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Expression of chick semaphorin 5B during neuronal development Legg, Arthur Terrence 2003

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Expression of Chick Semaphorin 5B During Neuronal Development by ARTHUR TERRENCE LEGG B . S c , The University o f British Columbia, 1998  A THESIS 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 OF THE REQUIREMENTS OF THE D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department o f Anatomy and Cell Biology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A A p r i l 2003 © Arthur Terrence Legg, 2003  U B C Rare Books and Special Collections - Thesis Authorisation Form  Page 1 o f 1  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada  http://www. library. ubc. ca/spcoll/thesauth. html  4/22/2003  Abstract During development, the formation o f a functional nervous system requires precise pathfinding o f axons to their targets. Growth cones at the leading edge o f these axons use the information supplied by a variety o f cues i n the environment to navigate their course. The semaphorins, comprising a large family o f neuronal guidance cues, were first identified in the grasshopper limb bud and were shown to be important for proper T i l axon pathfinding. Most o f the studies from invertebrates and vertebrates have demonstrated an inhibitory role but more recent studies have shown them to have a b i functional nature, also acting as attractive cues. The transmembrane semaphorin, chick semaphorin 5B ( c S E M A 5 B ) , is unique from other semaphorin in that it contains both an inhibitory S E M A domain and a region o f thrombospondin type-1-like repeats that have been associated with neuronal outgrowth. To determine whether this semaphorin plays a role i n axon guidance in the developing chick nervous system, its expression was analyzed using a variety o f techniques including in situ hybridization, R T - P C R , and immmunocytochemistry. Expression o f c S E M A 5 B is first clearly identified at E 5 i n the spinal cord, D R G s , retina, and in a variety o f neuroepithelia associated with the tectum, ventricular regions, and the olfactory system. Expression within the spinal cord is dynamic being first broadly expressed in both dorsal and ventral regions at E 5 with the ventral expression persisting through E l 1. A t this later stage the expression is associated with large-diameter cells i n the lateral motor column. Expression within the retina occurs along the retinal ganglion cell layer and is relatively uniform i n distribution and is maintained from E5 through E 1 0 , while the expression i n the tectum appears to occur i n a gradient with highest levels i n the anterior region. Results from these studies, along with  ii  some important in vitro studies performed by others i n our lab, have suggested that c S E M A 5 B is important for the neuronal pathfinding within the spinal cord, retina, and developing tectum.  Table of Contents Abstract List o f Figures List o f Tables List o f Abbreviations Acknowledgements INTRODUCTION A x o n guidance Semaphorins Genetic Analysis o f Semaphorins Semaphorin Receptors Genetic Analysis o f Semaphorin Receptors Semaphorin Signaling Transmembrane Semaphorins Class 5 Semaphorins Hypothesis MATERIALS A N D METHODS R N A Work Probe sequence Synthesis o f DIG-labeled R N A probe In Situ Hybridization studies Tissue preparation Pre-hybridization In Situ Hybridization Post-hybridization. Nissl stained sections Northern Analysis R T - P C R studies Affinity Purification o f c S E M A 5 B Antibody Purification o f GST-fusion protein Affinity column preparation Antibody purification procedure Antibody Staining Antibody staining o f dissociated cultures Western Blots Cytoskeletal extractions RESULTS Structure o f c S E M A 5 B Construction o f an R N A probe Northern Studies R T - P C R Studies In Situ Hybridization Studies c S E M A 5 B expression in the Spinal C o r d c S E M A 5 B expression in D R G c S E M A 5 B expression i n the Retina  :  ii vi vii viii xi 1 1 1 5 7 10 12 17 18 20 22 22 22 22 23 23 24 24 25 25 26 26 27 27 28 29 29 30 30 31 33 33 33 38 41 42 42 45 45  iv  c S E M A 5 B expression in the Tectum Epithelial expression Immunohistological Studies Westerns c S E M A 5 B distribution in the Spinal Cord Distribution o f c S E M A 5 B in D R G s c S E M A 5 B distribution in the retina DISCUSSION Role o f S E M A 5B in developing spinal cord Functional Studies Role o f S E M A 5 B in retina Functional Studies Role o f c S E M A 5 B in tectum Role o f S E M A 5 B in the neuroepithelium Future directions REFERENCES  49 52 57 57 60 68 68 72 73 79 81 82 83 85 88 89  v  List of Figures Figure 1: Semaphorins and Semaphorin Receptors  3  Figure 2: Semaphorin Signaling  13  Figure 3: Semaphorin 5B Structure and amino acid sequence  34  Figure 4: Analysis and Alignment o f the c S E M A 5 B R N A Probe  36  Figure 5: Northern and R T - P C R Results  39  Figure 6: In situ hybridization studies in the spinal cord  43  Figure 7: In situ hybridization studies i n the retina  47  Figure 8: In situ hybridization studies i n the tectum  50  Figure 9: In situ hybridization studies i n ventricular regions  53  Figure 10: In situ hybridization studies i n nasal regions  55  Figure 11: Antibody production and western blots  61  Figure 12: Semaphorin 5B distribution in Spinal Cord  63  Figure 13: Dissociated Spinal Cultures  66  Figure 14: Semaphorin 5B distribution i n the Retina  69  Figure 15: Models for c S E M A 5 B functions i n axon guidance  75  Figure 16: A M o d e l for c S E M A 5 B function i n migration  86  vi  List of Tables Table I: Summary o f c S E M A 5 B expression i n all regions  58  vn  List of Abbreviations bp  basepairs  BCIP  5-bromo-4-chloro-3-indolyl-phosphate  CNS  central nervous system  Cy3  indocarbocyanine  CUB  complement-binding  DH  dorsal horn  DRG  dorsal root ganglion  ECM  extracellular matrix  EN  external nuclear layer  EP  ependymal layer  DEPC  diethyl pyrocarbonate  FITC  fluorescein isothiocyanate  GCL  retinal ganglion cell layer  GPI  glycosylphosphatidylinositol  GST  glutathione S-transferase  IN  inner nuclear layer  kb  kilobase  kD  kilodalton  mg  milligram  MgCb  magnesium chloride  MgS04  magnesium sulphate  MICAL  flavoprotein monooxy-genase  ml  milliliter  mM  millimolar  NBT  4-nitro blue tetrazolium chloride  NGF  nerve growth factor  Npn  neuropilin  NT  neurotrophin  OFL  optic fiber layer  ON  optic nerve  ONH  optic nerve head  OTK  off-track  PBS  phosphate buffered saline  PCR  polymerase chain reaction  PLMC  presumptive lateral motor column  PMSF  phenylmethylsulfonyl fluoride  PNS  peripheral nervous system  RGC  retinal ganglion cell  RT-PCR  reverse-transcriptase polymerase chain reaction  SDS  sodium dodecyl sulfate  SGC  stratum griseum centrale  SO  stratum opticum  ng  microgram  ul  microliter  uM  micromolar  ventral horn white matter  Acknowledgements I would firstly like to thank Dr. Timothy P. O'Connor for allowing me to conduct m y research in his lab, for his excellent guidance throughout the entire duration o f this degree, his passion for science, and for his friendship i n and out o f the lab. I would like to thank m y committee members, D r . W . Tetzlaff and Dr. C . Roskelley for their suggestions and scientific advise. I would like to thank Dr. Jennifer Bonner for her scientific guidance, her friendship while sharing an office. I would like to thank m y fellow graduate students i n Anatomy as they made this whole process a lot o f fun. I would like to thank m y parents who have supported me i n whatever endeavors I have gone through. Finally, I would like to thank m y L o r d Jesus Christ who gives me strength and guidance always.  xi  Introduction: Axon guidance: The generation o f a functional nervous system requires precise pathfinding o f axons to their appropriate targets. H o w neurons accomplish this feat while making relatively few errors is unclear, however, the precise temporal and spatial distribution o f guidance factors is a requirement. Guidance cues evoke stereotyped responses from neurons including attraction or repulsion, and can act locally i f bound to cell membranes and the E C M , or at a distance i f they are secreted. The growth cone, a highly dynamic, actin-rich sensory structure found at the leading edge o f the axon, is responsible for interpreting these signals and translating them into a number o f growth cone behaviors including outgrowth, turning, fasciculation, retraction, and stalling (Suter and Forscher 2000). When first describing a growth cone over a century ago, Ramon y Cajal proposed that this structure responds to guidance factors to direct axonal growth. This has proven to be true and in the subsequent century, numerous guidance cues have been identified, and their effects on growth cone behaviors have been characterized. Even though a number o f families o f guidance cues have been identified, the precise mechanisms underlying growth cone behavior and response to these guidance cues are not resolved. Semaphorins: To date the major families o f guidance proteins identified include the Ephrins, Slits, Netrins, and Semaphorins (Grunwald and K l e i n , 2002). These guidance cues can localize to the membrane, as is the case with ephrins and some members o f the semaphorin family, or they may be secreted proteins like netrins, slits, as w e l l as some o f the semaphorins. Different families are typically associated with a particular response,  1  either attractive or inhibitory, but many exhibit bi-functional guidance properties. O f these guidance cues, semaphorins comprise the largest family. W i t h more than 20 different members identified to date, the semaphorins are subdivided into eight different classes based on domain structure and species o f origin (figure 1; Semaphorin Nomenclature Committee 1999). Semaphorins comprise a diverse family o f secreted and membrane associated proteins characterized b y the presence o f a conserved semaphorin (Sema) domain, 500 amino acids i n size. The semaphorins may be bound to the membrane through either a transmembrane domain (classes 1, 4, 5, 6) or through a glycophosphatidyl inositol linkage (class 7), or they may be secreted as is the case with classes 2, 3, and V (figure 1). The first identified semaphorin, grasshopper Sema-la (previously named fasciclin IV), was initially implicated i n axon guidance using antibody perturbation experiments (Kolodkin et al., 1992, 1993). Subsequent purification o f a secreted semaphorin, chick Collapsin-1 (Sema3A), demonstrated the chemorepulsive nature o f this semaphorin as acute addition led to rapid actin depolymerization and growth cone collapse o f D R G neurons i n vitro (Luo et al., 1993). Following this initial characterization, a number o f studies went on to demonstrate the repulsive nature o f semaphorins for a variety o f neuronal populations (Matthes et al., 1995; Tanelian et al., 1997; Bagnard et al., 1998; Chedotal et al., 1998; Shoji et al., 1998; Winberg et al., 1998b; Y u et al., 1998; de Castro et al., 1999; M i y a z a k i et al., 1999; Rabacchi et al., 1999; Roos et al., 1999, Steup et al., 1999; Y u et al., 2000; Tamamaki et al., 2003). In addition to functioning as repulsive guidance cues, semaphorins have also been found to function as attractive cues (Wong et al., 1997; Bagnard et al., 1998; Song et al., 1998; de Castro et al., 1999; W o n g et al., 1999; Bagnard et al., 2000; Fujioka et al., 2003). For  2  Figure 1 : Semaphorins and Semaphorin Receptors Genes encoding semaphorins are highly conserved from invertebrates to humans. There are at least 20 known semaphorins that can be divided into 8 subclasses based on structural similarities. Classes 1, 2, and 5 are invertebrate semaphorins, classes 3 through 7 are vertebrate semaphorins, and class V are viral associated semaphorins. Common to all semaphorins is a 500-amino acid length semaphorin domain at their amino termini (orange), which is highly conserved between invertebrates and vertebrates. The carboxyl regions o f the semaphorins are more variable. Classes 2 through 4, 7, and a subset o f V contain an Ig domain (blue/white), while class 5 members contain a thrombospondin type-1 repeat domain (purple). The semaphorins are further distinguished biochemically as being secreted, membrane glycosylphosphatidylinositol (GPI)-anchored, or transmembrane molecules. A schematic representing the two classes o f semaphorin receptors, the neuropilins and plexins, depicts the various domain structures associated with each. There are two classes o f neuropilins, both o f which have a very small intracellular domain, which interact with the secreted class 3 semaphorins and the plexins, and are only found in vertebrates. The plexin family has four classes ( A - D ) in vertebrates, with known interactions with semaphorins from classes 3, 4, and 7. There are two classes ( A & B ) i n invertebrates that are known to interact with semaphorins from class 1. K n o w n interactions between the receptors and semaphorins are shown with lines (red).  3  example, while Sema3A is repulsive for cortical axons, another semaphorin, Sema3C acts as an attractive guidance cue for these same neurons (Bagnard et al., 1998). Similarly, in the developing grasshopper limb, the T i l neurons turn away from their normal pathway to contact cells ectopically expressing Sema-la, suggesting an attractive response (Wong et al., 1999). In addition, the activation o f the c G M P pathway in Xenopus spinal neurons can convert Sema3A repulsion to attraction (Song et al., 1998). Additional examples o f both repulsive and attractive roles for these proteins in vitro have been demonstrated (Raper, 2000). Genetic Analysis of Semaphorins: A l o n g with many in vitro studies describing the function o f semaphorins, a number o f studies have described a role for semaphorins in vivo. Observations on a number o f mutants from classes 1 and 2 i n invertebrates, and from classes 3 and 6 in vertebrates, have led to many conclusions about the in vivo function o f these semaphorins (Behar et al., 1996; Taniguchi et al., 1997; Shoji et al., 1998; Catalano et al., 1998; Winberg et al., 1998; Y u et al., 1998, 2000; R o y et al., 2000; White et al., 2000; Bahri et al., 2001; Leighton et al., 2001; Ginzburg et al., 2002). There are three semaphorins (Cesema-la or smp-1, Ce-sema-lb  or smp-2, Ce-sema-2a or mab-20) in C. elegans and  mutants for each have been described (Roy et al., 2000; Ginzburg et al., 2002). While the majority o f defects associated with Ce-sema-2a include errors i n morphogenesis and epithelial cell-to-cell contacts, these embryos also display errors in axon guidance and cell migration (Roy et al., 2000). Mutants exhibit a number o f fasciculation and pioneer guidance defects i n at least one o f the D A and D B motor neurons, as well as errors i n neuroblast cell migration (Roy et al., 2000). A mutation i n either the Ce-sema-la or Ce-  5  sema-lb also results i n a number o f defects i n epidermal cell morphogenesis and cell-tocell contacts (Ginzburg et al., 2002). In addition to errors i n morphogenesis, and Ce-sema-lb  Ce-sema-la  mutants exhibit mild defects in the guidance o f P L M L and P L M R axons,  including premature stopping and unusual branching (Ginzburg et al., 2002). In Drosophila,  null mutations i n D-Sema-la  result in premature stalling o f motor axons and  failure o f these axons to defasciculate ( Y u et al., 1998). In addition, ectopic expression of D - S e m a - l a i n muscle cells results in motor axons avoiding these targets ( Y u et al., 1998). Expression o f a truncated form o f D - S e m a - l a i n these flies is able to rescue this phenotype, suggesting that the cytoplasmic domain o f this transmembrane semaphorin is not required for this function (Raper, 2000). Recent studies on D-Sema-la  mutants have  demonstrated that this semaphorin is also required for proper axon guidance and synapse formation in the adult Giant Fibre system (Godenschwege et al., 2002). Rescue experiments in these mutants further demonstrated that D - S e m a - l a is required both preand post-synaptically (Godenschwege et al., 2002). Ectopic expression o f D-Sema-2a i n muscles prevents motoraxons from correctly pathfinding to these targets (Winberg et al., 1998). The converse is true in D-Sema-2a mutants, where many motorneurons are observed to incorrectly invade muscle fibers that normally express D-Sema-2a (Winberg etal., 1998). In addition to genetic analysis o f semaphorins i n invertebrates, vertebrate mutants have also been described. M i c e lacking Sema3 A exhibit a number o f defects i n sensory and motor axon patterning i n the developing spinal cord (Behar et al., 1996; Taniguchi et al., 1997), however, the overall morphology o f the C N S i n these mice is relatively normal (Catalano et al., 1998, Ulupinar et al., 1999). This may be due to functional redundancy  6  o f different semaphorins. For example, sympathetic neurons are repulsed by multiple semaphorins from the class 3 family in vitro (Adams et al., 1997; Takahashi et al., 1998). Homozygous mutant mice lacking Sema6A, the mouse homologue o f invertebrate D Sema-la, display a number o f aberrant thalamocortical projections (Leighton et al., 2001). These neurons normally express Sema6A, which suggests that this guidance defect is acting cell-autonomously and that this semaphorin serves as a guidance receptor in these neurons (Leighton et al., 2001). Thus, while the studies on the Sema3A knock-out mouse are not conclusive, other in vivo studies from invertebrates and mice support the in vitro findings and demonstrate that a number o f different semaphorin classes normally function in axon guidance during development. Semaphorin  receptors:  Two families o f transmembrane proteins, the neuropilins (Npn) and the plexins have been identified as receptors for semaphorins. The first receptors discovered belong to the neuropilin family o f proteins, Npn-1 and Npn-2. They were first identified as semaphorin receptors i n an expression screen which exploited their high affinity for semaphorin-3A (Kolodkin et al., 1997; H e and Tessier-Lavigne, 1997; Fujisawa and Kitsukawa, 1998; K o l o d k i n 1998). Subsequent analysis demonstrated that this interaction was conserved for other class three members, however, different class members were shown to interact with different combinations o f neuropilins for signaling (Nakamura et al., 2000). Specifically, Sema3A was shown to signal through a receptor complex consisting o f Npn-1, while Sema3F signals through a complex consisting o f Npn-2, and  7  Sema3C signals through a hetero-dimeric complex consisting o f both Npn-1 and Npn-2 (Nakamura et al., 2000). Npn-1 was originally characterized as a cell surface molecule with a restricted expression pattern in the optic tectum o f Xenopus (Takagi et al., 1991). When over expressed in embryonic mice, Npn-1 leads to axon defasciculation and sprouting (Kitsukawa et al., 1995). The Npns are structurally characterized by the presence o f two extracellular complement-binding ( C U B ) domains, followed by the presence o f two coagulation factor V / V I H homology domains, a M A M domain, and a small cytoplasmic region (figure 1; Chen et al., 1997). The C U B domain was shown to be the interacting domain for Sema3A from analysis o f various Npn-1 deletion mutants (Nakamura et al., 1998). M a n y observations apart from the biochemical interaction studies have demonstrated that Npn-1 is required for functional Sema3A receptors. Antibodies to Npn-1 block Sema3A induced collapse o f neurons in vitro (He and Tessier-Lavigne, 1997; K o l o d k i n et al., 1997). In addition, D R G neurons isolated form Npn-1 null mice do not undergo growth cone collapse i n the presence o f semaphorin 3 A (Kitsukawa et al., 1997). The small cytoplasmic tail o f Npn-1 interacts with a S E M C A P protein, neuropilin interacting protein (NIP) which is thought to induce clustering, however, it is the extracellular region o f Npn-1 which is thought to mediate its function in semaphorin binding (figure 2 C ; C a i and Reed, 1999). Based on its structure and some key observations, it appears as though at least one additional transmembrane protein is required for Sema3A activity (Nakamura et al., 1998). Presently, an invertebrate equivalent for neuropilin has not been found.  8  Another family o f transmembrane proteins, the plexins bind specifically to semaphorins either alone, or in combination with the neuropilins (Comeau et al., 1998; Winberg et al., 1998). There are nine known plexins, which subdivide into four families based on structure; p l e x i n - A l - 4 , B l - 3 , C l , and D l (Tamagnone et al., 1999). Plexins are transmembrane proteins characterized b y the presence o f an extracellular semaphorin domain and cysteine-rich region, as well as a conserved plexin-specific cytoplasmic domain (Tamagnone et al., 1999). There are 3 known classes o f semaphorins which bind directly to plexins. In Drosophila,  sema-la and sema-lb bind p l e x i n - A l directly, while in  vertebrates Sema4B binds p l e x i n - B l , and Sema7A binds p l e x i n - C l (figure 1; Winberg et al., 1998; Lange et al., 1998; X u et al., 1998). Neuropilins and plexins interact to form a receptor complex for the secreted class 3 semaphorins. Initial results demonstrated that plexins form stable complexes with N p n 1 and Npn-2 when ectopically expressed i n C O S - 7 cells (Takahashi et al., 1999). When co-expressed in C O S - 7 cells, neuropilins and plexins induce collapse i n the presence o f Sema3A, while expression o f either one alone does not (Takahashi et al., 1999). Overexpression o f a truncated p l e x i n - A l lacking the intracellular domain inhibits Sema3A induced collapse i n cultured sensory neurons (Takahashi et al., 1999). In addition to the Npns and the plexins, another family o f receptor molecules shown to contribute to semaphorin receptor complexes includes L l , a member o f the immunoglobulin superfamily o f adhesion molecules. Analysis from L l -deficient mice demonstrated that cortical neurons i n these mice were unresponsive to Sema3A but not to Sema3B or Sema3E (Castellani et al., 2000). Further analysis demonstrated that the extracellular domains o f L l and Npn-1 directly associate to form a receptor for Sema-3A  9  (Castellani et al., 2000). Interestingly, a soluble form o f L I , with an attached F c domain, can convert Sema3A-induced axonal repulsion into attraction in vitro (Castellani et al., 2000, 2002). Studies on plexin associated proteins from Drosophila have implicated the renamed Dtrk receptor off-track ( O T K ) , as a binding partner required for Plexin A l signaling (Winberg et al., 2001). This was supported from experiments which demonstrated that otk mutants displayed similar phenotypes to loss-of-function mutations o f either Semala or PlexA (Winberg et al., 2001). Additionally, otk loss-of-function mutations interact genetically with S e m a l a and P l e x A , and reduced levels o f otk suppress Sema-la gain-of-function phenotypes (Winberg et al., 2001). Finally, recent work by Giordano et al. 2002 has demonstrated that Plexin B I (the Sema4D Receptor) and Met (the Scatter Factor 1/ Hepatocyte Growth Factor Receptor) associate in a complex when co-expressed i n M L P 2 9 epithelial cells. Binding o f Sema4D to Plexin B I stimulates the tyrosine kinase activity o f Met, resulting in tyrosine phosphorylation o f both receptors (Giordano et al., 2002). Cells that lack Met expression do not respond to Sema4D (Giordano et al., 2002). This same cells w i l l respond to Sema4D is they are then forced to express Met (Giordano et al., 2002). These results suggest that the M e t receptor may also contribute to a receptor complex for various Sema4D. Genetic Analysis of Semaphorin  Receptors:  A number o f mutants have been described for both the neuropilin and the plexin families o f receptors. There are two plexins (plx-1 ,plx-2), i n C. elegans, but no obvious neuropilin genes (Fujii et al., 2002). P l e x i n A (plx-1) null-mutants exhibit a number o f  10  morphological defects including the displacement o f r a y l , one o f nine male-specific genital sensilla (simple sense organ) in the tale, and a partial loss o f seam cells which lead to a discontinuous outer alae, a cuticular structure running longitudinally along the lateral surface o f the body w a l l (Fujii et al., 2002). Interestingly, suppression o f CeSema-la and Ce-Sema-lb displayed a similar phenotype toplx-1, whereas  Ce-Sema-2a  mutants exhibit a distinct phenotype (Fujii et al., 2002). In Drosophila, PlexA loss-offunction mutant phenotypes phenocopy those o f Semala including a failure o f ISNb growth cones to defasciculate from one another at any or all three o f the ISNb choice points (Winberg et al., 1998). A l s o , PlexA and Semala loss-of-function mutations interact genetically, while PlexA loss-of-function suppresses Semala  gain-of-function  phenotypes (Winberg et al., 1998). Interestingly, over-expression o f Plex A i n all neurons leads to axon guidance defects in all parts o f the motor projection and within the C N S (Winberg et al., 1998). Mutations i n PlexB also result i n axon guidance phenotypes (Hu et al., 2001). The ISNb neurons i n flies overexpressing PlexB fail to innervate their normal muscle targets ( H u et al., 2001). In mice lacking neuropilin-1, the phenotype is similar to that o f Sema3A but it is much more severe, and mice arrest developmentally (Kitsukawa et al., 1997). These animals display severe abnormalities in cranial and spinal neurons, along with aberrant efferent projections within the P N S including abnormal limb innervation (Kitsukawa et al., 1997). M i c e with mutations i n neuropilins-2 also display a number o f neuronal defects within the C N S and P N S , including severe defasciculation o f oculomotor neurons (Giger et al., 2000, M a r i n et al., 2001). M i c e that lack plexin-A3 have a number o f axon guidance defects including an inability o f the ophthalmic branches o f the trigeminal nerve  11  to fasciculate (Cheng et al, 2001). These mice also display a number o f guidance defects associated with the hippocampus including aberrant dentate granule cell and mossy fibre projections (Cheng et al., 2001). Semaphorin  Signaling:  Recent studies have begun to identify the downstream signaling targets involved in transmitting the semaphorin signal (reviewed in Castellani and Rougon, 2002). A number o f these studies have implemented the Rho GTPases i n semaphorin signaling. Dominant negative R a c l was shown to inhibit Sema3A-induced collapse o f D R G neurons, while a constitutively active R a c l partially mimicked the Sema3A growth cone collapse (Jin and Strittmatter, 1997). Both plexins and neuropilin signaling cascades result in the activation or inhibition o f specific Rho GTPases including Rho A , R a c l , R n d l , and R h o D (figure 2). In Droshophila,  P l e x B was shown to bind activated R a c l , sequestering it from  p21 activated kinase ( P A K ) its downstream effector (figure 2 A ; Driessens et al., 2001, H u et al., 2001). In addition to an interaction with activated R a c l , P l e x B also binds R h o A creating a scenario were signaling through P l e x B leads to collapse by simultaneously inhibiting the R a c l pathway, while leading to activation o f the R h o A pathway (figure 2 A ; Driessens et al., 2001, H u et al., 2001). This model was also supported from in vitro studies on interactions between Sema4D and plexin B I , which demonstrated that clustering o f this plexin leads to an interaction with active Rac-1, that the collapse induced by Sema4D is characteristic o f Rho activation, and that there was again an inhibition o f P A K activity (figure 2 B ; V i k i s et al., 2000; R o h m et al., 2000; Driessens et al., 2000). Again, R a c l and R h o A activities are regulated through distinct  12  Figure 2: Semaphorin Signaling A series o f schematic representations o f signal transduction pathways involved i n semaphorin activity is shown. In (A), the invertebrate Semal A interacts with Plexin A l and O T K to recruit kinases including enabled. In (B), the vertebrate class 4 transmembrane semaphorins signal through the class B plexins associated with M E T . This activation leads to a recruitment o f the Rho family o f small GTPases that signal to either inhibit, or induce collapse. The guanine exchange factor L A R G interacts with plexin B l and activates the Rho A pathway. In (C), the secreted semaphorins, signal through a clustered neuropilin/plexin complex to recruit R n d l , which activates P A K leading to collapse. Recruitment o f R h o D to the same binding region inhibits this R n d l activity. Signaling through plexin A l also leads to activation o f a number o f other pathways involving C R M P s , F y n , Cdk5, and a pool o f G S K - 3 . Receptor interactions with L l are thought to modulate semaphorin signaling through changes i n c G M P levels. In (D) bi-directional signaling o f transmembrane semaphorins may occur through a number o f possible candidates.  13  B ScmalA  Recruitment Of Other Kinases  1 / 1  Decreased motility j  Actio Turnover  Increased 1 motility  / / Decreased Motility/ Actin Turnover  Local Protein Translation  PLEXIN SIGNALLING (transmembrane semaphorins)  Tubulin Dynamics  Reorganization of Cytoskeleton  NEUROPILIN/PLEXIN SIGNALING (secreted semaphorins)  SEMAPHORIN BI-DIRECTIONAL SIGNALING  14  binding sites where Plexin B l directly interacts with activated R a c l (figure 2 B ; H u et al., 2001; V i k i s et al., 2002). Recent work b y Aurandt et al. (2002) has implicated the activation o f Rho by a Rho-specific nucleotide exchange factor L A R G , in response to stimulation by Sema4D (figure 2B). The L A R G protein was shown to interact directly with the C-terminal region o f Plexin B l through its P D Z binding domain (Aurandt et al., 2002; Perrot et al., 2002). Sema3A signals through clustering o f a neuropilin/plexin A l complex (figure 2C). There appear to be differences i n the downstream effectors o f Plexin A l compared to plexin B l (figure 2 B , C ) . W h i l e R a c l activity is important for signaling through plexin A l , it does not appear to interact with this plexin (figure 2 C ; K u h n et al., 1999; R o h m et al., 2000). While R a c l does not interact with plexin A l , both the Rho-like GTPases R n d l and R h o D were shown to bind to the intracellular region o f plexin A l , and actually compete for the same binding region (figure 2 C ; R o h m et al., 2000; Zanata et al., 2002). Interestingly, while R n d l activation by plexin A l results i n cytoskeletal collapse, binding o f R h o D has an antagonist effect, blocking R n d l activity (figure 2 C ; Zanata et al., 2002). Plexin and neuropilin receptor complexes also induce activation o f L L M kinase and subsequent inhibition o f cofilin, resulting i n a reduction in actin turnover and retraction o f the growth cone (figure 2 C ; A i z a w a et al., 2001). The Collapsin response mediator proteins ( C R M P s ) are another family o f proteins which were first identified by their possible involvement i n the Sema3A-induced collapse o f D R G growth cones (Goshima et al., 1995). Further evidence came from a study demonstrating that an a n t i - C R M P antibody blocks collapse induced by Sema3 A (Wang and Stittmatter, 1997). A recent study examining the role o f C R M P s in Sema3A  15  signaling demonstrated that they are linked to Plexin A l through activation o f Fes/Fps (Fes) tyrosine kinase which then phosphorylates plexin A l and C R M P in a F e s / C R M P / C R A M ( C R M P - a s s o c i a t e d molecule) complex (figure 2 C ; Mitsui et al., 2002). The activation o f Fes appears to be required for Sema3A-induced collapse o f C O S 7 cells expressing P l e x A l , Npn-1, and Fes (Matsui et al., 2001). Signaling through Sema3A also appears to activate a pool o f glycogen synthetase kinase 3 ( G S K 3 ) , a seronine/threonine kinase implicated i n a variety o f growth factor signaling cascades (figure 2 C ; Eickholt et al., 2002). This activation occurs at the leading edge o f the growth cone (Eickholt et al., 2002). Other downstream effectors o f semaphorin signaling include the flavoprotein monooxy-genases ( M I C A L S ) , which have been shown to interact directly with P l e x A in Drosophila  (figure 2 A ; Terman et al., 2002). The M I C A L family o f proteins have  multiple domains which are known to be important for interactions with actin, intermediate filaments, and cytoskeletal-associated adaptor proteins, and may thus mediate the cytoskeletal alterations characteristic o f semaphorin signaling (Terman et al., 2002). A n additional kinase dependent pathway i n Sema3A signaling involves the src kinase F y n and the Thr/Ser kinase cyclin-dependent kinase 5 (Cdk5: figure 2 C ; Sasaki et al., 2002; Pasterkamp and K o l o d k i n , 2003). Evidence from Xenopus neurons suggests that Sema3A signaling also requires local protein synthesis i n the growth cone, as the inhibition o f translation in the axon prevents Sema3 A induced collapse and turning (figure 2 C ; Campbell and Holt, 2001). In addition to functioning as ligands, transmembrane semaphorins also contain intracellular regions which have been shown to interact with a variety o f targets that may be involved in various signaling cascades. In Drosophila,  Sema-la is involved i n  16  synapse formation and may signal bi-directionally v i a Enabled (figure 2 D ; Godenschwege et al., 2002). Other possible interacting proteins include the synapse associated protein PSD-95, S E M C A P - 1 , and E V L (ena vasp regulator) protein (figure 2 D ; Inagaki et al., 2001; Wang et al., 1999; Klostermann et a l , 2000). While the list o f possible candidates involved in semaphorin signaling is constantly increasing, a clear picture as to how the semaphorins are able to pass on guidance information to the cytoskeleton is unclear. Even more unclear is whether the transmembrane semaphorins are able to signaling bi-directionally, and whether this is crucial for their role in axon guidance. The studies on bi-directional Sema-la signaling in Drosophila would suggest that the answer to the last two questions is yes (Godenschwege et al., 2002).  Transmembrane Semaphorins: The majority o f research on semaphorins i n vertebrates has focused on characterizing the role o f the class 3 secreted semaphorins during neuronal development. Recently, more work has begun to focus on the role o f transmembrane semaphorins i n development, however little is known about their function. Most o f the studies on vertebrate transmembrane semaphorins have looked at the role o f the class four semaphorin Sema4D (CD100), a semaphorin important i n the immune system (Hall et al., 1996; reviewed i n Suzuki et al., 2003). Recent experiments on the vertebrate semaphorin Sema6A, a transmembrane semaphorin homologous to the insect semaphorin class 1, have demonstrated its ability to repel sympathetic and D R G neurons in vitro ( X u et al., 2000). Analyses from mice lacking Sema6A transcript suggest that it is also important for proper development o f the thalamocortical projection in vivo (Leighton et al., 2001). Based on research showing that a transmembrane semaphorin, semaphorin l a has the  17  potential to be attractive for Til neurons in the developing grasshopper limb, our lab focused on determining whether transmembrane semaphorins might play a similar attractive role in vertebrates (Wong et al., 1999). Using a series of degenerate primers to the grasshopper semaphorin 1 a semaphorin domain, an embryonic chick cDNA library was screened to determine if other semaphorins with a conserved semaphorin domain might convey a similar function. One of the candidate genes that was identified corresponded to a class 5 transmembrane semaphorin, chick Semaphorin 5B (cSEMA5B). Class 5 Semaphorins: The class 5 family members are distinguished from other semaphorins by the presence of a unique protein domain containing seven thrombospondin type-1 repeats on their extracellular region, located 3' of the semaphorin domain. This domain is of particular interest as previous work has show that thrombospondin type-1 repeats can induce outgrowth of rat cortical neurons in culture, and is responsible for the attachment of various cell types, both neuronal and non-neuronal, when grown on a substrate containing thrombospondin (Neugebauer et al., 1991; O'Shea et al., 1991 Osterhout et al., 1992; Adams and Tucker, 2000). This class of semaphorins was first described in mice, and contains two known family members in vertebrates, Sema5A and Sema5B (Adams et al., 1996). These two family members are 58% identical and 72% similar to each other, with Sema 5 A spanning 1077 amino acids, and Sema5B spanning 1093 amino acids (figure 4; Adams et al., 1996). The semaphorin domains in these mice are 64% identical to one another and are most similar to invertebrate semaphorin 1 domains (-60-66% similar). In mice, Sema5 A and Sema5B are differentially expressed in embryonic and  18  adult tissues. Though expressed i n similar regions, Sema5A is expressed primarily in mesodermal cells, while Sema5B is exclusively in the neuroepithelium (Adams et al., 1996). In addition, northern blot analysis on mouse tissue shows that the highest expression levels o f Sema5B were found in brain tissue expressed as a single transcript, 5.9kb i n size (Adams et al., 1996). The expression o f semaphorin 5B has also been examined in the forebrain o f rats over a development period ranging from E15-P7 (Skaliora et al., 1998). S E M A 5 B is broadly expressed i n all neuroepithelia throughout the brain at each o f the ages examined. In particular, epithelial regions, such as the ventricular zone, which normally corresponds to high areas o f neuronal proliferation, show strong expression (Skaliora et al., 1998). S E M A 5 B is distributed uniformly and is strongly expressed in cortical ventricular and subventricular zones at postnatal day 0 (Skaliora et al., 1998). This wide spread epithelial expression suggests that S E M A 5 B might play a general role i n these proliferative tissues, possibly with cell migration (Skaliora et al., 1998). Interestingly i n the rat, S E M A 5 B is expressed i n discrete patches within the striatum and may be involved i n the correct patterning o f cells within this region o f the brain. A t P7, S E M A 5 B is highly expressed in the rat visual cortex (Skaliora et al., 1998). In Drosophila, only one member o f this class o f semaphorins exists (Khare et al., 2000; Bahri et al., 2001). It is most similar to Sema5B but is not a homologue o f either vertebrate class 5 members, and was thus given the designation o f Semaphorin 5 C , (DSema 5 C ; Khare et al., 2000; Bahri et al., 2001). In flies, D S e m a 5 C is found at segment boundaries in regions giving rise to muscle attachments (Bahri et al., 2001). DSema 5 C exists as two different protein isoforms that share a similar expression pattern.  19  The D S e m a 5 C mutant flies are homozygous viable and display no obvious embryonic phenotypes (Bahri et al., 2001). A s no detailed analysis was performed on the nervous system, a clear role for this semaphorin in Drosophila has not been established. A possible role for class V semaphorins in axon guidance was suggested from studies examining the well known neurological disorder i n humans, Cris-du-chat syndrome (Simmons et al., 1998). These studies reveal that the S E M A 5 A gene accounts for 10% o f the deleted region associated with this disorder. In addition, recent studies on Sema5 A in mouse have demonstrated its ability to repulse retinal axons in vitro (Oster et al., 2003). In these studies, Oster et al. 2003 examined the expression o f this semaphorin i n the developing retina and found it to be specifically expressed in the optic disk and along the optic nerve. They were able to demonstrate that the inhibitory response o f R G C to Sema5A was maintained i n the presence o f L l , laminin, or netrin-1 signaling in vitro (Oster et al., 2003).  Hypothesis: While the studies in both the mouse and rat have described the expression o f Semaphorin 5 B , they both have a number o f limitations i n that they have not examined the distribution o f this semaphorin protein, they have only looked at particular regions or over a small developmental range, and they have not provided much insight as to the role o f the class 5 semaphorins. In order to determine a functional role for this semaphorin, its expression was examined i n the developing chick from E 2 through until E l 7 . The examination o f c S E M A 5 B expression was completed using a series o f in situ hybridization studies, and with antibodies raised against the N-terminal region o f the protein. I hypothesize that c S E M A 5 B is expressed during in regions associated with  20  neuronal outgrowth and guidance, including the spinal cord, retina, and tectum. Based on its expression along with the results from functional in vitro studies performed by others in our lab, I propose that c S E M A 5 B is an inhibitory guidance cue that contributes to proper axon outgrowth and guidance within these structures. In addition, I hypothesize that c S E M A 5 B is expressed along many neuroepithelia and that it plays an inhibitory role in these highly proliferative regions, to facilitate there migration from these epithlial layers towards target tissue i n the developing C N S .  21  Methods RNA Work: Probe sequence: A c D N A sequence corresponding to the C-terminal cytoplasmic region of cSEMA5B was produced from the full-length cSEMA5B (obtained from an E10 chick cDNA library, Clontech) sequence cloned into the pLitmus29 vector. The following primers were generated to isolate this sequence from pLitmus29: Primerl (forward): A G C T G T C T G A T C C C C A T G A G 5  3  Primer 2 (reverse): ' G A A A A G A A C A T G C A C A A A C C C ' 5  3  The corresponding PCR product was subcloned into the vector bluescript29-KS, containing a T3 and T7 promoter sequence for the production of R N A . Synthesis of DIG-labeled R N A probe: To synthesize DIG-labeled antisense and sense R N A probes, the following reagents (Roche) were mixed in the following order at room temperature to produce a 20ul reaction mixture: 9.5jul of sterile DEPC treated dH 0, 4 pi of 5x transcription buffer, 2  2/tl 0.1 M DTT, 2/d of nucleotide mix (pH 8.0), 1/d of Linearized bluescript-cSEMA5B plasmid (1 pg/ul), 0.5 pi Placental ribonuclease inhibitor (40 U/ul), ljul of T7 or T3 R N A polymerase (10-20 U/ul). The resulting mixture was incubated at 37° C, for 2 hours. After the reaction was completed, a 1 pi aliquot was removed and separated on a 1.5% agarose/0.5XTBE gel containing 0.5 pg/ml EtBr in order to estimate the amount of probe synthesized. To remove any remaining plasmid 2 pi of ribonuclease-free DNase 1 (10 U/ul) was added to the reaction mixture and it was incubated at 37° C for 15 min. A n R N A isolation column (GIBCO) was used to isolate the resulting R N A probe. As an  22  alternative method, R N A was isolated with the addition o f a volume o f 100 u.1 D E P C treated d H 0 and 10 ul o f 4 M L i C l (DEPC-treated) to the reaction. The R N A was 2  precipitated with the addition o f 300 ul o f ethanol followed by an incubation on ice for 30 minutes. The resulting mixture was spun in a microfuge for 20min. The pellet was washed with 70% ethanol, and spun down for another 2 minutes. A l l o f the E t O H was removed with an aspirator and pellets were allowed to air-dry briefly for 2-5 minutes. The R N A pellet was re-dissolved in D E P C treated d H 0 at -0.1 ug/ul (-100 pi) and 2  stored at -80° C .  In situ hybridization Studies Tissue Preparation: Embryonic chicks isolated at various stages, were submerged in a solution containing 4% paraformaldehyde in DEPC-treated P B S , and were washed at 4°C for a period o f 8 firs. Chicks at E 7 or older were first fixed v i a pericardial infusion, b y inserting a needle into the heart and injecting P B S for 2 minutes to clear the blood followed b y 10 minutes o f Paraformaldehyde to ensure rapid fixation. These embryos were then sectioned into numerous parts before submersion i n 4% paraformaldehyde solution at 4°C for 8 hrs. Following the fixation, the resulting embryos were submerged in a solution containing 30% sucrose in DEPC-treated P B S and were left on a rotator at 4°C overnight for cryo-protection. The embryos were submerged in O . C . T (optimal cutting temperature) compound (Tissue-Tek), and placed i n a metal tray preteated overnight with 0 . 2 N N a O H . Trays containing embryos submerged i n O . C . T compound were frozen i n a small vial o f 2-methylbutane contained in a bath o f liquid nitrogen. The  23  tissue was sectioned into lOum thick sections using a microtome (Bright Instruments) at - 2 0 ° C . The sections were placed on slides and dried i n an oven at 40°C overnight. Prehybridization: Slides were washed 2 X for 5minutes i n DEPC-treated P B S followed by two washes with DEPC-treated P B S containing l O O m M glycine. Sections were treated with DEPC-treated P B S containing 0.3% Triton® X - 1 0 0 and followed by two washes with DEPC-treated P B S . The sections were permeabilized for 20 minutes at 37°C with T E buffer (lOOmM T r i s - H C L , 5 0 m M E D T A , p H 8.0) containing l u g / m l Proteinase K (RNase free). Following permeabilization, the sections were fixed with DEPC-treated P B S containing 4% paraformaldehyde at 4°C for 5 minutes and washed twice more with DEPC-treated P B S . Slides were then placed i n RNase-free slide containers that contained 0 . 1 M triethanolamine ( T E A ) buffer, p H 8.0, containing 0.25% (v/v) acetic anhydride (Sigma), and the slides were allowed to shake for 10 minutes. Slides were washed again 2 X 5min with P B S then the sections were incubated at 42°C for 1 hour i n prehybridization buffer D I G Easy H y b solution (Roche Molecular Biochemicals) containing l m g / m l denatured and sheared salmon sperm and l m g / m l yeast t - R N A . In situ hybridization: A wax pen ( P A P ) was used to encircle the sections to prevent leakage o f hybridization buffer during the hybridization step. The slides were incubated i n the presence o f 200ul D I G Easy hybridization buffer (Roche) containing 50ng o f D I G labeled anti-sense or sense R N A probe. The sections were covered with a layer o f parafilm to prevent evaporation and were placed i n a humid chamber at 42°C for 24hrs or overnight.  24  Post Hybridization Procedure: Following the hybridization step, the sections were put through a series o f washes in a shaking water bath. The slides were washed twice for fifteen minutes i n 2 X S S C buffer at 37°C, followed by two washes with I X S S C buffer for 30 minutes at 37°C, and twice more for 45 minutes i n a 0.1 X S S C buffer at 37°C. Sections were washed on a shaking platform 2 X for 10 minutes in buffer 1 (lOOmM T r i s - H C l ( p H 7.5), 150mM N a C l ) at room temperature. Sections were covered for 30 minutes with blocking solution (buffer 1 containing 0.1% Triton X - 1 0 0 and 2 % normal sheep serum (Sigma)) at room temperature. The blocking solution was decanted and sections were incubated for 2 hrs in a humid chamber with buffer 1 containing 0.1% Triton X-100, 1% sheep serum, and a 1:500 (weight) dilution o f sheep anti-DIG-alkaline phosphatase (Fab fragments; Roche) Sections were washed 2 X 10 minutes in buffer 1 on a shaking platform at room temperature. Sections were incubated for 10 minutes with buffer 2 (lOOmM T r i s - H C l (pH 9.5), lOOmM N a C l , 50 m M M g C l ) , and were then covered with the color solution 2  ( l O m L buffer 2 containing 200pl o f nitroblue tetrazolium ( N B T ) / 5-bromo-4-chloro-3indolylphosphate (BCfP) stock solution (Boehringer Mannheim)) i n a humid chamber for -12-24 hrs until desired color was achieved. The color reaction was stopped with the addition o f buffer 3 ( l O m M t r i s - H C l ( p H 8.1), I m M E D T A ) followed by a brief wash with distilled H2O. Sections were mounted with CytoSeal 280 (Richard-Allan) and overlaid b y a coverslip. N i s s l Stained sections: For sections prepared with a nissl-stain, 1 g cresyl violet acetate dissolved i n 990 m l o f d d H 0 and the mixture was heated to 35°C and stirred for several hours at 35°C. 2  25  The solution was filtered, allowed to cool at room temperature, and the p H was adjusted to 3.5 with glacial acetic acid. Sections prepared as outlined in tissue preparation above, were then submerged i n this cresyl violet solution and stained for 5-15 minutes. The sections were washed with 10 dips i n water, followed by 10 dips in 70% ethanol. Sections were dehydrated through ethanol by immersing the slides i n 2 changes o f 95% ethanol for 2 m i n each.  Northern Analysis Total R N A from spinal cord, tectum, and gut tissues o f various chicks ranging in age from E3 to E l 1 was T R I z o l extracted ( G i b c o B R L ) . A total o f 30u.g o f R N A , as measured by the absorbance at 280 nm, was electrophoresed i n a 1% denaturing formaldehyde agarose gel for 3 hours at 70Volts. Electrophoresed R N A was then transferred to H y b o n d - N nylon membrane (Amersham) overnight i n 10X S S C transfer buffer. The membrane was baked at 80°C for 2 hrs and incubated i n prehybridization buffer (50%) w/v deionized formamide, 5 X Denhardt's solution, 1%SDS, denatured salmon testes D N A ) at 42°C for 2-4 hrs. Membranes were then hybridized i n the presence o f anti-sense or sense R N A probes overnight at 42°C followed by a series o f washes in S S C buffers o f varying concentrations. The membranes were developed using a standard protocol from the D I G Northern Starter K i t (Roche).  R T - P C R studies S U P E R S C R I P T ™ II RNase FT Reverse Transcriptase was used to synthesize first strand c D N A for all R T - P C R studies (Invitrogen). Total R N A from spinal cord, tectum, and gut tissues o f various chicks ranging i n age from E3 to E l 7 was T R I z o l extracted ( G i b c o B R L ) . A total o f 1 ug o f R N A was incubated in the presence o f 50ng o f random  26  primers and 1 p i o f l O m M d N T P mix, to a total volume o f 12ul with ddEbO. The mixture was heated to 65°C for 5min followed by a quick chill on ice. A total o f 4pl o f 5 X FirstStrand Buffer, 2 p l 0.1 M D T T , and l u l o f RNase inhibitor were added to the mixture. Following incubation at 25°C for 10 minutes, the mixture was incubated at 42°C for 2 minutes and 1 p i o f S U P E R S C R I P T II enzyme was added. The mixture was then incubated for 50 minutes at 42°C and the reaction was stopped b y heating to 70°C for 15 min. A total o f 2pl o f first-strand c D N A mixture was used i n the P C R reaction, and the primers used were the same as used to make the R N A probe. Following the P C R reaction, the product was subjected to separation on a 1% agarose gel i n the presence o f E t B r to confirm the size (figure 5B). Affinity Purification of cSEMA5B A n t i b o d y Purification o f GST-fusion protein: The p G E X plasmid containing c S E M A 5 B peptide corresponding to the region o f antibody recognition and cloned downstream o f G S T , was supplied by D r . Wenyan Wang. E coli D H 5 a cells transformed with p G E X vector containing either the c S E M A 5 B - glutathione-S-transferase fusion protein ( G S T ) or G S T protein alone were used to inoculate 25ml o f L B supplemented with 50fig/fil o f ampicillin (amp). These 25ml cultures were grown overnight in the orbitial shaker at 37°C. The resulting cultures were used to inoculate 500ml o f L B supplemented with 50/^g//il o f amp. This culture was grown for 1.5-2 hrs at 37°C until reaching an optical density(600) o f 0.6-1.0. Protein expression was induced by the addition o f I P T G ( O . l m M final) followed by incubation at 37°C for 5 hours. The resulting bacteria were collected b y centrifugation at 8000 rpm at 4°C for 10 minutes and the resulting pellet was re-suspended i n 9ml o f M T - P B S (150mM  27  N a C l , 16 m M N a H P 0 , 4 m M N a H P 0 4 (pH 7.3)). The suspension was sonicated six 2  2  4  times at 30seconds each, with 30 seconds on ice between each sonication. Following sonication, 1ml o f 10% triton-X-100 was added and the suspension was rocked at 4°C for 5 minutes. The resulting lysates were spun down at 8000 rpm for 10 minutes at 4°C and pellets were discarded. The supernatant was added to a 2 m l slurry containing 160 m g (80mg/ml) o f Glutathione beads (Sigma G4251) pre-incubated for 30 minutes in M T P B S and washed 4 X with 15ml o f M T - P B S . The supernatant and beads were incubated at room temperature for 10 minutes. The mixture was centrifuged at 1000 rpm for 1 minute and the supernatant was removed. The beads were washed 5 X in 15 m l M T - P B S with 1% triton-X-100 followed b y 2 washes with M T - P B S (no Triton). The c S E M A 5 B - G S T fusion proteins (or G S T alone) were eluted by three washes with 2 m l o f elution buffer ( 5 0 m M Tris HC1 p H 8.0, l O m M Glutathione). Each elution wash was kept separate. 15ul o f the resulting elutions were run on a 12% polyacrylamide gel to determine i f elution was successful (figure 4). The OD go for each elution fraction were measured to 2  determine the peptide concentration ( 1 0 D - 0.5mg/ml). Affinity column preparation: The column was prepared as outlined by the AminoLink® K i t (Pierce Biotechnology). A 500 u.1 solution o f c S E M A 5 B peptide at 4mg/ml was diluted 1:3 in coupling buffer and loaded onto an equilibrated AminoLink® column. The resulting slurry was mixed at 4°C for 6hrs in the presence o f AminoLink® Reductant solution to bind the c S E M A 5 B peptide to the column. The remaining active sites in the column were bound using a Tris-based quenching buffer in the presence o f AminoLink® Reductant solution. Following a series o f washes with 15mls o f a I M N a C l buffer ( p H  28  7.5), the column was stored at 4°C in P B S buffer ( p H 7.3) containing 0.05% sodium azide. Antibody purification procedure: Following equilibration o f the column with 10ml o f P B S (pH 7.3) containing 0.05% sodium azide, 2.0ml o f sera was loaded onto the column and allowed to enter the gel bed. Sample was allowed to circulate though the column overnight at 4°C to allow for maximum binding o f antibodies. The column was then washed with 30ml o f A m m o L m k wash solution. Antibody was eluted with Immunopure® I g G elution buffer and the elution fractions were monitored by absorbance at 280 nm. Fractions containing A b were separated on a 12% polyacrylamide S D S gel and stained with coomassie blue to confirm the correct sizes for heavy and light chain regions (figure 12). A n t i b o d y Staining Embryo's were fixed in 3.7% paraformaldehyde v i a pericardial infusion (ie P B S in the heart for ~2-3minutes, followed b y 20min o f 3.7% para at room temperature). The fixed embryos were then imbedded i n O C T compound and sectioned into 1 Oum thick sections using a microtome at - 2 0 ° C (Bright Instruments). Sections were mounted onto pre-cleaned Superfrost® /Plus microsope slides (Fischer), and washed 3 X 5 minutes i n D  P B S ( A ) containing 0.05% Triton X-100. Sections were then washed 2 X 1 0 minutes i n P B S ( B ) containing 0.1% Triton X and 0.005% bovine serum albumin ( B S A ) . To block sections, slides were incubated for 1 hour P B S ( B ) containing 10% goat serum and 1 % B S A . Sections were then washed 2 X 1 0 minutes i n P B S ( A ) followed b y 2 X 10 minutes i n P B S ( B ) . Sections were incubated overnight at 4°C with primary antibody diluted appropriately i n P B S ( B ) usually 1:1000 ( l O p l i n 10ml). Sections were again  29  washed 2 X 10 minutes in P B S ( A ) followed by 2 X 10 minutes i n P B S ( B ) . Section were then incubated for 1 hour at room temperature with a Cy3 conjugated goat anti rabbit fluorescent secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, P A ) diluted at 1:500 in P B S ( B ) . Sections were washed 2 X 1 5 minutes i n P B S ( A ) . Sections were then covered in antifade (Molecular Probes) containing glycerol and P B S , covered with a coverslip and sealed using nail polish.  Antibody staining of dissociated cultures Spinal Cords were isolated from E8 chick and subjected to trituration through a small bore pipette i n the presence o f D M E M - F 1 2 . The resulting suspensions were plated on poly-L-lysine and laminin (lng/ml) coated coverslips in the presence o f N G F (20ng/ml), and were allowed to grow for 12 hrs at 37°C i n the presence o f CO2. After 12hrs cultures were fixed in 3.7% paraformaldehyde and stained as per protocol. Primary mouse anti-NeuN antibody was used at a dilution o f 1:100, while a n t i - c S E M A 5 B rabbit antibody was used at a dilution o f 1:1000. Pre-absorption o f antibody with fusion protein: A total o f 100u.g o f purified fusion protein was incubated with l O u l o f affinity purified antibody, and lOOul o f N e u N antibody i n a final volume o f 10ml o f P B S . The next day, cells were stained as per protocol using the pre-absorbed affinity purified antibody and N e u N Antibody.  Westerns Blots Approximately 100-200mg o f various tissues including spinal cord, tectum, retina, and gut were isolated from chick at various stages in microcentrifuge tubes. These tissues were homogenized using a homogenizing pestle in the presence o f lOOuL o f  30  RIP A buffer (150mM N a C l , 5 0 m M Tris p H 7.4, 5 m M E D T A , 5.0% N P - 4 0 , 1.0% sodium deoxycholate ( D O C ) and 0.1 % sodium dodecyl sulphate (SDS), aprotinin, leupeptin, phenylmethyl-sulfonyl floride (PMSF)). The total volume was brought up to 500(4,1 (200mg/ml) with the addition of RIP A buffer. The Cells were incubated i n RTPA buffer on ice for 20 minutes. Supernatants were collected and assayed for protein concentrations according to the manufacturer's instructions (BioRad). A total of 40ptg of protein was loaded and separated on a 12% SDS-poly acrylamide gel electrophoresis ( P A G E ) gels and then electro-transferred to Hybond E C L Nitrocellulose membranes (Amersham Pharmacia, Piscataway, N J ) . Membranes were blocked with 3% milk protein i n Tris buffered saline (TBS)-Tween-20 for 1 hour at room temperature. Membranes were probed with of rabbit a n t i - c S E M A 5 B antibody (1:1000) in TBS-Tween-20 overnight at 4°C. Blots were incubated with o f anti-rabbit horse radish peroxidase (1:2000; Jackson ImmunoResearch Laboratories, West Grove, P A ) in 1% milk in TBS-tween-20 for 1 hour at room temperature. Antibody was visualized by enhanced chemiluminescence ( E C L ) according to manufacturer's instructions (Amersham Pharmacia, Piscataway, N J ) . Cytoskeletal Extractions: For cytoskeletal fractions, the tissue isolation was the same as above, however, homogenized tissue was incubated on ice with constant rotation for 20 min i n 500/xl o f cytoskeletal buffer ( 5 0 m M N a C l , l O m M Pipes p H 6.8, 3 m M M g C l , 300 m M Sucrose, 2  0.5% triton-X 100, 1.2mM P M S F , 10/ig/ml aprotinin, leupeptin; H i n c k et al., 1994) to extract Triton insoluble proteins. The resulting lysates were spun down at 14,000 rpm for 10 minutes at 4°C and the insoluble pellet and supernatant were separated. A total o f 100/xl o f SDS IP buffer ( 1 5 m M Tris p H 7.5, 5 m M E D T A , 2 . 5 m M E G T A , 1% SDS) was  31  added to the pellets and pellets were boiled for 10-15 minutes at 100°C. The resulting suspension was brought up to 400/i.l with the addition o f cytoskeletal buffer. Protein assays were performed for both the soluble and insoluble fractions according to the manufacturer's instructions (Biorad) to determine the amount o f protein. A total o f 25pg o f protein from both fractions was loaded and separated on a 12% P A G E gel as described above.  32  Results The structure of SEMA5B: A n analysis o f c S E M A 5 B sequence shows that it is 1092 amino acids in length (figure 3). It is 77% identical and 87% similar to mouse Sema5B based on alignment using N T I Vector Suite 6.0 software. The structural domains o f the protein include the semaphorin domain spanning approximately 500 amino acids, followed by seven thrombospondin repeats, a transmembrane region spanning 20 amino acids, and a short cytoplasmic region o f 90 amino acids (figure 3A). Sequence analysis suggests that there may be a putative proprotein convertase cleavage sequence, I C K R / K R , within the fifth thrombospondin repeat (figure 3B). A potential cleavage product is supported from westerns using an a n t i - c S E M A 5 B antibody which recognizes two distinct products (figure 1 I D , E ) . Precedence for the cleavage o f semaphorins has been established for both the class 3 and 4 semaphorins (Adams et al., 1997; Elhabazi et al., 2001). Adams et al. 1997 demonstrated that furin-dependent proteolytic processing o f Sema3a is required for an enhanced repulsive functionality. Similarly, Sema4D is cleaved by a metalloprotease-dependent process and exists as both a transmembrane and diffusible molecule i n the immune system (Elhabazi et al., 2001).  Construction of an R N A Probe: In mice, the cytoplasmic tails o f the class 5 semaphorins consist o f 80 (Sema5 A ) or 91 (Sema5B) amino acids. This region shows a lesser degree o f similarity between 5 A and 5 B , than do other regions o f the protein (Figure 4 A ) . Indeed, an alignment o f Sema5A and Sema5B nucleotide sequences demonstrated that the cytoplasmic region shows the greatest degree o f divergence and thus provided an excellent sequence for the  33  Figure 3: Semaphorin 5B Structure and amino acid sequence A schematic o f c S E M A 5 B displaying the conserved regions between mice and chick. These include the signal sequence (blue), semaphorin domain (orange), thrombospondin repeats (green), and transmembrane domain (red) ( A ) . A l s o present on the schematic is the representation o f an affinity purified A b used for the immuno studies, and a nonradioactive R N A probe used in the in situ and Northern studies. A western blot displays the specificity o f the affinity purified A b , as both a monoclonal H A antibody and the c S E M A 5 B antibody specifically recognize a recombinant c S E M A 5 B protein, containing an H A tag, which is over-expressed i n H E K 293 cells.  In addition, the amino acid  sequence o f c S E M A 5 B is displayed along with the conserved regions as indicated (B).  34  SEMA5B -1092 AA IN LENGTH -N-TERMTNAL SIGNAL SEQUENCE -500 AA SEMA DOMAIN -SEVEN THROMBOSPONDIN REPEATS -TRANS MEMBRANE REGION  0^  ^  -100 AA CYTOPLAMSIC REGION r — i -EXPRESSED IN THE BRAIN AND SPINAL CORD anti-HA  anti-S5b  RNAPROBE  The Amino Acid Sequence for cSEMASB  B  (l)MWSRLKAISLSLPSLFLL(AJHLSASONVTEYSEAJEHOOCV^KEHPTIAF EDLKPWVSNFTYPGVHDFSOLALDANRNQLIVGARNYLFRLSLHNVSL IOATAWGSDEDTRRSCQSKGKTEEECONYVRVLIVlYGKKVFTCGTNAFS PVCSSROVGNLSKHDRINGVARCPYDPRHNSTAV1TSRGELYAATVIDFSGRDPA1YR  SLGNVPPLRTAQYNSKWLNEPNFIAAYDIGLFTYFFFRENAVEHDCGKTVYSRVARV CKNDIGGRFLLEDTWTTFMKARLNCSRAGEIPFYYNELOSTFYLPEQDLIYGVFTTN VNSIAASAVCAFNLSAITOAFNGPFRYOENPRSAWLPTANPIPNFQCGTLSDDSPNEN LTERVLODAORLFLMNDVVOPVTVDPYVTODSIRFSKLVVDIVQGKDTLYHVMYIG TEYGTtLKALSTTNRSLRSCYLEEMOILPDGOREAIKSLOILHSDRSLFVGLNNGVLK IPLERCSMYRTEGECLGARDPYCGWDNKOKRCTTIEDSSNMSLWTONITECPVKNL TTNGRLGPWSP WQPCEHSDGDSTGSCMCRSRSCDSPRPRCGGRSCEGARIEVANC SRNG4 WTPWSSWALCSTSCGIGFQVRQRSCSNPAPRYGGRVCVGQSREERFCNENSPCPLPIFWSSWG PWNKCSVNCGGGIHSRQRTCENGNTCPGCAVEYKTCNPESCPEWRRNTPWTPWMPVNITQN  GARQEQRFRYTCRAQLSDPHELQFGRKKTESRFCPNDGSAMCETDSLVDDLLKTGKJSASll iSGGWSFWGAWSSCSRDCELGFRIRKRTCTNPEPKNGGLPCVGSAMEYQDCNPHPCPVKG SWSCWTPWSQCSATCGGGHYQRTRTCTNPAPSSGEDICIGLHTEEALCNTHPCEGGWSEWSE  WSLCNEEGIQYRSRYCEVHSPDSSQCVGNSJQYQDCLYNEIPYILPASS1DESTNCGGFSUH  UATGVSCFFGSSIXTFfVIYVYCORCOROSOESTVIHPTTPNHLHYKGNTTPKNE KYTPMEFKTLNKNNLIPDDRTNFYPLQQTNVYTTTYYPSTLNKYDYRPEASP GRTFTNS)(1092) Signal Sequence Antibody peptiderecognition sequence — Sema domain sequence  AAA  AAA  Tsp type-l-like repeat sequences —AAA Transmembrane sequence AAA R N A probe sequence (AAA) Putative cleavage sequence AAA  35  F i g u r e 4: A n a l y s i s and alignment of c S E M A 5 B R N A Probe A n alignment o f the c S E M A 5 B R N A probe sequence along with c S E M A 5 B , Sema5B, and Sema5A using Vector N T I suite 6.0 (A). Letters i n yellow represent identical amino acids for all three sequences, while letters in light blue represents identical amino acids for c S E M A 5 B and Sema5B. A high divergence between Sema5A and Sema5B in mice over the probe region is indicated by a lack o f blue or yellow lettering (A). The probe sequence was amplified from full-length c S E M A 5 B v i a P C R , and is 350bp i n size when run on a 1% agarose gel at 9 0 V (B). A double digest using NotI and Smal releases the probe sequence from the bluescript29 K S vector i n the first lane, while a PvuII digest confirms the correct orientation o f the probe i n the second lane (C). Both the antisense and sense R N A probes are ~350bp i n size when run on a 1% agarose gel (D). The amount o f probe is determined to be lOOng/u.1 when compared to a series o f standards o f known DIG-labeled R N A s concentrations, using the same color development process employed i n the i n situ hybridization procedure (E). The R N A isolated from tissue runs as two bands as most o f the R N A present is r R N A which runs at ~4.8kb and ~ 2kb i n size, corresponding to the 28S and 18S bands (F). The antisense probe recognizes dot blots o f total R N A on a nylon membrane following the northern blot process (G). The sense probes also cross react with the total R N A , but to a much lesser degree (G).  36  ChickSEMA5B probe  VBYVYCQRCQRQSQESTVIHPTT  Chick SEMA5B PASSIDESTN Mouse Sema5B PASSVEETTS  CGGFSLIHLIATGVSCFFGSSLLTFV1YVYCQRCQRQSQ.ESTVIHPTT CGGFNLIHLIVTGVSCFHJSGLLTLAgYgSCQHCQRQSQEST^HPAT  Mouse SemaSA V § V f R ^ S V E E K R C G E F N M F H | F H H A ^ H M B W Q QQ2 B i Consensus PASSIDEST CGGF LIHLIATGVSCFILS LLTLVIYVYCQRCQRQSQESTVIHPTT v  ChickSEMA5Bprobe  Chick SEMA5B Mouse Sema5B Mouse SemaSA  p  s s  G C L L T L  Y T Y C  R Y  S H  T V I H P V  „ —HYKGNTTPKNEKYTPMEFKTLNKNNLIPDDRTNFYPLQQTNVYTTTYYPSTLNKYDYR HL  p u H L — HYKGNTTPKNEKYTPMEFKTLNKNNL IPDDRTNFYPLQQ7NVYTTTYYPSTLNKYDYR PNHL—HYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQTNVYTTTYYPSPLNKPSjR PAALNSSITNHINKLDKYDjVEAIKAFNKNNLILB^NK((NPHLTGKTY|NAYF(DLNNYDEY  Consensus PNHL  HYKGNTTPKNEKYTPMEFKTLNKNNL IPDDRTNFYPLQQTNVYTTTYYPSTLNKYDYR  Chick SEMA5B probe p E A S P G R T F T N S ChickSEMA56 P E A S P G R T F T N S Mouse Sema5B P E A S P G Q R C F P N S Mouse Sema5A Consensus P E A S P G R T F T N S  (97AA) (1092AA) (1093AA) (1077AA)  RNA Standard  Antisense  Sense -350 b p ,  Antisense  G  37  production o f a specific R N A probe to distinguish between the two family members (Adams et al., 1996). U s i n g this information, a chick probe corresponding to the final 300 bp, or the intracellular region o f c S E M A 5 B , was used for the in situ hybridization and Northern experiments (figure 4 A ) . Further sequence analysis demonstrated that this region is not only divergent between the class 5 semaphorins, but is distinct from other known semaphorin sequences. Following amplification o f the desired probe sequence using P C R , the resulting product was sub-cloned into a Bluescript29-KS expression vector to produce both sense and anti-sense probes (figure 4 B - D ) . Initial analysis o f the probes was done by spotting total R N A , isolated from 293 cells expressing recombinant c S E M A 5 B , onto a nylon membrane, followed by a Northern blot procedure (figure 4F,G). The presence o f strongly hybridized spots on the blot probed with the anti-sense R N A , demonstrated that these probes provided a suitable tool for use i n the Northern studies (Figure 4F, G ) . In addition, a quantification protocol using dot blots, demonstrated that sufficient quantities o f the DIG-labeled R N A probes were available for in situ hybridization studies (Figure 4E). N o r t h e r n Studies: In order to determine whether c S E M A 5 B exists as a single transcript in chick, a Northern analysis was performed on spinal cord tissue obtained from an E 9 chick. A single band at ~5kb suggests that c S E M A 5 B does exist as a single transcript (Figure 5A). Northern analysis on R N A isolated from 293 cells expressing a full-length recombinant form o f c S E M A 5 B also displayed a single band o f similar size, confirming the findings from chick tissue (Figure 5 A ) . This single transcript product is consistent with Northern  38  F i g u r e 5: N o r t h e r n a n d R T - P C R Results Chick S E M A 5 B is expressed as a single transcript o f ~5kb as confirmed from total R N A isolated from cells expressing recombinant c S E M A 5 B and from E 9 S C tissue (A). R T - P C R using primers specific to c S E M A 5 B confirms the expression o f this semaphorin in spinal cord tissue isolated from chick ranging i n age from E3 until E l 7 (B). H E K - 2 9 3 cells expressing recombinant c S E M A 5 B were used as a positive control.  39  NORTHERN RESULT E9 SC 293 CellsCSEMA5B 4.8kb  3kb  B  RT-PCR RESULT sa  a. Ok re  SC TISSUE  E3  E10  293-  E17cSEMA5B  350b^  40  analyses o f other class 5 semaphorins in Drosophila and mice (Adams et al., 1996; Khare et al., 2000). In mice, Northern blot analysis o f m R N A isolated from embryos revealed two Sema5A transcripts o f 5.5 and 9.4 kb, while Sema5B is expressed as a single transcript o f 5.9kb (Adams et al., 1996). In Drosophila, the class 5 semaphorin DSema5C is expressed as a single transcript ~5kb i n size (Khare et al., 2000). A s only a probe to the cytoplasmic region was used i n these studies on c S E M A 5 B , it does not rule out the possibility for the existence o f splice variants that lack this intracellular region. R T - P C R studies: A s the Northern studies were technically difficult, and the sensitivity was low, R T - P C R was employed. Using primers specific to c S E M A 5 B , R T - P C R was performed on a variety o f different chick tissues from a wide range o f developmental stages. R T P C R analysis demonstrated expression o f c S E M A 5 B i n the neural tube or spinal cord as early as E 3 and as late as E l 7 (figure 5B). Expression o f c S E M A 5 B at E 3 was not confirmed using Northern blot analysis neither was any message observed i n the in situ hybridization studies from chicks at this age. This is likely due to the greater sensitivity o f the R T - P C R technique, and that less material is required. The spinal cord expression at E l 7 was also the latest stage examined for all o f the studies presented, and corresponds with a stage when the majority o f spinal cord development is complete (Eide and Glover, 1997). Other tissues examined included the tectum, retina, and regions o f the gut (Data not shown). A l l o f these areas were shown to express c S E M A 5 B , and confirmed the findings from both the Northern and in situ hybridization studies.  41  In situ Hybridization Studies: In order to gather more information as to the distribution o f c S E M A 5 B message, in situ hybridization was utilized. Based on the results presented above, in situ hybridization studies were performed on chicks ranging in age from E 2 until E l l . From these in situ hybridization studies, c S E M A 5 B expression was first clearly observed in a variety o f tissues at E 5 . This expression was broad and included the gut, epidermis, spinal cord, brain, olfactory and ventricular epithelium, and the retina. W h i l e many tissues express c S E M A 5 B , this thesis focuses on its expression i n the developing nervous system.  c S E M A 5 B expression in the Spinal Cord: Strong c S E M A 5 B expression is observed in the spinal cord at stage 26 (E5; figure 6A). A t this age, the expression i n the spinal cord is broad, being present in both dorsal and ventral regions including the mantle layer (figure 6A). Though this expression is broad, it is restricted to the grey matter with no observable message i n the surrounding white matter (figure 6A). While the dorsal and ventral horns are not distinguished yet, a number o f sensory axons have already reached the dorsal funiculus, an area associated with expression o f c S E M A 5 B (figure 6 A ; Airman and Bayer 1984). There is also expression along the entire dorsal-ventral axis o f the medial ependymal ( E P ; ventricular epithelium) layer, an area associated with a large population o f mitotic cells (figure 6 A ; Bellairs and Osmond, 1998). B y stage 28 (E6) however, the expression o f c S E M A 5 B becomes more restricted in appearance, with the highest levels observed i n large cells o f the ventral horn associated with presumptive lateral and medial motor columns (figure 6B, D ) . Again, there is expression in the ependymal layer (figure 6B).  42  Figure 6: In situ hybridization studies in the spinal cord In situ hybridization studies using a probe for c S E M A 5 B clearly confirm its expression in the spinal cord including the mantle layer, D R G s , and the dorsal surface epithelium (SE) at stage 26 (E5; A ) . A t E 6 , the expression o f c S E M A 5 B increases ventrally i n presumptive lateral motor columns ( P L M C ) and is present along the ventricular ependymal (EP; epithelial) layer as indicated by the arrow heads (>; B ) . A t E 9 c S E M A 5 B message is highest i n the ventral horn ( V H ) , with lower levels i n dorsal regions including the dorsal horn ( D H ; C ) . In contrast, the white matter ( W M ) is negative (C). A t higher magnification, the ventral expression in the S C at E 6 is clearly cellular in the presumptive lateral motor column (D). The expression i n D R G s at E 6 is uniform (E, F ) . Scale bar in ( A , B , C ) is 100pm; scale bar in (D,E,F) is 20pm.  43  44  The intensity o f c S E M A 5 B expression in the ventral spinal cord continues to increase through E 9 (figure 6C). The majority o f c S E M A 5 B expression is associated with cells located in the lateral motor column region (figure 6C). While much o f the spinal cord patterning has taken place at this stage, a number o f primary afferent axons are still invading the dorsal grey matter with many reaching the lateral motor column at E10 (Shiga et al., 2000). Observations from E10 spinal cord sections indicate that this expression o f c S E M A 5 B is maintained during these latter axon pathfinding events (data not shown). c S E M A 5 B expression in D R G : Expression o f c S E M A 5 B was also apparent within dorsal root ganglia. This expression was evident as early as E5 (Figure 6 E , F). Unlike the dynamic expression i n the spinal cord, the expression in the D R G s remained uniform and constant throughout development. A s there are a variety o f different sensory neurons and glial cells present at all o f the ages examined, characterization o f the cell types expressing c S E M A 5 B awaits further analysis. Different populations o f cells are localized to different areas within the D R G , and while high magnification on D R G s demonstrates that not all o f the cells express c S E M A 5 B , the expression is uniform and not biased for a particular area associated with a specific cell type (figure 6F; Eide and Glover, 1997). This coincident expression o f a semaphorin i n both the spinal cord and D R G neurons is similar to the expression o f Sema3A in embryonic chick (Shepherd et al., 1996). Expression of c S E M A 5 B in retina: In addition to the spinal cord and D R G s , cells associated with the retina also express c S E M A 5 B . Expression can be clearly observed at stage 26 (E5), in an inner  45  layer o f the retina, presumably along the retinal ganglion cell ( R G C ) layer ( G C L ; Figure 7). The expression is confined to the cell body layer and is not associated with the nerve fibre layer (figure 7C-F). Unlike the uneven distribution o f c S E M A 5 B message within the spinal cord, the expression associated with the retina is uniform. Expression is absent in the optic nerve head ( O N H ) and along the optic nerve ( O N ; Figure 7A). This region shows exclusive expression o f Sema5A in mice (Oster et al., 2003). The cells expressing c S E M A 5 B are likely ganglion cells, as the other populations o f cells associated with the R G C layer, including some amacrine and Muller glial cells, do not have their nuclei exclusively i n this region ( M e y and Thanos, 2000). Interestingly retinal axons likely receive inunction from the radial glial cells o f M u l l e r which have been shown to express different guidance cues, for example the netrin receptor D C C (Gad et al., 2000). Retinal ganglion cells encounter other guidance cues, such as laminin, through interactions with the glial endfeet and basal lamina present i n the nervefibre layer at the vitreo-retinal border (figure 15B; Stier and Schlosshauer, 1999). Interactions between radial glia and the retinal axon growth cones are assumed to occur within the optic stalk, along the optic tract, and within the optic tectum (Silver, 1984; Thanos and M e y , 2001). A t later stages including E 9 , this c S E M A 5 B expression becomes more intense and continues to be associated with the inner most cell layer o f the retina (Figure 7E). A t these older ages, the distribution o f c S E M A 5 B message remains uniform along both the temporal/nasal axis and the rostral/caudal axis. A t these stages, it is still not clear as to what cell-types are expressing c S E M A 5 B , but the expression overlaps the normal distribution o f R G C and amacrine cells ( M e y and Thanos, 2000).  46  Figure 7: In situ hybridization studies in the retina c S E M A 5 B expression within the retina is relatively uniform in the nasal temporal plane. This expression at stage 26 (E5) is associated with the R G C layer ( G C L ) as indicated from the arrows ( A , B , C , D ) . Notably, c S E M A 5 B expression is absent from the optic fibre layer ( O F L ) and the optic nerve head ( O N H ) as indicated with an asterisk ( A , C , D ) . A t a later stage, c S E M A 5 B expression is still associated with the G C L in the E 9 retina (E). The sense probes do not have message associated with this layer (F). (G) is an example o f a Nissl-stained retina used in histological analysis o f the tissue. The associated retinal layers identified include the optic fibre layer ( O F L ) , ganglion cell layer ( G C L ) , inner nuclear layer (IN), external nuclear layer (EN), and retinal pigmented epithelium (RPE). Scale bar for (A, B ) is 100pm; scale bar for (C-G) is 10pm.  47  c S E M A 5 B expression i n the T e c t u m : A t early stages o f development, the tectum is the most prominent anatomical and functional structure o f the avian brain. Results from the in situ hybridization studies demonstrate that c S E M A 5 B is expressed in the tectum at E5 (figure 8), just prior to the arrival o f the first retinal fibres at E 6 , when maximal cell proliferation is occurring i n the tectum (Halfter, 1987). Similar to the expression o f c S E M A 5 B in the spinal cord, the distribution o f message i n the tectum is asymmetrical (Figure 8). The highest expression appears to be associated with anterior regions, getting less intense i n posterior regions (Figure 8A). Anterior/ventral tectum represents the entry for incoming retinal afferents (figure 16C). In middle regions o f the tectum, this expression occurs along the inner most ependymal (EP; neuroepithelium) lining o f the ventricle, a layer highly associated with cell proliferation at this stage (figure 8 A ; M e y and Thanos, 2000). There is also additional expression in a central layer at E 6 and E 7 , which later forms the stratum griseum centrale ( S G C ; figure 8 B , C ) . This layer is comprised o f large multipolar neurons, often referred to as ganglion cells, which are generated early i n comparison to other neurons in the tectum (figure 8 B , C ; M e y and Thanos, 2000). While this layer represents the principal tectal efferent neurons, it also acts as a target for incoming retinal afferents growing along the superficial, stratum opticum layer ( M e y and Thanos, 2000). Tectal expression was observed at later stages but is not included i n this report. The expression in the tectum overlaps a period o f maximum cell proliferation as well as innervation from retinal afferents.  49  Figure 8: In situ hybridization studies in the tectum Expression o f c S E M A 5 B is present at stage 26 (E5) along the ependymal (EP; epithelial) layer as indicated by arrowheads (>; A - C ) . The anterior expression (<) o f c S E M A 5 B is greater than the posterior expression (*) along the E P ( A - C ) . In sections from the dorsal tectum, there is additional expression o f c S E M A 5 B in the presumptive stratum griseum centrale ( S G C ) layer as indicated (#; B , C ) . There is no expression along the stratum opticum (SO) the most superficial layer associated with optical fibres. Scale bar in A is 100pm; scale bar in ( B , C ) is 10pm.  50  51  E p i t h e l i a l expression: In all o f the regions examined there is an obvious expression o f c S E M A 5 B in the associated neuroepithelia. This expression is consistent with observations from mice and rat, where Semaphorin5B is ubiquitously expressed in most neuroepithelia i n the brain and other structures (Adams et al., 1996; Skaliora et al., 1998). Observations from embryonic chick brain show that c S E M A 5 B is expressed i n the ventricular ependymal (epithelium) layer at stage 26 (E5; Figure 9). The expression, as seen in the ependymal layer o f the spinal cord, is relatively uniform along the anterior-posterior axis. Sections from rostral and caudal regions o f the brain again display a similar pattern o f expression within the ventricular epithelium (Figure 9 A , B ) . This expression is consistent with brain sections from rat where S E M A 5 B is found uniformly distributed i n most o f the epithelia within the brain, including the ventricular zones (Skaliora et al., 1998). H i g h expression o f c S E M A 5 B is also observed i n the olfactory epithelium in transverse sections from E 5 and E 6 chick (Figure 10). During embryogenesis many studies have demonstrated that the olfactory epithelium is the site o f origin o f several types o f cells which migrate along the olfactory nerve (ON) towards the brain (Dryer and Graziadei, 1994; Drapkin and Silverman, 1999; Wray, 2001). The expression o f c S E M A 5 B decreases i n the surrounding lateral olfactory tissue layers. These layers serve as target regions for a number o f cells produced along the olfactory epithelium. Thus, this assortment o f migrating cell populations and various neuronal pathfinding events associated with the olfactory epithelium during these stages correlates with expression o f CSEMA5B.  52  Figure 9: In situ hybridization of ventricular epithelium c S E M A 5 B expression i n the ventricles at stage 26 (E5) is highest in the neuroepithelium as indicated by the arrowheads ( A - C ) . This expression is uniform along anterior-posterior plane ( A ) . A similar, uniform, epithelial-associated expression is seen at lower levels within the brain (B). Scale Bar in ( A , B ) is 100pm; scale bar i n C is 20pm.  53  Figure 10: In situ hybridization studies in nasal regions A t stage 26 (E5), expression o f c S E M A 5 B i n the nasal epithelium is relatively uniform (A, B ) . A t higher magnification, the c S E M A 5 B distribution is greatest i n the inner epithelial layer, decreasing i n the outer layers (C, D ) . Scale bar i n ( A , B ) is 100u,m; scale bar in (C,D) is 10u.m.  55  56  In addition to the tissue regions mentions above, there was expression seen i n the surface epithelium located dorsally to the spinal cord. This observation was consistent in both the in situ hybridization studies as well as from antibody studies (figures 6 A & 12 A ) . Again, similar staining patterns for semaphorins have been observed from studies on secreted sema3A (Shepherd et al., 1996). There was also distribution o f c S E M A 5 B expression in the epithelium associated with the gut (data not shown). A summary o f the expression o f c S E M A 5 B from all tissues examined clearly demonstrates the association o f this expression with the neuroepithelia i n these regions (Table I). Immunohistological studies : Using an affinity purified antibody (see materials and methods) raised against the N-terminal region o f c S E M A 5 B , the distribution o f c S E M A 5 B protein was analyzed (figure 3). Based on the expression o f semaphorin from the in situ hybridization studies, chicks were examined from E5 through until E l 1 to determine whether the protein distribution mimicked that o f R N A . Initial studies performed using serum from nonaffinity purified antibody revealed expression i n the spinal cord similar to the R N A distribution from in situ hybridization studies, and seemed to support both the findings from the in situ hybridization studies, as well as the use o f this antibody i n further studies (data not shown). Westerns: In order to confirm the specificity o f the antibody, a series o f western blots from a variety o f different tissues were produced and analyzed. Initial blots suggested that the non-affinity purified antibody recognized a number o f different antigens on the blot (data not shown). Based on these westerns the antibody underwent an affinity purification  57  Table I: Summary of c S E M A 5 B expression in all regions A summary o f the noted expression o f c S E M A 5 B fro the in situ hybridization studies highlights the broad expression o f this semaphorin in the developing neuroepithelium as well as within the C N S including the spinal cord and tectum. Abbreviations: N / C , not completed; n/a, not applicable.  58  6i  V  u  ii  1  g  £ 41 "S • 1—t  > 5  bD  1  "§) co  2 §  > -o  o  ll  <a JGTJ  o o o Q Z  a  CO  CO  is  g  O  4>  . J  >  g> o  G  iS ii  pi bD  a _o CJ  .o  'co co  11  CJ  3S> .3  co  h  eggs  CO  CJ o 5 o < Z  co  s  < £ < & Z Z  ~a "a z zJS i | | 2  o  |  ^  z  z  1»  'a, •  a o  CO  CO  CO CO  Vi Vi Oi  u <u a a a a ^> ^>  CO  U  co <U  u u § Sou  co  —* co —i co ccs en  ja a, ja  l-  ii  I* <U  iH  ii  >.  >i 03 0 3  e o  <9 * <9 »* o  OH CU  If  ^3 .2 •-3 co  5  bO  c jg <U Jg "3 a  pq  a .2.2  a,  <u  bO  cj  — — PL,  ,1  ft  03  8^  < <> ^  111  •s  1  CU <U  JEJ  •Sou  2  O  CO  a  t-l  03  .2  .8  OH 0H  t  co  (U  ww  bO  bD bD c ct  o o o 5J  ii  CU  .8 CD bt>  §  o  CO  CO  CJ  U O  z z  <<ZZ  a> co  1  |W  Q j l co co co S3 ii ii ii O B >»>»>» C  (U cu 4>  a CJ o oo a cc  o  ,8) \<  1§  00  t5  in "« 1^- o >n *o r> W WW WWWW  -s o 03  'gn|| .2 cj™ .9 | H 0i VOH 5 to  co  CJ  03  .9 Pi  co  U U  CO  CO  o  t5 ON  w  00  in W  W  OO  ^ ^ ^ ^  0O  —i ^  VO  o  cu >  o  t^- ON <n  w w ww  1  <u  w  00  W  —H  ON  ww 03 CO 03  procedure, outlined in the materials and methods (figure 11). Brain fractions and spinal cord fractions were isolated at embryonic days 8 and 9, and the protein fractions were separated on SDS-polyacrylamide gels. The resulting blots showed a significant decrease in the number o f bands present. There were two prominent bands one at 90 k D and one at 130kD (the predicted molecular weight; figure 1 ID). Further isolation o f the soluble fraction from the insoluble cytoskeletal fraction revealed a single band i n the insoluble fraction (containing membrane fraction) at 130kD (figure 1 I E ) . This band corresponds with the predicted molecular weight for c S E M A 5 B and confirms the specificity o f the affinity-purified antibody. The staining was then performed i n non-detergent conditions in order to prevent any antibody from entering the cells. The identity o f the 90kD band remains unclear, however, a possible cleavage binding site within the fifth thrombospondin repeat would produce a secreted product o f ~90kD in predicted size. The possibility for this product presents an interesting situation where c S E M A 5 B might exist in a transmembrane form or as a secreted molecule that may act long range. c S E M A 5 B distribution i n S p i n a l C o r d : After a series o f trials using a variety o f fixation procedures and antibody exposures, consistent labeling o f c S E M A 5 B was obtained. Within body sections at embryonic days E 6 and E 7 , the expression is observed in the spinal cord and the surface epithelium located dorsally to the spinal cord (figure 12 A ) . A closer examination o f the expression in the spinal cord at this stage suggests that it is broadly distributed i n the spinal cord in both dorsal regions, including lamina II and III and i n ventral regions including the presumptive lateral motor column (figure 12A). Though the expression is  60  F i g u r e 1 1 : A b production and western blots Isolated G S T (A) and c S E M A 5 B (B) peptides were separated on a 12% polyacrylamide S D S gel and stained with coomassie blue to confirm the correct size, ~26kDa and 35kDa respectively. Following A b purification through a G S T and c S E M A 5 B column, the final elutions containing c S E M A 5 B A b s , were confirmed by separating the heavy and light chain A b peptides on a 12% polyacrylamide S D S gel (C). Total protein was isolated from E9 brain and Spinal cord tissue using R I P A buffer (D). Affinity purified c S E M A 5 B A b recognizes two distinct bands at 90kD and 130kD (D).  Separating the soluble  fractions (right lane) from the insoluble fractions containing the membrane (middle two lanes) reveals a 130kD band i n the insoluble component o f similar size to the c S E M A 5 B expressing 293 control cells (left lane: E ) .  61  MW  MVC  62  F i g u r e 12: Semaphorin 5 B distribution i n S p i n a l C o r d A t E 7 , the distribution o f c S E M A 5 B protein in the grey matter is wide spread (A) with highest levels i n the ventral regions (B). The dorsal surface epithelium (SE) also stains for c S E M A 5 B . E10 Spinal cord sections stained without detergent show staining in the spinal cord with highest levels in a subset o f cells i n the same location as presumptive motor neurons (D, E ) . The staining associated with D R G s is relatively uniform at all stages examined ( A , D , F ) . Sections stained with the pre-immune serum show no significant staining (C). Scale bars in ( A , C , D ) are 100pm; scale bar i n ( B , E , F) are 10p.m.  63  broad, the levels o f c S E M A 5 B are higher in ventral regions, as is evident from the more intense levels o f fluorescence in this region o f the spinal cord (figure 12 A , B ) . Similar to the regions identified in the in situ hybridization studies, the expression is highest in cells located within the presumptive lateral motor column. In addition, there is some staining associated with the ependymal layer along the dorsal ventral axis. A s the spinal cord continues to develop, there are significant changes in the distribution o f c S E M A 5 B . The dorsal expression o f semaphorin diminishes, particularly in medial regions corresponding to lamina III. The ventral expression at this age remains strong at E8 and E 9 along with staining along the ventral ependymal layer (figure 13B; data not shown). A t later stages including E10 and E l 1, the expression continues in ventral regions however, becoming localized to a population o f large diameter cells in the ventral horn. The identity o f these cells is unclear, but overlaps with the normal distribution o f motor neurons (figure 12 D , E ; Eide and Glover, 1997). In addition, spinal cord cells that have been dissociated were also labeled with c S E M A 5 B antibody along with a neuronal marker N e u N to further characterize these cells (figure 13). From these experiments it is clear that many o f the cells labeled with a n t i - c S E M A 5 B antibody are neuronal, as they are also recognized by the N e u N antibody (figure 13 A , B , D , E ) . Interestingly, a small population o f cells that express c S E M A 5 B are not N e u N positive, and based on morphology appear to be glial i n origin (figure 13 A , B ) . When the antibody is pre-incubated with c S E M A 5 B fusion protein prior to staining, the cells no longer stain for c S E M A 5 B further confirming the A b ' s specificity (figure 13 C , F).  65  F i g u r e 13: Dissociated S p i n a l Cultures A n t i cSema5B antibody (A) labels neuronal cells (C) as well as glial cells (*). Spinal cords were isolated from E8 chick and subjected to trituration with a small bore pipette. The resulting suspension o f cells was plated on coverslips coated with poly-L-lysine and laminin. Cells were doubled labeled using anti Sema5B antibody (green; A ) and a neuronal anti N e u N antibody (red; C ) . Cells stained with A b s that are pre-incubated with c S E M A 5 B peptide, are devoid o f c S E M A 5 B staining (B). These same cultures stain for N e u N (D). Scale bars i n ( A - D ) are 10p.m.  66  *  1 * • *  A  T\  c  • •  • •  41  B  D  67  The high levels o f c S E M A 5 B i n this region o f the spinal cord suggests that it might be involved in either the sorting o f motorneurons or in the maintenance o f proper patterning within the spinal cord. Distribution of c S E M A S B in D R G s : The in situ hybridization studies showed that expression o f c S E M A 5 B in the D R G s is uniform and maintained from E5 through E l O . The distribution o f c S E M A 5 B within D R G s , as demonstrated b y immuno-histochemical studies using a n t i - c S E M A 5 B antibody, is consistent w i t h the distribution o f c S E M A 5 B message (compare figures 6 A , E ; 12A, F). Again, c S E M A 5 B is highly expressed in D R G s at all stages examined. D R G ganglion were isolated from E8 chick and were dissociated and labeled using c S E M A 5 B antibody. A strong axonal and cell body punctate staining confirms the presence o f c S E M A 5 B in the D R G neurons (data not shown).  A t E 6 when D R G expression is  clearly apparent, there are different sized classes o f sensory neurons that occupy different spatial domains within the D R G . However, there was no apparent restriction o f the labeling with either population o f neurons, leaving the identity o f these neurons unknown. c S E M A 5 B distribution in the retina: The distribution o f c S E M A 5 B protein i n the retina is consistent with the R N A studies and is associated with the retinal ganglion cell layer. This expression is identified at E7 and continues through E 9 and E l O (figure 14). W h i l e the distribution o f this semaphorin in the retina remains relatively uniform throughout development, the levels o f expression appear to change (figure 14 A , E ) . W h i l e not quantified, it is clear that the intensity o f fluorescence increases with an increase in age, particularly at E 9 and E l O  68  Figure 14: Semaphorin 5B distribution in the Retina The distribution o f c S E M A 5 B is identified in the R G C layer as early as E 7 ( A , B ) . A t later stages (ElO), c S E M A 5 B expression remains uniform and is associated with the R G C layer (D, E , F). A t these later stages, the intensity o f the staining increases as compared to E 7 . Staining using pre-immune serum from rabbit was used as a control (C, F). The staining is visualized with an anti-rabbit, C y - 3 secondary A b . Scale Bar i n A is 100u.m; Scale bar i n B is lOum; Scale bars i n (C-F) are lOurn.  69  E9  E7  GCL  A  D  B  -  C  GCL  70  (figure 1 4 D , E ) . Though the distribution o f c S E M A 5 B associated with the ganglion cell layer would suggest that the staining is neuronal, this has not been confirmed from dissociated cultures.  71  Discussion: Ten years after the identification o f the first semaphorin i n the grasshopper limb (Kolodkin et al., 1992, 1993), our understanding o f this family o f proteins has greatly advanced. While much effort has gone into the characterization o f the secreted vertebrate semaphorins, recent work has begun to characterize vertebrate transmembrane semaphorins. The results presented i n this thesis, in conjunction with some important functional studies from our lab, have allowed us to gain some insights into the role o f one transmembrane semaphorin in chick, S E M A 5 B . What is particularly intriguing about this semaphorin is the presence o f a typically repulsive S E M A domain and a region o f thrombospondin repeats associated with neuronal outgrowth. The possibility o f two regions associated with opposite guidance responses located on the same protein makes this semaphorin an interesting choice for study. These studies have demonstrated that the expression o f c S E M A 5 B i n chick is consistent with expression o f this semaphorin in other organisms including mice and rat. W i t h i n the developing nervous system, the majority o f c S E M A 5 B expression is associated with the neuroepithelium. This expression occurs along the epithelium o f olfactory system, i n the ventricular zone o f the brain and spinal cord, and i n the tectal epithelium (table I). During the ages examined, all o f these regions are associated with the proliferation o f a number o f different cell types, many o f which are neuronal. While much o f the literature on semaphorins suggests that they are involved in axon guidance, a number o f studies have implicated a role for semaphorins in the migration o f a variety o f different cell types (Hu et al., 1996; de Castro et al., 1999; Sugimoto et al., 2001; Ginzburg et al., 2002; Comoglio and Trusolino, 2002; Tsai et al., 2002; Tamamaki et al.,  72  2003). A s most semaphorins are inhibitory guidance cues, one can speculate that perhaps in these tissues, this semaphorin may be required for the induction o f migration o f these cells away from their proliferative centers, and into the surrounding tissue. In addition to this broad epithelial expression o f c S E M A 5 B , there are also a variety o f cells within the D R G s , spinal cord, and retina which express c S E M A 5 B and are not associated with an epithelial layer. Expression o f c S E M A 5 B i n these regions overlaps with the normal distribution o f neuronal cells and correlates with a number o f key axon guidance decisions and processes. Indeed, evidence collected from a number o f functional in vitro studies demonstrates that different neuronal populations associated with these regions are inhibited by c S E M A 5 B . Based on our knowledge on semaphorin function from in vivo and in vitro studies, these results support a role for this semaphorin in neuronal development i n the chick.  Role of S E M A 5B in developing spinal cord: The expression o f c S E M A 5 B within the grey matter o f the spinal cord is dynamic, being first expressed broadly i n both dorsal and ventral regions, but later being restricted to ventral regions. This dynamic expression correlates with some key axon guidance decisions i n these regions. A t this stage o f development various populations o f sensory and motor neurons make projections either to or away from the developing spinal cord. Primary D R G sensory neurons follow highly stereotyped pathways in reaching their specific target areas i n the spinal cord. The first sensory afferents reach the spinal cord at embryonic day 4.5 (E4.5), just prior to the first observed expression o f c S E M A 5 B i n both dorsal and ventral grey matter (Vaughn and Grieshaber, 1973; Altman and Bayer, 1984). These incoming sensory afferents then grow rostrocaudally along the dorsalateral regions  73  o f white matter, waiting for about 48 hrs before finally invading the grey matter at its most dorsal aspect (Vaughn and Grieshaber, 1973; Altman and Bayer, 1984). A s semaphorins are generally characterized as inhibitory guidance cues, the expression o f c S E M A 5 B in the dorsal grey matter at this stage may contribute to the prevention o f these sensory afferents from invading the dorsal grey matter. Other semaphorins, including Sema3A, have also been implicated i n the guidance o f sensory afferents during these pathfinding processes (Tanelian et al., 1997; Pasterkamp et al., 2000). While all o f the sensory afferents enter the spinal cord at the dorsal horn, different neuronal populations are instructed to terminate at different regions in the spinal cord based on their sensory function (figure 15). These D R G neurons are divided into distinct populations that express different markers and serve different sensory modalities. The neurotrophin(NT)-3-responsive, T r k C positive, large-diameter D R G afferents involved i n proprioception, project to the ventral spinal cord to synapse on the dendrites o f motor neurons (figure 15 ( A , E l O ) ; Pasterkamp et a l , 2000; F u et al., 2000). The smalldiameter, T r k A positive, nerve growth factor ( N G F ) responsive D R G neurons that are implicated in thermoreception and nociception, synapse most dorsally i n laminae I and II, while those implicated i n mechanoreception terminate in laminae III-IV (figure 15 ( A , E l O ) ; Pasterkamp et al., 2000; Zhang et al., 1994). This dorsal migration o f T r k A neurons has been associated with the presence o f the repulsive Semaphorin 3 A expressed in ventral regions o f the spinal cord at this point in development (Fu et al., 2000). Indeed, in vitro studies from chick D R G s have shown that T r k A neurons are inhibited by semaphorin 3 A , which even causes collapse o f their growth cones in vitro (Luo et al., 1993). In contrast, T r k C neurons do not respond to sema3A, which correlates with their  74  Figure 15: A Model for c S E M A 5 B function in guidance Spinal Cord ( A ) : A schematic representation o f the dynamic c S E M A 5 B distribution within the developing spinal cord highlights a possible role for this semaphorin in axon guidance from E4.5 to E 1 0 (A). Sensory afferents first arrive at the dorsal grey matter just prior to c S E M A 5 B expression at E 5 . A s D R G axons are inhibited by c S E M A 5 B in vitro this broad expression i n the grey matter may contribute in preventing these axons from invading dorsal regions, leading to axonal growth along the rostral/caudal axis through E6.5. A s the spinal cord develops the dorsal expression o f c S E M A 5 B decreases and coincides with the entry o f sensory axons into the dorsal funiculus at E6.5/E7. Variable inhibitory phenotypes o f D R G axons to c S E M A 5 B in vitro, suggests that these neurons have differential responses to this semaphorin. These studies demonstrate that many axons ceased growth after contact with cells expressing c S E M A 5 B , while other axon avoided these cells. D R G axons may target to different regions o f the spinal cord depending on their response to c S E M A 5 B (see E10). A t later stages o f development, the ventral expression is associated with large diameter cells in the lateral motor column at lamina FX. Again, functional in vitro studies on sympathetic neurons demonstrated that they are strongly inhibited by c S E M A 5 B . This inhibitory response to c S E M A 5 B may contribute to the ventral exit o f motor neurons as well as preventing sympathetic axons from invading the ventral spinal cord. Different anatomical structures associated with the spinal cord include: the laminae I-IX, ventral horn ( V H ) , dorsal horn (DH), white matter ( W M ) , dorsal root ganglia ( D R G ) , sympathetic ganglia (SG).  75  Retina (B): A schematic representation o f c S E M A 5 B expression in the retina, along with the expression o f other known guidance cues at the optic nerve head highlights a role for this semaphorin i n axon guidance in the retina (B). The expression o f c S E M A 5 B at E5 corresponds with the migration o f retinal ganglion axon from the ganglion cell layer ( G C L ) , to the optic fibre layer ( O F L ) and towards the optic nerve head ( O N H ; Inset 2). Retinal axons which express the known plexin interacting protein, L l , are directed away from a layer o f c S E M A 5 B inhibition and grow along a layer o f basal lamina containing laminin which promotes neurite outgrowth. Expression o f c S E M A 5 B is absent from the retinal axons and its presence i n the G C L , i n conjunction with the results from in vitro studies, suggests that it may actually help to prevent axons from straying away from the O F L into other retinal layers. U p o n reaching the optic nerve head, axons encounter new guidance cues including netrin-1 and possibly c S E M A 5 A (based on the expression o f Sema5A in mouse; Inset 1). The attractive guidance cue netrin-1 is expressed by a ring o f epithelial cells surrounding the O N H and helps to attract these incoming retinal axons to this structure. Interestingly in the presence o f laminin the normally attractive netrin switches to an inhibitory guidance cue which helps to steer axons into the optic nerve head and away from the retina through the optic nerve. The expression o f Sema5A along the O N H and O N is thought to act as an inhibitory sheath which encases the retinal axons along their path towards the chiasm (Oster et al., 2003). This continuous inhibition from SEma5 A is supported from studies which demonstrated that the inhibitory response o f . R G C axons to Sema5A is maintained in the presence o f L l and netrin-1 (Oster et al., 2003). Thus, the possibility o f this mutually exclusive expression o f class 5 semaphorins  76  may contribute to the guidance o f retinal axons. Different retinal layers are identified i n Inset 2: basal lamina ( B L ) , optic fibre layer ( O F L ) , ganglion cell layer ( G C L ) , inner plexiform layer (IPL), inner nuclear layer (IN), outer plexiform layer ( O P L ) , external nuclear layer (EN), receptor layer ( R L ) , pigment epithelium (PE). Tectum (C): A schematic representing the expression o f c S E M A 5 B i n the tectum displays the increasing posterior to anterior gradient. This expression o f c S E M A 5 B overlaps with the arrival o f the first retinal fibres at E 6 , i n the ventral/anterior region o f the tectum. A gradient o f expression suggests that different populations o f retinal afferents may be directed to a particular region o f the tectum depending on their expression o f the receptor for c S E M A 5 B . Those neurons which are inhibited by c S E M A 5 B the most w i l l travel to anterior regions, while neurons unresponsive to c S E M A 5 B may continue to the posterior tectum.  77  78  ability to migrate ventrally i n the presence o f this inhibitory semaphorin. Studies from mice lacking Sema3A do not show any major guidance errors associated with these neurons, suggesting that other factors contribute to this process, including c S E M A 5 B (Taniguchi et al., 1997). Support for the involvement o f cSEMA5J3 i n these guidance events is based on the dynamic expression o f this semaphorin in the grey matter and from functional in vitro studies in the lab. A s demonstrated in the in situ hybridization and antibodies studies, the dorsal expression o f c S E M A 5 B declines at approximately stage 31 (E6.5-E7). This decrease in dorsal expression corresponds with the first entry o f T r k A  +  afferent  collaterals into the dorsal gray matter (figure 15 A ; Pasterkamp et al., 2000; F u et al., 2000). Thus a decrease i n this inhibitory semaphorin might help to facilitate the entry o f sensory axons into dorsal regions o f the grey matter. Contrastingly, there is a continuous ventral expression through E l 1 (table I). A s the spinal cord develops, this ventral expression becomes more discretely localized to a small population o f large diameter cells that overlap with the normal distribution o f motor neurons. Motor neurons undergo sorting and must pathfind to specific targets either i n the periphery. This discrete c S E M A 5 B expression in motor neurons may function i n guiding the motorneurons from the spinal cord at these later stages o f spinal cord development (E8 and later; Eide and Glover, 1997).  Functional Studies: A number o f in vitro studies in our lab have demonstrated that c S E M A 5 B inhibits the growth o f E7 D R G neurons. What is particularly interesting is that D R G neurons show variable guidance behaviors in response to c S E M A 5 B . In the presence o f small  79  groups o f 293 cells expressing cSEMA5J3, some o f the D R G neurons avoid these c S E M A 5 B expressing cells, while some o f the D R G neurons do not avoid the cells but rather cease their growth after contacting them. The existence o f these multiple phenotypes suggests that perhaps different D R G neurons respond differently to c S E M A 5 B . Further tests looking at the different neuronal populations in vitro would be able to determine this. While the response o f D R G neurons to c S E M A 5 B is somewhat variable, the same is not true o f sympathetic neurons. Based on the expression studies, these neurons do not express c S E M A 5 B (figure 12 D ) . From the functional in vitro studies, it is obvious that these neurons are strongly inhibited by c S E M A 5 B , completely avoiding the c S E M A 5 B expressing cells. The contrast in these neuronal responses to c S E M A 5 B may play an importance i n the chick. Incoming D R G neurons, even though they do not invade the dorsal grey matter at E 5 , are not completely repelled from it but rather grow rostrally/caudally until entering 48hrs later. This creates a scenario where incoming D R G axons are not repelled away from the dorsal gray matter, but rather they may cease their growth until they receive further instruction. A s the levels o f c S E M A 5 B decrease, then perhaps these axons can continue to migrate into the gray matter and synapse in their target regions. Discrete ventral expression in different cells may prevent certain D R G axons from forming a synapse with these cells. In addition, the expression o f c S E M A 5 B in ventral regions may result in the pathfinding o f motor axons away from the spinal cord to the periphery, and its presence in the spinal cord may prevent the sympathetic neurons from incorrectly invading these regions. A model for a possible role o f c S E M A 5 B in the spinal cord is presented (figure 15A).  80  Role of SEMA5B in retina: While the expression o f c S E M A 5 A has not been described i n chick, the results from expression studies i n mice suggest a situation where mutually exclusive expression o f these two class 5 semaphorins may correlate with sorting and pathfinding o f retinal ganglion cells ( R G C ) at this stage. In mice, retinal distribution o f Sema5B has not been described, however studies on the retinal expression o f Sema5A have demonstrated that it is specifically localized to the optic nerve head ( O N H ) and along the optic nerve ( O N ; Oster et al., 2003). Studies on c S E M A 5 B i n chick have demonstrated that it is broadly expressed along the G C L i n retina but is absent from the optic fibre layer, O N H , and O N (figure 7 A ) . Taking the results from both organisms suggests that their expression is mutually exclusive. These results are supported from studies i n mice and rat describing the expression o f these two class members are sometimes mutually exclusive. The expression o f c S E M A 5 B occurs at a time when axons are i n the midst o f a number o f important axon guidance decisions. The first fibers leave the eyeball and grow along the optic stalk at about stage 21 (E 3.5; Thanos and M e y , 2001). A x o n s first arrive at the chiasm from stage 22-24 (E3.5-E4), then advance through the contralateral optic tract towards the tectum, where they first arrive at the anterior pole at E 6 (Halfter, 1987). Within the retina differentiated ganglion cells extend axons exclusively within the innermost retina layer, the optic fiber layer, thereby avoiding misrouting into outer retina layers (Stier and Schlosshauer, 1999). These retinal axons likely receive instruction from the radial glial cells o f M i i l l e r until the growthcones approach the glial endfeet present in the nerve fibre layer at the vitreo-retinal border (Stier and Schlosshauer, 1999). These axons are then directed to grow towards the optic fissure, likely receiving their instruction  81  from both the glial endfeet and the basal lamina present (Halfter and Schurer, 1998). Interactions between radial glia and the retinal axon growth cones are assumed to occur within the optic stalk, along the optic tract, and on the optic tectum (Silver, 1984; Thanos and M e y , 2001). During this period o f growth within the retina, this instruction is the result o f the interaction between the growth cones and a number o f molecules which are required for proper guidance (Thanos and M e y , 2001). A role for semaphorins i n the guidance o f retinal ganglion cells was identified i n Xenopus. Retinal ganglion cells expressing the neuropilin-1 receptor are responsive to Sema3A, and are guided to their correct projection by the presence o f Sema3 A i n the optic chiasm (Campbell et al., 2001). In the chick, the role o f semaphorins i n guiding retinotectal axons remains elusive. Based on the expression o f S E M A 5 B , along with observation from Sema5 A expression i n mice, it seems a likely possibility that the combination o f these two semaphorins might work in concert to guide neurons from the retina and through the optic disk towards the chiasm (figure 15B).  Functional Studies: A s is the case with neurons associated with the spinal cord, a number o f in vitro studies i n the lab have examined the response o f retinal ganglion neurons to c S E M A 5 B . In these studies it has been demonstrated that retinal ganglion cells avoid cells expressing c S E M A 5 B as compared to controls. In addition, Oster et al. (2003) demonstrated that these same neurons are inhibited by Sema5 A , helping to support a role for this class o f semaphorins as an inhibitory guidance cue for retinal afferents. A possible model for the role o f c S E M A 5 B i n the retina is presented (figure 15B).  82  Role of cSEMA5B in tectum: Another known model for studying axon guidance is the development of retinotectal projections. The primary retinotectal projection of chicken maintains a specific, topographical order at structural levels (Thanos and Mey 2001). Within the tectum, a decreasing anterior to posterior gradient of cSEMA5B expression suggests that it may be involved in proper topographical projection of incoming retinal afferents. The existence of a gradient cues within the tectum was first proposed in Sperry's chemoaffinity hypothesis, based on his experiments with the retinotectal projection in frog (Sperry, 1963). Support for tectal gradients of guidance cues came from in vitro studies using a co-culture system of retinal explants and tectal membranes of anterior or posterior tectal origin (Halfter et al., 1981; Bonhoeffer and Huf, 1982; Bonhoeffer and Huf, 1985). These experiments demonstrated that temporal axons of the retina prefer to grow on anterior tectal membranes while those of the nasal retina did not. From a variety of studies it is clear that axons from retinal ganglion cells in the temporal regions of the retina will target to the anterior regions within the tectum, while axons from R G C in the nasal retina will target to the posterior tectum. Subsequent studies have gone on to identify a number of different molecules that are expressed in gradient in the tectum (reviewed in Thanos and Mey, 2001). Presently the most intensely studied family of guidance cues in the retinotectal projection is that of the ephrins, ligands for the Ephrelated tyrosine kinase receptors (Knoll and Drescher, 2002). Initial experiments characterizing the ephrins demonstrated that they were expressed in a gradient fashion in the tectum, with high levels located in the posterior tectum (Cheng et al., 1995; Drescher  83  et al., 1995). H i g h levels o f Ephrin-A2 and Ephrin-A5 i n the posterior tectum act as ligands for Eph-A3 expressed i n the retina (Cheng et al., 1995; Drescher et al., 1995). The receptor Eph-A3 is also distributed i n a gradient that increases from nasal to temporal i n retinal axons (Drescher et al., 1995). The ephrins are inhibitory and thus, axons from temporal regions o f the retina associated with high levels o f E p h - 3 A are more inhibited by the ephrin ligands in the tectum, and are restricted to anterior regions o f the tectum associated with lower ligand levels (Sefton and Nieto, 1997; Pittman and Chien, 2002). In the case o f c S E M A 5 B , the expression gradient occurs i n the opposite direction o f the ephrins, with highest levels in anterior regions at E 6 (figure 8 A ) . This expression o f c S E M A 5 B correlates with the arrival o f the first retinal afferents at the anterior/ ventral regions o f the optic tectum at E 6 (figure 15C). Retinal axons advance over the tectal surface and then descend into the stratum griseum et fibrosum superficiale (SGFS) to connect with tectal targets, from E 7 to E12-13 (Thanos and M e y 2001). Expression o f c S E M A 5 B within the tectum during these processes suggests that it may play a role i n axon guidance. This is again supported from the in vitro studies i n the lab which demonstrated that retinal ganglion afferents avoid cells expressing c S E M A 5 B . A s the ligand for c S E M A 5 B is unknown, it is difficult to predict whether a subset o f retinal neurons would likely respond to this semaphorin upon reaching the tectum. A l s o , it has not been demonstrated whether different retinal ganglion cell types have variable responses to c S E M A 5 B in vitro. Interestingly, many axons within the retina have presumably been pre-exposed to c S E M A 5 B before reaching the tectum, based on the retinal expression. This leads to the question as to whether this w i l l affect their response  84  to c S E M A 5 B within the tectum? A model summarizing a possible role for c S E M A 5 B in the tectum is presented (figurel5C).  Role of S E M A 5 B in the neuroepithelium: In addition to a role i n pathfinding, the expression o f c S E M A 5 B in the neuroepithelium overlaps with normal areas o f cell proliferation and sorting. Within most o f these structures, new cells migrate from inner layers to outer layers. In all o f these areas the expression o f c S E M A 5 B is greatest along the inner layer, decreasing i n the outer layers. This expression, i n conjunction with the in vitro studies and known evidence on semaphorins, suggests that c S E M A 5 B may play a role i n the migration o f these cells from the proliferative inner layer. Recent evidence on the class 3 semaphorins have implicated them in the migration o f glial cells associated with the optic nerve (Sugimoto et al., 2001; Tamamaki et al., 2003). These studies demonstrate that the migration o f glial precursors along the rat optic nerve is guided by gradients o f diffusible cues produced i n the optic chiasm, including Sema3A (Sugimoto et al., 2001). Within this population o f glial precursors, it was demonstrated that different cell types would selectively respond to different cues, and thus some were responsive to Sema3 A while others were not (Sugimoto et al., 2001). Thus, the possibility that c S E M A 5 B is able to contribute to the migration o f neuronal and glial precursors within these proliferative epithelial layers, is likely. In order to examine this aspect o f c S E M A 5 B function, further studies in vitro using assays to assess cell migration might help to support this hypothesis. A model for the role o f c S E M A 5 B in cell migration within the various epithelia is presented (figure 16).  85  F i g u r e 16: A M o d e l for c S E M A 5 B function i n migration  A schematic representation of cSEMA5B function during cell migration from the various neuroepithelia. Cell migration plays a major role for proper CNS development in vertebrates. Many neuronal progenitors, post-mitotic neurons, and glial progenitors born in proliferative zones within the nervous system, must migrate away from these regions towards their final destination in the cortex and other areas of the CNS. A number of key players involved in this migration process have been identified. The broad expression of cSEMA5B in a number of proliferative zones suggests that it contributes to this process of cell migration in these tissues. As it has been identified as ah inhibitory guidance cue, cSEMA5B expression along the inner most ependymal layer (EP) may help to drive migration awayfromventricular regions towards the target tissue. The levels of cSEMA5B decrease in more superficial layers including the intermediate zones (IZ), target regions, and outer layers (OL).  86  Ventricle  87  Future Directions: The experiments presented in this thesis have described the expression of c S E M A 5 B from E5 to E l 1. Though they have provided us with key insight into the role and expression of this semaphorin, there are a number of experiments which would help in better characterizing its expression. While sections from different levels of tectum have demonstrated that it exists in an increasing posterior to anterior gradient, a clear gradient in whole tectum has not been established. Thus, performing whole-mount in situ hybridizations would help to define the expression of c S E M A 5 B in an intact chick. 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