e s , it had been p roposed that N H E R F prote ins are respons ib le 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 of th is , p o d o c a l y x i n mutants l a c k i n g the P D Z b ind ing 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 , more i n t r i g u i n g l y , I have now s h o w n that p o d o c a l y x i n is actual ly respons ib le for l o c a l i z a t i o n o f N H E R F 1 in M C F - 7 ce l l s (see chapter 3). W h i l e on ly about 2 0 % of p o d o c a l y x i n is m i s l o c a l i z e d in the absence of interact ion w i th N H E R F , v i r tua 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 ap ica l surface, in ce l l s ec top ica l l y express ing p o d o c a l y x i n mutants incapable o f interact ing 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 ap ica l l oca l i za t ion of both p o d o c a l y x i n and N H E R F ear ly in c e l l po la r i za t ion , suggests that p o d o c a l y x i n , not N H E R F 1 , is the ma jo r p layer in the process 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 important imp l i ca t ions for the numerous act iv i t ies o f these s c a f f o l d i n g proteins. N H E R F 1 and N H E R F 2 are capable of interact ing w i t h a w i d e var iety of m o l e c u l e s , i n c l u d i n g ion transporters, s i g n a l l i n g proteins, and receptors, such as ep idermal g rowth factor receptors and |32-216 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 Podocalyxin-Null 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 ESCs that specifically expressed podocalyxin and G F P in a Cre recombinase-dependent 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, l ikely 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 podxt1' mice, with the intention of 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 defect , express ion i n podocytes was expected to rescue these m i c e , thereby enab l ing analysis of adult mice l a c k i n g podoca lyx in in other tissues. D u e to delays in product ion o f cond i t iona l p o d o c a l y x i n express ing m i c e (chapter 4 ) , a new strategy was in i t iated in order 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 in podocytes . Podxl was inserted into an express ion vector d o w n s t r e a m of the p o d o c y t e - s p e c i f i c NPHSJ promoter , and transgenic m i c e were generated us ing this construct (chapter 5) . T h e transgene was expressed spec i f i ca l l y in podocytes, so podxt'+transgene+'~ m i c e were crossed w i t h podxt'transgene'1' m i c e , and these were in terbred to generate m i c e express ing p o d o c a l y x i n on ly in k idney . S t r i k i n g l y , podocytes in these m i c e d i sp layed re la t i ve ly no rmal foot processes and 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 inappropr ia te l y reta ined 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 . T h i s p r o v i d e d c o n c l u s i v e ev idence that podoca l yx in is both necessary and suff ic ient fo r podocyte morphogenesis , again demonstrating podoca l yx in ' s role in generation o f ce l l surface projections. H o w e v e r , although podocyte architecture was repaired in these transgenic m i c e , they sti l l suffered perinatal lethal i ty , suggest ing that p o d o c a l y x i n m a y play addi t ional essential roles in other tissues. L o s s o f podoca lyx in may cause vascular defects, such as decreased p e r m e a b i l i t y fo r t ransendothe l ia l m i g r a t i o n , increased g loba l s t i ck iness o f vascu la r endothel ia l ce l ls to hematopoiet ic ce l l s , or co l lapse of b lood vessels due to interactions between o p p o s i n g membranes in thei r l u m e n s . P late le ts l a c k i n g p o d o c a l y x i n may generate clots inappropriately , or they may be decreased in number due to megakaryocyte abnormal i t i es . L o s s o f p o d o c a l y x i n f r o m the l i n i n g s o f body cav i t ies m a y lead to 218 increased organ damage due to inappropr iate adhesion o f organs to one another. There m a y even be neuronal defects in p o d o c a l y x i n - n u l l animals . Thus , the absence o f rescued m i c e is actual ly a more fasc inat ing outcome than that w h i c h was predicted. It w i l l be in te res t ing to assess other d e v e l o p m e n t a l defects that m a y be caused by loss o f p o d o c a l y x i n . 6.2 Significance of Results Express ion o f podoca lyx in in a variety of tissues, inc lud ing podocytes, vasculature, some hematopoiet ic ce l l s , boundary elements separating organs, and a subset of neuronal ce l l s , a l o n g w i th altered express ion in numerous mal ignant s i tuat ions, makes it a potent ia l ly impor tan t p rote in f o r fur ther a n a l y s i s . T h e absolute requ i rement fo r p o d o c a l y x i n express ion in order for m i c e to surv ive beyond the f i rst day of l i fe makes it even more int r igu ing . 6.2.1 Podocalyxin in Normal Development A d h e s i o n and m o r p h o g e n e s i s are both e x t r e m e l y impor tan t ac t i v i t i es th roughout deve lopment . A d h e s i o n , or lack thereof, regulates proper m i g r a t i o n o f c e l l s , w h i l e morphogenesis is cr i t ica l fo r the generation of many spec ia l i zed ce l l types. P o d o c a l y x i n is c lear ly important in fac i l i ta t ing fo rmat ion of intr icate podocyte foot processes, but it m a y a lso p lay a ro le in generat ion o f neuronal processes and the m e g a k a r y o c y t i c extensions i n v o l v e d in platelet product ion . In addi t ion , it may be invo l ved in g lobal ce l l m i g r a t i o n and m o v e m e n t o f h e m a t o p o i e t i c ce l l s across endothe l ia l barr iers w h e n 2 1 9 c o l o n i z i n g new niches. Fur thermore , the coat ing o f p o d o c a l y x i n on mesothel ia l ce l l s l i n i n g body cavi t ies l i k e l y protects organs f r o m damage. T h i s is especia l ly ev ident in 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 h i c h are i n e f f i c i e n t at re t rac t ing p h y s i o l o g i c o m p h a l o c e l e s late in d e v e l o p m e n t . 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 t h r o u g h o u t development and in the adult may regulate adhesion and ce l l morphogenesis in a variety of 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 th a w ide variety of cancers. It is upregulated in hepatoce l lu la r c a r c i n o m a and a subset of test icu lar cancers , and it is a lso f o u n d in leukemias (Chen et a l . , 2 0 0 4 ; K e l l e y et a l . , 2 0 0 5 ; Schopper le et a l . , 2003) . Importantly , it is upregulated or mutated in h igh ly aggressive breast cancers , W i l m s ' tumours , and prostate cancers (Casey et a l . , 2 0 0 6 ; Somas i r i et a l . , 2 0 0 4 ; S tanhope -Baker et a l . , 2004) . A l t h o u g h the result of p o d o c a l y x i n dysregulat ion in these mal ignant situations is not yet c lear , its assoc ia t ion w i t h the most aggress ive cases and its role i n regu la t ing c e l l adhesion suggest that it may faci l i tate metastasis. M o r e o v e r , so l id tumours, w h i c h require increased nutrients, and therefore increased b lood f l o w , may become more aggressive as a resul t o f 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 u n d e r l y i n g m e c h a n i s m s behind increased p o d o c a l y x i n express ion and its effects in these disease condit ions is therefore part icular ly important. 2 2 0 6.3 Future Directions A l t h o u g h I have gained some important insights into the funct ions of p o d o c a l y x i n , the m e c h a n i s m s behind its act i v i t ies , its roles in deve lopment , and the consequences o f aberrant express ion , this w o r k has generated many more questions. In m y o p i n i o n , the most in te res t ing avenues to pursue i n c l u d e unders tand ing where p o d o c a l y x i n is absolutely essential throughout development , how it affects cancer progression, and how it can induce morpho log ica l changes in the absence of the majority of its cy top lasmic ta i l . 6.3.1 Understanding Podocalyxin Mechanistically F i n d i n g an in vitro model system for assessing p o d o c a l y x i n funct ion (chapter 3) was a major step f o r w a r d , and the induct ion o f m i c r o v i l l u s f o r m a t i o n can now be used to elucidate mechanisms of podoca lyx in - induced morpho log ica l changes. De le t ion of al l but six jux tamembrane a m i n o ac ids of p o d o c a l y x i n ' s intracel lu lar d o m a i n was expected to complete ly b lock interactions wi th al l k n o w n cy top lasmic interaction partners, i n c l u d i n g N H E R F proteins and ezr in . W h i l e N H E R F certainly does not interact w i t h this de let ion mutant (F igure 3 - 1 5 ) , absence of podoca lyx in/ez r in interact ions must be c o n f i r m e d . A l t h o u g h two o f the three a m i n o ac ids k n o w n to be i n v o l v e d in d i rect ly b i n d i n g ez r in were retained in the podoca lyx in Ata i l mutant, I C A M - 3 , w h i c h also interacts direct ly w i t h ez r in , fa i l s to b ind when on ly the f i rst eight a m i n o acids are retained in the c y t o p l a s m i c tai l (Schmieder et a l . , 2 0 0 4 ; Serrador et a l . , 2002) . T h u s , the R E H Q R S G S sequence o f I C A M - 3 is insuf f i c ient fo r interact ion w i t h ez r in , suggest ing that the p o d o c a l y x i n ta i l mutant that inc luded on ly C C H Q R F (Atai l ) w o u l d also be un l ike l y to b ind . H o w e v e r , to 221 f o r m a l l y prove this , G S T - p u l l d o w n exper iments w i l l be per formed us ing G S T - t a g g e d N ' te rmina l ezr in and lysates f r o m cel ls expressing p o d o c a l y x i n mutants. A s s u m i n g that the Ata i l mutant cannot interact w i th ez r in , it is possible that an addit ional protein interacts w i th the extracel lular or t ransmembrane regions of podoca l yx in in order to l i nk it to the act in cy toske leton , or at least to transduce the s ignal required fo r act in reorganizat ion and m i c r o v i l l u s fo rmat ion . A l t h o u g h m u c h of p o d o c a l y x i n ' s extracel lu lar d o m a i n is not conserved , the jux tamembrane stalk retains about 7 0 % s imi la r i t y across species , so this is a l i k e l y candidate region fo r a prote in -prote in interact ion d o m a i n . A l t h o u g h f i n d i n g an extracel lu lar b ind ing partner fo r p o d o c a l y x i n may be d i f f i cu l t , the question of whether or not there is one is an important one to address. S p e c i f i c de le t ions o f 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 j u x t a m e m b r a n e ext race l lu lar sta lk , the cys te ine -bonded g lobu la r d o m a i n , and port ions o f the m u c i n d o m a i n should be generated in an effort to p inpoint regions o f importance. In add i t ion , reagents cou ld 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 ia l ic ac id residues cou ld be removed us ing neuraminidase, or O - g l y c o s y l a t i o n cou ld be b l o c k e d entirely . T h u s , many exper iments c o u l d be per formed in order to p inpo int the mechan ism by w h i c h podoca lyx in regulates ce l l morpho logy . 6.3.2 Podocalyxin's Role in Development T h e unexpected lack o f rescue of p o d o c a l y x i n k n o c k o u t m i c e express ing t ransgenic p o d o c a l y x i n spec i f i ca l l y in k idney was quite in t r igu ing . If the k idney defect was real ly 2 2 2 the on ly cause o f perinatal lethal i ty in podoca l yx in -de f i c ien t an imals , re introduct ion o f the protein into podocytes should have repaired the defect and rescued the mice . T h u s , either p o d o c a l y x i n was expressed inadequately in t ransgenic a n i m a l s , or the k i d n e y defect is not the o n l y major abnormal i t y in 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 seem to be expressed s u f f i c i e n t l y to repai r the defect , as d ramat ic m o r p h o l o g i c a l differences were noted between podxl'1' an imals wi th and without the transgene, al though it was possible that there were some sl ight di f ferences between these mice and w i ld type l ittermates (chapter 5) . T h u s , a l though podocy te -spec i f i c expression o f p o d o c a l y x i n d id not enable analysis o f defects in adult an imals , w i th the appropriate assays it may st i l l be possible to f i n d other defects in podoca lyx in -def ic ient animals 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 podoca l yx in has been associated wi th numerous types of cancer, conf i rmat ion that it is a causal factor in cancer progression has yet to be obtained. Th i s w i l l be investigated us ing two approaches. F i rst , M C F - 7 cel ls ectopica l ly expressing podoca lyx in (chapter 3) w i l l be in jected 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 ce l ls are k n o w n to generate tumours , and overexpress ion o f p o d o c a l y x i n may enhance metastasis of these ce l l s . If d i f ferences are noted between cancer progress ion in m i c e injected w i t h p o d o c a l y x i n -transfected ce l ls and vector cont ro l ce l l s , then the exper iment w i l l be repeated us ing M C F - 7 ce l ls ectop ica l l y express ing 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 gain insights into exact ly how podoca l yx in affects cancer progression, mechanist ica l ly . T h u s , the c r i t i ca l d o m a i n s respons ib le fo r cancer progress ion w i l l be iden t i f i ed , and potent ial interact ing proteins w i l l be e x a m i n e d . Important ly , the p o d o c a l y x i n b i n d i n g 223 protein , ezr in is essential fo r numerous s igna l l ing pathways impl icated in metastasis and poor outcome in various cancers (Casey et a l . , 2006) . T h e second strategy for assessing p o d o c a l y x i n ' s i n v o l v e m e n t in cancer aggressiveness invo l ves overexpress ion of podoca l yx in in m a m m a r y t issue, and perhaps other sites, in transgenic m i c e . T h e C r e - l o x P strategy d iscussed in chapter 4 is be ing repeated us ing 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 induc ib le in any tissue, or at any t ime point , as l o n g as a C r e mouse is ava i lab le . F o r e x a m p l e , c ross ing p o d o c a l y x i n induc ib le transgenic mice to whey ac id ic protein ( W A P ) - C r e mice (Wagner et a l . , 1997) w i l l s p e c i f i c a l l y i n d u c e p o d o c a l y x i n exp ress ion in the m a m m a r y g l a n d . 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