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Characterization of Semaphorin 5B in avian visual system development Wood, Jacqueline Leigh 2006

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CHARACTERIZATION OF SEMAPHORIN 5B IN AVIAN VISUAL SYSTEM DEVELOPMENT  by  JACQUELINE LEIGH WOOD B.Sc, The University of British Columbia, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in  THE FACULTY OF GRADUTE STUDIES  (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA June 2006  © Jacqueline Leigh Wood, 2006  Summary  The development of the avian retinotectal projection is a well established model for the study of axon guidance and topographic map formation. Here we employed immunoflourescence to demonstrate that the guidance molecule, Semaphorin 5B, is expressed differentially across the tectum during the time that retinal ganglion cell axons extend into and establish their connections. Beginning on embryonic day 5, a robust anterior to posterior and dorsal to ventral gradient was observed. Highest expression was localized to the superficial SGFS cell layer and continued until embryonic day 12. In vitro analysis using a cell island assay indicated that retinal ganglion cell neurites make significantly fewer contacts and avoid cells expressing Semaphorin 5B in comparison to control cells. No temporal or spatial disparity in response was observed, as RGC'sfromall ages examined (E5-E8) and all locations of the retina, responded similarly to Semaphorin 5B. Interestingly, immunoflourescence examination revealed that Semaphorin 5B is also highly expressed within the developing RGC layer of the avian retina. Further analysis indicated that Semaphorin 5B protein is spatially restricted to RGC perikarya and was not observed in RGC neurites at this stage of development. Together this data implicates Semaphorin 5B as an inhibitory guidance molecule expressed in both the retina and optic tectum in regions adjacent to developing RGC neurites. This study identifies Semaphorin 5B as a novel RGC guidance cue and provides the first evidence of transmembrane semaphorin involvement in RGC axon arbourization and target selection.  u  TABLE OF CONTENTS ABSTRACT  ii  T A B L E OF CONTENTS  iii  LIST OF T A B L E S  v  LIST OF FIGURES  vi  ABBREVIATIONS  vii  ACKNOWLEDGEMENTS  ix  C H A P T E R I- INTRODUCTION  1  The growth cone  2  Avian visual system development i) Retinal development ii) Development of the tectum iii) Axis specification iv) R G C axon guidance v) Retinotectal map formation The semaphorin family Hypothesis  4 4 5 6 8 12 16 20  C H A P T E R II- M A T E R I A L S A N D M E T H O D S . . . Animal Care Dissection and Staging Western Analysis Immunoprecipitation Immunoflourescence of embryos Dissociated Cell Culture Maintenance of Cell Lines Co-culture Experiments Immunoflourescence of Cultured Cells Co-culture Quantification C H A P T E R III- RESULTS Semaphorin 5B Protein Expression in the Developing Optic Tectum Spatial Expression of Sema5B within the Developing Avian Tectum Temporal Pattern of Sema5B Expression in the Embryonic Tectum Retinal Ganglion Cell Fibre Response to Sema 5B In Vitro Localization of Sema 5B in the Developing Retina  iii  22 22 22 22 23 24 26 26 27 27 28 30 30 33 41 46 52  C H A P T E R IV- CONCLUSIONS A N D DISCUSSION Implications of Sema5B Expression in the Avian Tectum Implications of Sema5B in Topographic Mapping Across the Tectum Implications of Sema5B Repulsion of R G C Neurites In Vitro Implications of Sema5B in the Developing Avian Retina Final Remarks  58 59 64 66 68 70  BIBLIOGRAPHY  71  iv  LIST OF TABLES Table 1. Molecules involved in R G C axon guidance and topographic map formation.. ..10  LIST OF FIGURES Figure I-1.  Schematic representation of RGC topographic map formation across the avian optic tectum 13  Figure 1-2.  Schematic representation of the Semaphorin family of guidance cues  17  Figure III-1. Analysis of Sema 5B protein presence in tectal tissue using western blot. ...31 Figure III-.2. Immunoflourescence examination of Sema5B expression in the E8 avian tectum 34 Figure III-3. Comparison of Sema5B immunoflourescence staining in the dorsal versus the ventral tectum Figure III-4. Immunoflourescence analysis of Sema5B expression in the anterior versus the posterior tectum  37  39  Figure III-5. Temporal examination of Sema5B expression in the anterior tectum using immunoflourescence  42  Figure III-6. Temporal examination of Sema5B expression in the posterior tectum  44  Figure III-7. In vitro analysis of Retinal Ganglion Cell response to Sema5B  47  Figure III-8. Temporal and spatial analysis of RGC response to Sema5B  50  Figure HI-.9. Investigation of Semaphorin 5B expression within the developing avian retina using western blot and immunoflourescence  53  Figure III-10. Analysis of Sema5B protein expression along developing RGC fibers  56  Figure IV-1. Schematic representation of Sema5B expression in the developing avian retina and tectum w 65  vi  ABBREVIATIONS A-P - anterior-posterior axis CBF-1 - chick brain factor-1 CBF-2 - chick brain factor-2 CS-PG- chondroitin sulfate proteoglycan DMEM-dubelco's modified eagles medium D-V - dorsal-ventral axis E - embryonic day (E3-embryonic day 3, E8 - embryonic day 8, etc.) ECL-electrogenerated chemiluminescence EDTA-ethylenediametetraacetic acid FBS-fetal bovine serum G C L - ganglion cell layer GPI-linked - glycophosphatidyl inositol-linked HBSS- hank's balanced salt solution HEK-human embryonic kidney H H - Hamburger and Hamilton stage (HH-11 - Hamburger and Hamilton stage 11) HRP-horseradish peroxidase N-T - nasal-temporal axis N G F - nerve growth factor NGS-normal goat serum OCT-optimal cutting temperature medium ONH- optic nerve head OPC - oligodendrocyte precursor cell  vii  PBS-phosphate buffered saline PMSF-phenyl-methyl-sulfonyl-fiouride RGC- retinal ganglion cell ROBO-roundabout RIPA-radio irnrnunoprecipitation buffer SDS-sodium-dodecyl sulfate SDS-PAGE- sodium-dodecyl sulfate polyacrylamide gel electrophoresis Sema3A - semaphorin 3A Sema3D- semaphorin 3D Sema3E- semaphorin3E Sema4A- semaphorin4A Sema5A- semaphorinSA Sema5B- semaphorin 5B Sema6A — semaphorin 6A SGFS — sratum griseum et fibroseum SO - stratum opticum TBST-tris-buffered saline TSR-1 — type-1 thro mho spend in repeat  vm  AKNOWLEDGEMENTS I would like to extend my sincerest gratitude to the many people without whom completion of this thesis would not have been possible. Thank you to all the members of the O'Connor lab both past and present, who have made these past years so memorable. Your support, advice and encouragement have never gone without appreciation. Special thanks to Dr. W. Wang, Robyn Lett and Martin Williamson for your continued friendships and close collaboration. I must also thank Marcia McCoy, Patrick Lajoie, Julian Guttman and Aruna Somasiri for their attentive guidance as senior scientists. Many thanks also to my committee members: Dr. Vanessa Auld, Dr. Joy Richman and Dr. Wolfram Tetzlaff, who have all provided me with kind suggestions and guidance along the way. I would like to take this opportunity to convey my deep appreciation to my family, who all have continually encouraged me to pursue my dreams. I am profoundly lucky and sincerely grateful for all the opportunities you have given me. Nils - Danke auch fur deine Hilfe, deine Geduld und dein Verstandnis. Du bist jemand auf den ich mich immer verlassen kann. Most of all, I must thank Dr. Tim O'Connor for his unrelenting patience, support and guidance. I cannot express enough gratitude for your mentorship which has enabled me to realize my own abilities and love of science. Thank you. Finally, I would like to make a special dedication to a man whose integrity and desire to help others has inspired me and many others. Dr Webber you will not be forgotten.  ix  CHAPTER I: INTRODUCTION Formation of ordered and functional neural networks depends on the precise connection of neurons with their target. In order for growing axons to reach their final destination they mustfirstnavigate a specified path by making stereotypic, directional decisions to reach their targetregionand then must identify their correct target. Misguidance of these projections is rare, indicating that underlying mechanisms must be robust to ensure that the proper pathways are established (reviewed by Tessier-Lavigne and Goodman, 1996). To date there have been several proposed mechanisms to explain axon guidance (Weiss, 1937; Sperry, 1963; Letourneau, 1975). The majority of evidence supports the proposition that a combination of attractive and repulsive molecules direct neurite outgrowth and steering (Ramon y Cajal, 1892; Sperry, 1963, Serafini et al, 1994; Hopker et al., 1999). Isolation and characterization of guidance cues has resulted in the identification of many different families including the ephrins, netrins, slits and semaphorins (Kolodkin et al., 1991; Niclou et al., 1994; Serafini et al., 1994; Drescher et al., 1995; Nakamoto et al., 1996; Julien et al., 2005). Interestingly, many of these guidance molecules have also been shown to be critical in accurate target selection (Cheng et al., 1995; Drescher et al., 1995; Tanaguchi et al., 1997; Brown et al., 2000; Liu et al., 2004). For example, as ciliary nerve projections approach their target, they grow across the cornea and terminate before reaching the tunica fibrosi of the lens. These axons are repelled by the inhibitory guidance cue semaphorin 3, which is expressed throughout the lens at this time (Tanaguchi et al., 1997). In mice lacking semaphorin 3 ciliary nerve terminals grow past their usual termination site and into areas of the lens, indicating its normal expression pattern slows ciliaryfibersand ensures their appropriate target selection (Tanaguchi et al., 1997). An important structure that is critical  1  for steering and responding to these cues is the growth cone. First identified over 100 years ago, it is the sensory-motor apparatus at the axon tip that directs neurite extension (Ramon y Cajal, 1937; Gunderson and Barrett, 1979).  -The Growth ConeIn vitro turning assays using cultured neurons are a common method for analyzing growth cone steering mechanisms (de la Torre et al., 1997; Hopker et al., 1999; Campbell and Holt, 2001; Gomez et al., 2003). Using this assay, growth cones have been shown to turn towards a source of an attractive guidance cue (de la Torre et al., 1997; Hopker et al, 1999). Similarly this assay has been used to turn and direct growth cones down established gradients of repulsive cues (Hopker et al., 1999; Henley and Poo, 2004; Piper et al., 2006). This turning assay has been instrumental in analyzing the intracellular molecules associated with turning. Studies focused on identifying intracellular changes involved in growth cone steering have determined that both calcium and cyclic nucleotide levels can be affected during a growth cone response (Lohof et a, 1992; Song et al., 1997; Song et al., 1998; Song and Poo, 1999; Hong et al., 2000; Peterson and Cancela, 2000; Gomez et al, 2001; Nishiyama et al., 2003). It is generally accepted that these and other second messengers elicit their function by effecting changes in the growth cone cytoskeleton through second messenger signaling mechanisms (Henley and Poo, 2004; Suter et al., 2004; Anderson, 2005; Kali! and Dent, 2005). The cytoskeleton of the growth cone is comprised of a stable microtubule core restricted to the centre of the growth cone and a peripheral actin meshwork with filament projections termed filopodia (Tennyson, 1970; Dickson, 2002; Gallo and Letourneau, 2004;  2  Gordon-Weeks, 2004). Filopodia dynamically extend and retract from the growth cone, suggestive of active sampling of guidance cues (Aletta and Greene, 1988; Heidemann et al., 1990; Halloran and Kalil, 1994; Lauffenburger and Horwitz, 1996). Experiments using the actin depolymerizing agent cytochalasin have substantiated this hypothesis by showing that filopodial presence is necessary for proper axon guidance (Bentley and Toroian-Raymond, 1986; Chien et al., 1993). Growth cones treated with cytochalasin in vivo become disoriented and make a number of misguidance errors in the developing grasshopper limb bud (Bentley and Toroian-Raymond, 1986). Similar results have been observed in vertebrates, indicating that the filopodial requirement for proper axon guidance is conserved across species (Chien et al., 1993). High resolution time lapse imaging has also noted growth cone involvement in target selection (Harris et al., 1987; Bastmeyer and O'Leary, 1996). Upon reaching target tissues axons reduce their rate of extension and obvious morphological changes in the growth cone become visible. For example, in Xenopns, measurements indicate that retinal ganglion cells (RGC's) travel at an average rate of 60 um/h as they are guided toward the optic tectum but slow to approximately 16 um/h after reaching the optic tectum (Harris et al., 1987). This change in growth rate accompanies increased arborization, as growth cones develop numerous side and back branches (Harris et al., 1987). It is thought that these changes in growth cone structure and speed reflect the increased need for the axon to distinguish accurately between signals in the environment One of the key models for studying these processes of target selection and axon guidance in vivo is the avian retinotectal system (Thanos and Mey, 2001).  3  -Avian Visual System DevelopmentDuring visual system development RGC's of the neural retina differentiate, migrate to the vitreal surface and extend axons to their target, the optic tectum (Prada et al, 1981). RGC axons terminate topographically along the two axes of the tectum; the anterior-posterior (AP) and dorsal-ventral (D-V) axis (Goldberg, 1974; Grassland et al., 1975; Thanos and Bonhoeffer, 1983; Thanos et al., 1984). This topographic mapping of retinal fibers is a crucial developmental event and is necessary for the maintenance of spatial information within the visual system (Drager and Hubel, 1975). This system then, provides an ideal model to investigate the functional mechanisms of topographic map formation.  i) Retinal Development Early in nervous system development primary optic vesicles arise as lateral outgrowths from the developing prosencephalon (Hamburger and Hamilton, 1951 ;Thanos and Mey, 2000). As they develop, these vesicles evaginate toward the dermis while remaining attached to the neural tube through the optic stalks (Hamburger and Hamilton 1951; Thanos and Mey, 2000). In later stages these stalks house the developing RGC axons that will form the optic nerve. Upon contact with the dermis, the neuroepithelium of the otic vesicles are induced to differentiate into either neural retina progenitors or the underlying retinal pigmented epithelium (Daniotti et al., 1994). The first retinal cells to terrninally differentiate are RGC's. Exiting the cell cycle at embryonic day 2 (E2), RGC's then begin migrating to the presumptive ganglion cell layer (GCL; Prada et al., 1981; Snow and Robson, 1994; Watanabe et al., 1991). Upon reaching the inner limiting membrane, RGC's form the GCL and begin extending axons toward the optic disc. Migration of other retinal cells  4  follows, creating a laminated neural epithelium (Mey and Thanos, 2000). The fully stratified retina is established by E16 and contains six layers: the GCL, the RGC nerve fiber layer, the internal plexiform layer, the internal nuclear layer, the external plexiform layer and the external nuclear layer (Ramon y Cajal, 1936; reviewed by Mey and Thanos, 2000). After migration is complete, amacrine and horizontal cell bodies are located in the inner nuclear layer and photoreceptors in the external nuclear layer. RGC axons located in the fiber layer will project to their target, the optic tectum, which develops simultaneously.  ii) Development of the Tectum The avian optic tectum is a bilobed structure arising from the dorsal mesencephalon (Hamburger and Hamilton, 1951). The major recipient of RGC fibers, it is analogous to the mammalian superior colliculus (Brown et al., 2000). Anterograde tracing studies using horseradish peroxidase have determined that the topographic mapping of RGC's is observed in mammalian as well as avian systems (Drager and Hubel, 1975). In the avian system, migration and stratification of the optic tectum occurs in a complex pattern. Beginning as a pseudostratified neuroepithelium, tectal larnination follows three waves of proliferation and migration (La Vail and Cowan, 1971b). Eight histological layers are observed as early as E8 and the complete fifteen layer tectum is established by E12 (Ramon y Cajal, 1911; Huber and Crosby, 1933; Mey and Thanos, 2000; Nakamura and Sugiyama, 2004). The most superficial layer of the tectum, the stratum opticum (SO), contains RGC axons that project from the retina. These fibers remain in the SO until E10 when RGC fibers begin to penetrate into the underlying stratum griseumfibrosum(SGFS;  5  Thanos and Mey, 2001). SGFS invasion is followed by arborization and the formation of functional synapses. Interestingly, tectal development is not uniform along both the A-P and D-V axes. Throughout development, many processes including proliferation, differentiation and migration are more advanced in the anterior and ventral regions of the tectum (La Vail and Cowan, 1971a; Cowan et al., 1975; Mey and Thanos, 2000). This developmental asymmetry is also observed during retinotectal map formation with RGC's from the central retina reaching the most advanced, anterior and ventral tectal areasfirst(La Vail and Cowan, 1971a; La Vail and Cowan, 1971b;Cowan et al., 1975). The significant influence of the retinal and tectal axes on the establishment of the retinotectal projection has prompted a number of studies focused on elucidating the mechanisms underlying axis specification in the visual system (Marin and Puelles, 1994; Martinez et al., 1991; Bally-Cuif et al., 1994).  iii) Axis Specification  The location of RGC perikarya within the retina has been shown to influence the cell's size, shape, amount of dendritic arborization as well as the target position of their axons along the tectal axes (Thanos et al., 1992). To examine when the D-V and N-T axes of the retina are established, axes have been rotated at various developmental stages. Experiments conducted up to Hamburger and Hamilton (I111) stage 11 (approximately E1.52), showed that when retinas were rotated 180°, RGC's still maintained their original tectal innervation pattern (Dutting and Meyer, 1995). These findings indicate that RGC identity is established early in development, by HH stage 11 and prior to retinal neurogenesis.  6  Investigations aimed at identifying the molecules involved in this early patterning of the retina resulted in the isolation of a number of transcription factors including chick brain factor-1 (CBF-1), chick brain factor-2 (CBF-2; Azuma et al., 2005; Takahashi et al, 2003; Schulte and Cepko, 2000). These two factors are expressed early in development as opposing gradients, CBF-1 being expressed along the nasal pole and CBF-2 along the temporal pole of the retina (Takahashi et al., 2003). Research has shown that CBF-1 and CBF-2 expression is essential for the specification of the N-T axis of the avian retina (Takahashi et al, 2005). Experiments assessing retinotectal map formation following ectopic CBF expression demonstrated that misexpression of CBF-1 and CBF-2 resulted in the abnormal mapping of RGC axons along the A-P axis of the tectum (Takahashi et al., 2003). These results indicate that the N-T axis of the retina is specified by CBF transcription factor expression and is crucial for proper RGC target selection. Recent research has determined that CBF transcription factors are required for the differential expression of a number of molecules across the retina (Takahashi et al., 2003;. Of particular interest is the CBF-1 mediated restriction of the guidance receptor EphA3 (Takahashi et al., 2003). CBF-1 expression represses CBF-2 and subsequent EphA3 protein expression in the nasal retina, enabling RGC's across the N-T axis to respond differently to ephrinA guidance cues (Takahashi et al., 2003). While the transcription factor responsible for D-V axis specification has yet to be identified, it is likely to mediate its effects through a similar mechanism effecting differential EphB protein expression (McCafferey et al., 1993; Mey at al., 1997). Although retinal identity is a critical factor in retinotectal development, targeting of retinal axons across the tectum also relies on tectal polarity. To examine the mechanism  7  underlying tectal axis specification, studies have focused on identifying the key events leading to tectal polarity. One significant factor appears to be the location of the isthmus, an identifiable tissue located at the posterior border of the tectum. Transplantation of the isthmus into the diencephalon induces ectopic expression of the tectal markers En and Wnt-1 and the subsequent differentiation into tectal tissue (Marin and Puelles, 1994; Martinez et al., 1991). More recently, experiments have determined that Fgf8 is the signaling molecule secreted from the isthmus that is responsible for the induction of En and Wnt-1 expression (Brand et al., 1996; Crossley et al., 1996; Meyers et al., 1998; Reifers et al., 1998; Martinez et al., 1999; Shamim et al., 1999). Furthermore, En has been shown to regulate the expression of the tectal guidance cues ephrin A2 and ephrin A5 (Logan et al., 1996). These findings suggest that the isthmus is an organizing centre that induces the expression of RGC guidance cues across one axis of the tectum (Freidman and O'Leary, 1996). In order to determine when this tectal polarity is established, studies have assessed retinotectal map formation following rotation of the optic tecta. A 180° rotation of the avian tectum prior to HH stage 15 (E2.5), successfully re-oriented the A-P axis resulting in normal topographic map formation (Itasaki et al., 1991). In contrast, tecta rotated at later stages retained their original axis specification causing the retinotectal map to be inverted along the A-P axis (Matsuno et al, 1991; Ichijo et al., 1990).  iv) RGC Axon GuidanceBefore RGC fibers reach their target, the optic tectum, they must first make a number of directional decisions (Goldberg and Coulombre, 1972; Snow et al., 1991; Brittis et al., 1992; Rager et al., 1992; Deiner et al., 1997; Maclennan et al., 1997). For this reason the  8  avian visual system is commonly used to study axon guidance as well as topographic map formation. RGC's located near the optic fissure in the central retina are the first to reach the GCL layer (E2-E3) and begin axonogenesis (Goldberg and Coulombre, 1972; Rager et al., 1992). As RGC axons grow they remain adjacent to the inner limiting membrane, interior to their perikarya and do not penetrate underlying retinal layers (Radius and Anderson, 1979; Thanos and Mey, 2001; Oster et al., 2004). The first fibers to arrive at the optic disc originate near the choroid fissure and enter the optic nerve head at E3 (Thanos and Bonhoeffer, 1983; Halfter, 1987). Axons from more peripheral regions of the retina grow towards and fasiculate with central fibers, arriving at the optic disc later in development (Thanos and Bonhoeffer, 1983). The inhibitory molecules preventing deviation of RGC axons into other layers of the neural retina have yet to be identified. RGC axon growth is also directed away from the lens and towards the optic disc. Studies to elucidate the mechanisms involved in directing RGC growth within the retina have identified a number of contributing guidance molecules (refer to Table 1). Chondroitin sulfate proteoglycans (CS-PG's) are expressed by neuroepithelial cells along the peripheral borders of developing RGC axons (Snow et al., 1991; Brittis et al., 1992). In vitro RGC neurites avoid areas of CS-PG expression and suggest CS-PG's act to direct RGC fibers toward the optic disc (Snow et al.,1991).  Additional experiments indicate that the  embryonic avian lens also has the capacity to repel RGC axons (Ohta et al., 1999). This guidance activity could not be attributed to the previously characterized retinal repulsive molecules Sema3 and CS-PG (Ohta et al., 1999). Furthermore, the cue netrin has been shown to attract RGC axons in vitro through the deleted in colorectal cancer receptor (DCC; Deiner et al., 1997). Immunoflourescence analysis of both avian and murine systems found  9  Table 1.1 Molecules involved in R G C axon guidance and topographic map formation Guidance Molecule Ll  Laminin  Location of expression  Effect on RGC axons  Phenotype in loss of function  Known Receptors  References  R G C axons  Fasciculation  Defasciculation  3,8,9,20  Retina  L l , TAG-1, P-Integrin, Integrins  CS-PG  Peripheral retina  Promotes growth Inhibitory  Netrin-1  O N H , optic nerve  Attractive  Ephrin-A2  Posterior tectum  Repulsive (temporal R G C ' s only)  Ephrin-A5  Caudal superior colliculus  Ephrin-A6  Nasal retina, caudal superior colliculus Dorsal tectum, optic chiasm  Repulsive (temporal R G C ' s only) Repuslive  Ephrin-Bl  Ephrin-B2  Dorsal tectum, optic chiasm  Sema3A  Tectum  Sema3D  Tectum (higher in ventral)  Sema3E Sema4A Sema5A Sema5B Sema6A  Slitl Slit2  Axon penetration into peripheral retina Optic nerve hypoplasia t arborization,, Abnormal retinotectal map Abnormal retinotectal map  2,3 ?  DCC  4,6,18,  EphAl-3  7,13,15  EphAl-5,7  15,20  EphA 1-5,7  15,20  Attractive (ventral R G C ' s only) Attractive (ventral R G C ' s only) Repulsive  Tipsilateral projections  EphB 1/2/3  15,  tipsilateral projections  EphB 1/2/3  15  Np-1, PlexinA1,A2,A3,A4, L l , VEGFR1,2  14  Repulsive  D - V tectal map defects  Repulsive Retina O N H and in optic nerve Retina Retina Optic chiasm, optic nerve Optic chiasm, optic nerve  1,10  Repulsive  Repulsive Repulsive  Wandering axons  Premature decussation Premature decussation  19 Nrp-1 Tim2  17  Plexin-B3  18  ? ?  18 18  Robol  11,16,  Robo2  11,16  References: 1. Reichardt et al. (1992); 2. Snow and Letourneau (1992); 3. Brittis et al. (1992); 4 Kennedy et al. (1994); 5. Brittis et al. (1995); 6.Deiner et al. (1997); 7.Feldheim (1998); 8.0tt et al. (1998); 9.Crossin et al. (2000); lO.Colognato and Krushel (2000); ll.Brose and Tessier-Lavigne (2000); 12. Niclou et al. (2000); 13.Wilkinson (2000); 14.Campbell et al. (2001); 15.Kn6ll and Drescher (2002); 16.Plump et al. (2002); 17.Steinbach et al. (2002); 18,Oster et al. (2003); 19.Liu et al.( 2004); 20..Inatani et al. (2005)  10  netrin to be expressed by neuroepithelial cells processes within the optic nerve head (Deiner et al., 1997; MacLennan et al., 1997). Loss of netrin expression in vivo resulted in optic nerve hypoplasia with RGC axons reaching, but not exiting the optic disc (Deiner et al., 1997). Following entrance into the optic nerve head RGC axons grow along the optic tract until they reach the optic chiasm. Spatial topography is maintained within the optic nerve during RGC axon growth. Central RGCfibersgrow along ventral stalk walls while more peripheralfibersgrow in more dorsal stalk regions (Rager and Oeynhausen, 1979, Navascues et al., 1987). Netrin-1 is expressed along the length of the optic nerve and is hypothesized to promote growth to the optic chiasm (Deiner et al., 1997; Oster et al., 2004). The adhesion molecule LI promotes fasciculation within the nerve, while expression of the repellent slits along optic nerve borders may prevent axon deviation (Erskine et al., 2000; Niclou et al., 2000; Oster et al., 2004). RGC axons start to arrive at the optic chiasm at E4 (Thanos and Bonhoeffer, 1983). Fibers then cross the midline and continue growth along the contralateral optic tract The guidance molecules Slit-1 and Slit-2 are implicated in ensuring correct timing of RGC axon decussation (Erskine et al., 2000; Plump et al., 2002).  Although single knockouts of either  slit-1 or slit-2 resulted in relatively few RGC pathfinding errors, double gene knockouts caused premature midline crossing (Erskine et al., 2000; Plump et al., 2002). Additional molecules implicated in proper chiasm formation include GAP-43, the transcription factor Zic2, EphBl, EphB-2 and the repulsive ligand Ephrin-B2 (Strittmatter et al., 1995; Kruger et al., 1998; Nakagawa et al., 2000; Walz et al., 2002; Murai and Pasquale, 2003; Williams et al., 2003; Pak et al., 2004; Inatani, 2005).  11  v) Retinotectal Map FormationFollowing decussation at the optic chiasm RGC fibers continue their growth towards the optic tectum. The first RGC axons to reach the tectum project from the central, most developed regions of the retina, arrive at the anterior tectal pole at E6 (Cowan et al., 1968; LaVail and Cowan, 1971a, LaVail and Cowan ,1971b; Thanos and Mey, 2001). Initial studies on avian visual system development determined that these RGC fibers map topographically on the optic tectum (DeLong and Coulombre, 1965; Goldberg, 1974; Crossland et al., 1975; Thanos and Bonhoeffer, 1983). These findings coincided with Sperry's early observations that 180 ° rotations of the eye within its orbit induced inverted responses to visual stimuli (Sperry, 1963). Sperry hypothesized that projections from the eye respond to chemotropic molecules differentially distributed across the optic tectum. Findings in recent years have verified this hypothesis by identifying attractive and repulsive guidance molecules involved in tectal patterning (Henkemeyer et al, 1996; Holash et al., 1997; Connor et al., 1998; Birgbauer et al., 2000). Axons of RGC's originating in temporal eye regions project to the anterior pole of the optic tectum, while nasal RGC's project to posterior tectal areas (Figure 1-1; DeLong and Coulombre, 1965; Goldberg, 1974; Crossland et al., 1975; Thanos and Bonhoeffer, 1983 ). Topographic mapping is also observed along the D- V axis, with dorsal RGC's projecting to ventral tectal areas and ventral RGC's to more dorsal regions (Pittman and Chien, 2002). Electrophoretic analysis of protein composition across retinal poles identified a novel guidance tyrosine kinase receptor expressed preferentially by temporal RGC axons (Drescher et al., 1995; McLaughlin and O'Leary, 1999). This protein, EphA3 was expressed as a  12  Figure 1-1.  13  Figure 1-1. Schematic representation of retinotectal mapping across the avian optic tectum. The retina (blue) has two, the dorso-ventral (D-V) axis and the nasal temporal (N-T) (AJB)axes. Likewise, the optic tectum (orange) has two, the anterior-posterior (A-P) and the dorsal-ventral axes (A,B)- RGC's target distinct regions of the tectum creating a topographic map along both tectal axes. RGC's located in the ventral retina project to the dorsal tectum while RGC's situated in the dorsal retina target the ventral tectum (A). In contrast, temporal RGC fires are restricted to the anterior tectum while nasal RGC axons grow through anterior and into posterior tectal regions (B). Arrows = RGC axons; 1) dorsal; V=ventral; A=anterior; P= posterior; N=nasal; T=temporal.  14  gradient along the nasal-temporal axis of the retina, with expression being highest in temporal eye regions (Cheng et al., 1995, Monschau et al., 1997; Inatani, 2005). Previous in vitro studies where retinal explants were cultured on alternating stripes of anterior and posterior tectal tissue demonstrated temporal RGC axon avoidance of posterior tectal substrates (Walter et al., 1987; Walter et al., 1990). In contrast, nasal neurites showed no obvious tissue preference (Walter et al., 1987; Walter et al., 1990). Interestingly, temporal RGC avoidance disappeared following posterior stripe heat treatment and suggested protein mediated posterior tectal targeting (Walter et al., 1987, Walter et al., 1990). Identification of the EphA receptor implicated it as a mediator of the stripe assay observations. As expected, interference with EphA signaling prevented temporal RGC axon avoidance of posterior tectal tissue stripes (Ciossek et al, 1998; Nakamoto et la., 1996). Together this data implicated the EphA protein in target selection of temporal RGC axons to the posterior optic tectum (Nakamoto et al., 1996). Later studies identified other Eph receptors differentially distributed across the retina. EphB2 is expressed in a D-V gradient with highest expression localizing to ventral RGC's axons (Henkemeyer et al, 1996; Holash et al., 1997; Connor et al., 1998; Birgbauer et al., 2000). This transmembrane protein binds two possible avian tectal ligands ephrin-Bl and ephrin-B2 (Holland et al., 1996; Braisted et al., 1997; Bruckner et al., 1997). Corresponding with receptor expression across the retina, tectal guidance cues have also been identified. Ephrin-A2 and ephrin-A5 are both transmembrane ligands that bind Eph A3 and repel RGC axons (Hornberger et al., 1999). Both ephnn-A2 and A5 are expressed in the optic tectum in an anterior to posterior gradient and prevent temporal axons from entering the posterior tectal pole (Nakamoto et al, 1996; Knoll and Drescher, 2002).  15  Immunohistochemistry has localized ephrin-A's to the superficial most cellular layer, the SGFS, during RGC innervation (Rodger et al., 2000). Located just below the developing SO, ephrin A's also function to restrict RGC axon arborization during SGFS penetration (Yates, 2001; Sakurai et al., 2002). Contributing to D-V tectal patterning is ephrin-Bl, a transmembrane protein that binds RGC EphB's to attract ventral RGC axons into the dorsal tectum (McLaughlin et al., 2003). Recent evidence also implicates ephrin B's as Afunctional molecules which like the ephrin-A's can control retinal fiber arborization in the developing tectum (McLaughlin et al., 2003). Many other guidance molecules are thought to be involved in RGC axon guidance and topographic map formation; however, these cues have yet to be identified. Recently, studies have implicated another molecule, a member of the semaphorin family in D-V patterning of the zebrafishtectum (Liu et al., 2004).  -The Semaphorin FamilyThe semaphorins are a large family of molecules implicated in cell migration, axon guidance, fasciculation, arbourisation and synapse formation (Pasterkamp and Kolodkin, 2003; Gherardi et a., 2004; Kruger et al., 2005). While the majority of semaphorins act as inhibitory or repellent molecules, permissive semaphorin cues also exist (Wong et al., 1997; Barres et al, 1999; Dent et al., 2004; Wolman et al., 2004). It appears that semaphorins typically confer their inhibitory activity through a conserved 500 amino acid sema domain (Gherardi et al., 2004). To date over twenty different semaphorin family members have been identified. Members are further classified into eight subclasses based on c-terminal  16  Figure 1-2 Invertebrate  Class  3  1  Members A a  A-F  b  4 A-G  Key:  Viral  Vertebrate 5  7  A,B  A  V A  Cytoplasmic Extracellular  5A  2a  PIxnA OTK  pixnB  PIxnBI PlxnB2 MET CD72 Tim-2  PIxnAI PlxnA2 W 'Nrp1/2 PIxnDI A PlxnA3 L1 PlxnA4 VEGFR1/2 I-M  r-v  7A  ¥ PlxnB3  • g  6D 1a,1b  • •  VA VB,  PIxnAI  PlxnCI PlxnCI  0  Sema TM ss TSR-1 IgG  Figure 1-2: Schematic representation of the Semaphorin family of guidance cues (A). All semaphorins contain a 500 amino acid domain termed the Sema domain (green). Family members are further classified into eight subclasses (1-7,V) based on c-terminal homology. Subclasses 1 and 2 are semaphorins characterized in invertebrates while class 3-7 are found in vertebrates. Secreted members are found in classes 2,3 and 7. In contrast, class 4-6 semaphorins are associated with the membrane through either GPI-linkage or transmembrane domains. ClassV denotes semaphorins identified in vaccinia or variola species. Arrows indicate known receptors (purple) for semaphorin class members. Semaphorin5B (B) is a member of the class five semaphorins, containing seven type-1 thrombospondin repeats (TSR-1; red). Antibodies (grey) used in our experiments were generated to an epitope on the N-terminus. The receptor for Sema5B is not yet known. PIxn= Plexin; Nrp= Neuropilin; Sema semaphorin domain; T M  transmembrane domain; SS= signal sequence;  TSR-1 ^ type-1 thrombospondin repeats; IgG = immunoglobulin domain.  18  homology and the presence of other structural domains (Figure 1-2 ; Nomenclature Comittee, 1999; Gherardi et al., 2004). Both secreted and cell membrane associated forms of semaphorins have been identified (Kolodkin et al., 1992; Kolodkin et al., 1993; Mathes et al., 1995;Qu et al., 2002; Xiao et al., 2003; Cafferty et al., 2006). The structural differences of these molecules permit different mechanisms of action. While secreted semaphorins diffuse away from the cell to create guidance cue gradients, those containing transmembrane domains or glycophosphatydil inositol- linkages (GPI-linked) require cell contact to elicit their responses (Kolodkin et al, 1993; Isbister et al., 2003; Kantor et al, 2004). Recently our lab has identified a previously uncharacterized transmembrane semaphorin, sema 5B, in the developing chick embryo. Class five semaphorins are characterized by the presence of seven type 1 thrombospondin repeats (TSR-1; Adams et al., 1996). Studies have demonstrated that these TSR-l's found in the class five semaphorins can serve as attractive or adhesive substrates, promoting neural outgrowth and synaptogenesis (Christopherson et al, 2005). Recent work has determined one mechanism whereby TSR-1 's can modulate classfivesemaphorin function. Research by Kantor et al. has shown that TSR-1 's can physically interact with the glycosaminoglycan of molecules such as CS-PG's and result in the conversion of the typically attractive Sema5A into an inhibitory axon guidance cue (Kantor et al., 2004). Analyses using in situ hybridization and northern blot in murine and rodent systems have identified complimentary expression patterns of SemaSA and Sema5B during embryonic development (Piischel et al., 1995; Adams et al., 1996). SemaSA mRNA is found throughout mesodermal tissues, specifically cells of the sclerotome, branchial arches, limb buds, lateral plate mesoderm, notochord as well as the developing CNS neurocpithelium (Adams et al.,  19  1996). Although little data on Sema5B expression exists, an early study suggests that expression may be confined to the developing nervous tissue (Adams et al., 1996). In neural epithelium, Sema5B was observed to be present as a gradient, with highest expression existing in rostral regions of the neural tube (Adams et al., 1996).  -Hypothesislb date the function of semaphorin 5B in development is not known. PreUminary evidence from our lab has suggested that Sema5B is expressed in the optic tectum. Since many characterized semaphorin members are involved in the process of axon guidance, we hypothesized that Sema5B may play a crucial role in RGC innervation of the optic tectum. With this thesis I hope to characterize the expression of sema5B in the avian visual system. Previous work has implicated two semaphorins in RGC guidance and retinotectal map formation in vivo. Recently, the influence of a class three secreted semaphorin in retinotectal map formation has been established. Sema3D is expressed as a gradient across the zebrafish tectum and is necessary for the guidance of RGC axons along the D-V axis (Liu et al., 2004). In addition, the class five member, Sema5A, is expressed by neuroepithelial cells along the optic nerve where it serves an ensheathing function (Oster et al., 2003). Sema5A is also expressed by oligodendroctyes and oligodendrocyte precursors (OPC) in the optic nerve and inhibits RGC axon outgrowth in later stages of development (Goldberg et al., 2004). Evidence that the class five homologue of Sema5B, Sema5A, is capable of directing RGC neurite outgrowth suggests a similar function of Sema5B. Interestingly, preliminary resultsfromour lab indicate that Sema5B is not expressed along the optic nerve but instead  20  in the avian retina and optic tectum. Confirmation of these results would provide the first evidence of a transmembrane semaphorin expressed in both components of the avian visual system, the retina and its target during periods of retinotectal map formation. This thesis will attempt to determine when and where Sema5B is expressed during avian visual system development in order to deduce how transmembrane semaphorin molecules influence neuronal axon guidance and target selection. This goal will be achieved by detailing the spatial and temporal expression pattern of the Sema5B protein in both the developing avian retina and optic tectum. In addition, Sema5B effects on RGC neurite guidance will be examined using an in vitro assay. Characterization of semaphorin 5B expression and function in the avian visual system model will suggest possible roles of Sema 5B in visual system development and provide crucial information for later in vivo analysis of function. Based on preliminary Sema5B analyses, I hypothesize that Sema5B will be expressed in the avian retina and optic tectum in areas adjacent to RGC axon development. As expression in these areas is most likely to influence retinotectal map formation, it expected that (1) Sema5B will be expressed as a gradient in both the retina and tectum. Furthermore, (2) Sema5B will act as an inhibitory guidance molecule repelling RGC neurites away from areas of expression in vitro. These findings would be the first evidence to implicate a transmembrane Semaphorin family member in both avian retinotectal guidance and map formation.  21  CHAPTER II: MATERIALS AND METHODS -Animal CareStudies were performed using White Leghorn chickens obtained from the Poultry Unit at the University of Alberta (Edmonton, Alberta). Following transport to our laboratory, some eggs were placed in an incubator where development proceeded at similar rates to those described elsewhere (Bellairs and Osmund, 1998). Additional eggs were stored at 10-12°C, in order to slow development for no longer that one week prior to use. All experiments were conducted in accordance with the Canadian Council for Animal Care guidelines and approved by University of British Columbia Animal Care Committee.  -Dissection and StagingEggs were incubated for the number of days necessary to attain the desired embryonic age. Embryos were removedfromegg, placed into Hanks Balanced Salt Solution (HBSS;Gibco Life Technologies, Grand Square N. Y., USA .) and sacrificed immediately by decapitation. Developmental ages were determined using the Hamburger and Hamilton staging system as presented in Bellairs and Osmund (1998).  -Western AnalysisThe desired tissue was dissected and homogenized on ice in a glass pestle. Undesired tissue such as the retinal pigmented epithelia, connective tissue and epidermis were removed from the neural retina, spinal cord and optic tectum prior to homogenization. Radio immunoprecipitation lysis buffer ( R1PA; 150mM NaCl, 50mM Tris pH 7.4, 1% sodiumdodecyl-sulfate (SDS), 5mM ethylenediaminetetraacetic acid (EDTA), 1% IgePalCA630, lOul/ml of aprotinin, lOul/ml of phenyl-methyl-sulfonyl-fluoride (PMSF), lul/ml of leupeptin 22  and lul/ml of pepstatin A; pH 7.4) was added to promote cell lysis and facilitate protein extraction. Homogenate was sonicated with a Branson 250 sonifier on ice at an output level or 4. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by mixing chick tissue homogenate 1:4 with Sample Buffer (0.5M Tris-HCl pH 6.8, 74mM Bromophenyl blue, 62mM sodium-dodecyl-sulfate, 0.6M 2 Mercaptoethanol, 10% glycerol). All samples were boiled at 100°C on a heating block (VWR scientific) for 5 minutes, cooled on ice and loaded onto SDS-PAGE gels (10% separating, 4% stacking). Gels were resolved at 100 volts for 2 hours. Sample protein was then transferred onto PVDF membranes (Amersham Biosciences, Pittsburg PA, U.S.A.) overnight at 4° in Transfer Buffer (20% methanol, 0.19M glycine, 25mM tris base). Following transfer, membranes were washed with tris-buffered saline (TBST; pH 7.6; 20mM tris base, 14M NaCl, 0.1% Tween-20) for 5 minutes at room temperature. Nonspecific antibody binding was minimized by incubating membranes for 30-50 minutes in a blocking solution of 5% powdered milk in TBST prior to antibody exposure. Sema 5B presence was detected using a N-terminal rabbit anti-Sema 5B antibody diluted in TBST and 5% powdered milk overnight at 4°C. Membranes were subsequently incubated with a horseradish peroxidase (HRP) conjugated donkey anti-rabbit antibody (Jackson Immunoresearch Laboratories, West Grove PA, U.S.A) (1:500 in lx TBST with 5% milk powder) for 1 hour at room temperature. Bound protein was visualized with electrogenerated cherniluminescence as per manufacturer's instructions (ECL, Amersham Biosciences, Pittsburg PA, U.S A).  -ImmunoprecipitationTissue samples were homogenized on ice using a glass pestle in 1ml of Phosphate Buffered Saline (PBS, pH7.2; 150mM NaCl, 20mM Na P0 ). Samples were sonicated and left for 1 hour 2  4  at room temperature. Tissue proteins were denatured by adding traditional western blot sample buffer (0.5M Tris-HCl pH 6.8, 74mM bromophenyl blue, 62mM sodium-dodecyl-sulfate (SDS), 23  0.6M 2 Mereaptoethanol, 10% glycerol) in a 1:4 ratio. Samples were then boiled at 100°C for 5 minutes and cooled to room temperature. Once protein was sufficiently denatured, samples were loaded into 3.5kDa dialysis tubing (Spectra Por). Pore size prevented movement of proteins greater than 3.5 kDA across the membrane. Dialysis tubing was sealed and suspended in IX PBS overnight at room temperature to prevent SDS precipitation. Following dialysis samples were concentrated using 30 kDA Millipore centrifugal filters (Amersham Biosciences, Pittsburg PA, U.S.A.). Retained protein was re-diluted into a volume of 1ml PBS. Samples were precleared with 22.8ug of Chrompure Rabbit whole Immunoglobulin G (Jackson Immuno Research Laboratories, West Grove PA, U.S. A) and 25ul of Protein G Agarose (Invitrogen, Carlsbad, CA, USA). Samples were mixed on a tabletop test tube rocker for lhour at 4°C. Antibody-protein complexes were removed by centrifugation. Samples were spun down at 13 000 RPM for 5 minutes using an Eppendorf 54151 centrifuge. Supernatants were removed and incubated overnight at 4°C with 7.5ug of a rabbit anti-Sema5B antibody and 25 pis of recombinant Protein G-Agarose (Invitrogen, Carlsbad, CA, USA). Protein-antibody complexes were pelleted using centrifugation (13000 RPM, 5minutes). The supernatant was removed, the pellet re-suspended in PBS, and sample rocked for 5 minutes before further centrifugation. To reduce sample contamination with non-specifically bound proteins this process was repeated five times. After sufficient clearance the pellet was resuspended in 45uls of western blot sample buffer and boiled for 5 minutes at 100°C. Identification of Semaphorin 5B protein presence in immunoprecipitated samples was evaluated using SDS-PAGE and electrogenerated chemiluminescence as detailed above.  -Immunoflourescence of EmbryosSacrificed embryos were immersed in paraformaldehyde (3.7% in PBS) for 3 hours at 4°C and then cryoprotected in a 20% sucrose PBS solution overnight. Tissue was flash frozen 24  in Tissue Tek optimal cutting temperature (OCT) medium (Pelco, Redding CA, U.S.A) using liquid nitrogen and stored at -20°C for 45 minutes prior to sectioning. Retinal and optic tecta were cut in either transverse or saggital sections as demonstrated in Figure HI-2A. Tissue was cut using a cryostat (Bright Instruments) into 15-20 um sections and mounted on Fisherbrand superfrost plus slides (VWR, Mississauga ON, Canada). All mounted tissue was kept at -20°C until processed. After sectioning slides were immediately placed in glass Coplin jars and washed with PBS containing 0.1% Tween 20 (PBT). Slides were treated with 2% Normal Goat Serum (NGS; Colorado Serum Company, Denver CO, USA) in PBT solution to reduce nonspecific staining Following two 5 minute PBT washes, antibody incubations were conducted in humid chambers and slides pre-treated with Liquid Blocker Super Pap Pens (Pelco, Redding CA, U.S.A ) to prevent evaporation. All primary antibody incubations were carried out overnight at 4°C. The Sema5B protein was detected using either a Rabbit anti-Sema5B antibody directed to an N-Tenninal epitope (1:1000 diluted in PBT) or a Rabbit anti-Sema 5B antibody (1:500 diluted in PBT) raised against a C-Terminal epitope. Retinal ganglion cells and post mitotic neurons were identified using an antibody (1:500 diluted in PBT) to the TUJ-1 epitope of a mouse 03-tubulin isomer (Chemicon, Temicula CA, U.S.A). Slides were washed with three exchanges of PBT and incubated for 1 hour with secondary antibodies at room temperature. Protein presence was visualized using goat anti rabbit (1:500) Cy3 (Jackson hnmunoResearch Laboratories, West Grove PA, U.S.A) and goat anti mouse (1:500) Alexa 488 (Molecular Probes, Carlsbad, CA, USA). After subsequent washes with PBT, slides were treated with antifade (4% propyl-gallate in glycerol), cover slipped (24x50mm;VWR, Mississauga ON, Canada) and sealed using generic nail polish. All slides were stored at 4°C when not in use to prevent fading of fluorescence. Immunolabelled tissue was imaged using a digital camera (Qimaging systems, McHenry II, U.S.A) mounted on a Zeiss Axioplan JJ microscope ( Carl Zeiss  25  Ltd, Toronto ON, Canada) with Northern Eclipse Software. Imunohistochemical controls (IgG or secondary antibody only) were carried out in parallel on adjacent sections.  -Dissociated cell cultureUnder sterile conditions, retinaltissuewas dissected out of the eye and separated from the retinal pigmented epithelia and vitreous humour. Dissociation protocol followed that outlined by Sanders et al.(2005). Briefly, chick retinas were transferred into HBSS containing 0.05% trypsin (Sigma) and incubated for 10 minutes in a 37°C waterbath. To stop digestion 10% Fetal Bovine Serum (FBS; Cansera, Etobicoke ON, Canada), and 1% Bovine Serum Albumin (BSA; Fisher scientific, FairlawnNJ, U.S.A.) was addedtothe HBSS/trypsin solution. Retinal tissue was dissociated using blunted glass pipettes followed by repeated aspiration with a p200 pipetteman. Following dissociation cells were centrifuged at 15 OOOrpm for 1.5 min on an Eppendorf 54151 centrifuge. The resultant pellet was washed and resuspended with 10% FBS in HBSS. Dissociated cells were seeded onto coverlips pretreated for 24 hours with 0.5mg poly-lysine-hydrobromide and 0.151 ug of mouse laminin. Cells were cultured in Dulbecco's Modified Eagles Medium (DMEM; Sigma, Oakville ON, Canada ) supplemented with 1.2% FBS, 0.01% sodium pyruvate, 1% imulm-transferrin-selenium-X (Invitrogen, Carlsbad, CA., U.S.A) and 0.1% nerve growth factor (NGF; Sigma, Oakville ON, Canada). This culture media enabled optimal retinal ganglion cell survival and outgrowth. Total culture time was approximately 48 hours. Dissociated retinal ganglion cells were processed for protein expression using immunocytochemistry as described below.  -Maintenance of Cell LinesHuman embryonic Kidney (HEK) 293 cells were transfected with a p-display plasmid containing the sema 5B gene (Invitrogen, Carlsbad, CA, USA). These experimental cells 26  expressed sema 5B protein, identifiable by a hemaglutinin (HA) tag. Controls were HEK 293 cells transfected with p-display vector alone. The p-display vector conferred both cell lines resistance to the antibiotic Geneticin. Therefore cell lines were maintained in DMEM supplemented with 1% Geneticin (Invitrogen, Carlsbad, CA, USA) to maintain transfected cell purity.  -Co-culture ExperimentsSterile 12mm coverlips (VWR, Missasauga ON, Canada) were individually placed in the wells of a polypropylene 24 well plate (Fisher, Pittsburg PA, U.S.A). Coverlips were bathed with 0.05mg poly-L-lysine hydrobromide (Sigma, Oakville, ON, Canada) and 0.161 pg of mouse laminin (Sigma, Oakville ON, Canada). After 48 hours of incubation at 37°C, the solution was removed and coverslips were washed three times with DMEM (Sigma, Oakville ON, Canada). After washing, 0.5ml of retinal ganglion cell culture media (DMEM supplemented with 1.2% fetal bovine serum (FBS; Cansera, Etobicoke, ON, Canada), 0.01% sodium pyruvate, 1% insulin-trarisferrm-selenium-X (Invitrogen, Carlsbad, CA, U.SA ) and 0.1% NGF (Sigma,Oakville, ON, Canada) was added to each well. Either experimental Sema 5B protein expressing Human embryonic Kidney (HEK) 293 or control vector only cells were suspended in DMEM and added to coverslips at similar densities White leghorn chicks E4-E10 were sacrificed and staged as outlined above. All dissections and culture procedures were conducted under sterile conditions. Tissue was dissected from the four axes of the chick eye: nasal, temporal, dorsal and ventral, as illustrated in Figure 1-1. The retinal pigmented epithelia and vitreous humour were separated from the neural retina Retinal tissue was then dissected into small pieces and placed in pre-treated wells. Explants were cultured with either 5B expressing or control cells for 24-72 hours in an incubator at 37°C with 5% carbon dioxide. 27  -Immunoflourescence of Cultured CellsAfter incubation cell culture experiments were fixed using formaldehyde (3.7% diluted in PBS) for 15 minutes. Cells were permeabilized with 0.1% Trtion-X 100 and 1% BSA in PBS. Cultures were incubated with a primary antibody for 1 hour at room temperature. For coculture experiments retinal neurites were visualized using a rabbit anti-neurofilament (1:1000) antibody (Sigma, Oakville ON, Canada). As experimental cell constructs also contained a Hemaglutinin (HA) tag on the N-terminus of Semaphorin 5B, experimental cell identity was confirmed using the mouse anti-HA (1:1500) antibody (Sigma, Oakville ON, Canada). Dissociated retinal ganglion cell cultures were immunolabelled with a tuj-1 (1:500) mouse anti P3 tubulin antibody (Chemicon, Temicula CA, U.S.A.) and a rabbit anti N-terminal Sema 5B (1:1000) antibody. After primary antibody treatment cultures underwent three 5 minute washes with PBT, followed by a 1 hour incubation with secondary antibodies at room temperature. Secondary antibodies used included goat anti rabbit (1: 1000) cy3 (Jackson ImmunoResearch Laboratories, West Grove PA, U.S.A) and goat anti mouse Alexa 488. Labelling was observed and imaged as discussed above.  -Co-culture QuantificationNeurite behaviour was assessed based on avoidance of co-culture HEK 293 cells. All analyses were conducted under double blind conditions. Each island of HEK 293 cells was assessed as being either i) contacted by neurites or ii) avoided by neurites. Islands with fewer than three cells, or islands located too far for neurites to have contacted them, were not included. A threshold of five neurites was required in order for behaviour to be considered under the contact category. The percentage of cell island contacts was determined for each individual explant .This percentage was calculated using the ratio of number of cell islands contacted over 28  the number of islands that the neurons could possibly contact. All explants for each group were averaged and are represented in Figures UI-8 as mean percent contact. Standard error of the mean was calculated for all data points and the statistical significance of data analyzed using either a student's t-test or Analysis of Variance (ANOVA) with Statview Software. StudentNeuwman Keuls post hoc analyses were conducted to ensure significance without interaction.  29  CHAPTER III: RESULTS -Semaphorin 5B Protein Expression in the Developing Optic TectumIn avian systems retinotectal map formation begins at E6 when pioneer RGC fibers reach the anterior and ventral tectal poles (Cowan et al., 1968; LaVail and Cowan, 1971a; LaVail and Cowan, 1971b; Mey and Thanos, 2000). To investigate Semaphorin 5B protein involvement in retinal ganglion cell guidance we first examined expression at E8, a time when retinotectal map formation is underway. Tectal proteins were extracted from E8 chick tecta with RIPA lysis buffer and analyzed using SDS-PAGE gel electrophoresis. Isolated proteins were probed with rabbit polyclonal antibodies generated against an N-Terminal peptide sequence unique to semaphorin 5B. The resultant blot identified a distinct band, 80 kDa's in molecular weight (Figure III-l A). Previous research in our lab has determined that Sema5B protein is also expressed in the developing avian spinal cord from E7-E11 (Wang et al., submitted). These results have been substantiated by a number of studies confirming similar expression patterns in murine and rodent systems (Adams et al., 1996; Pineda et al., 2005). As Sema5B protein expression has been repeatedly shown in the spinal cord at this age, E8 spinal tissue was selected as a positive control during western blot analysis. As expected, protein extracted from avian spinal cord also elicited an 80 kDa band, similar to that observed in tectal extracts (Figure III-l A). Primary antibody specificity was further confirmed by incubating peptide fragments containing the semaphorin 5B epitope with primary antibodies prior to western blot analysis (Figure III-IB). Pre-incubation was sufficient to eliminate the 80 kDa signal in both spinal cord and tectal tissues. Control blots probed with secondary antibodies only were also conducted and produced blots devoid of signal (Figure III-1C).  30  Figure III-l.  TECT  SC  SC  TECT  Anti Sema 5B IP / Anti Sema5B Blot  Secondary Control  31  Figure IH-1. Analysis of Sema 5B protein presence in tectal tissue. Protein extracted from E8 avian tecta (TECT) probed with an N-Terminal Sema 5B antibody identified a band at an estimated molecular weight of 80 kDa (A). Spinal cord tissue (SC) served as a positive control. Control blots (B,C) were conducted in parallel and tested antibody specificity. Pre-incubation of primary antibodies with the N-terminal epitope prior to blotting produced blots devoid of signal (B). No bands were observed in gels blotted with secondary antibodies only(C). Immunoprecipitation and subsequent blotting with the Sema5B antibody identified two protein products (D). Concentrated protein pulled down by immunoprecpitation (PELLET) contained a higher molecular weight, 130 kDa, protein. As indicated, the 80 kDa protein could not be immunoprecipitated and remained in the supernatant (SUPER). The intense signal is due to the heavy and light chains of the antibody used for immunoprecipitation.  32  Analysis of open readingframetranslational products has indicated that the full length semaphorin 5B protein is 1093 amino acids in length and has an estimated molecular weight of 110 kDa's (Adams et al., 1996). As our band was lower than expected, a more thorough analysis was carried out on E8 avian tectal tissue using immunoprecipitation to concentrate protein prior to western analysis. Precipitation and subsequent probing of tectal protein with Sema5B directed antibodies enabled the identification of an additional 130 kDa protein (Figure III-ID). Interestingly, the lower weight 80 kDa protein could not be precipitated and remained in the supernatant. Together these results indicate that the Sema5B protein is present within the tectum in a 130 kDa and 80 kDa form during the critical period of retinotectal map formation.  -Spatial Expression of Sema5B within the Developing Avian TectumConfirmation of Sema5B presence in the tectum during RGC innervation suggested a possible function in retinotectal map formation. To investigate a role for sema5B in tectal development the tectal anlagen were sectioned saggitally (Figure I1I-2A), permitting immunoflourescent analysis of expression along the A-P axis as illustrated in Figure III-2B. Examination corifirmed earlier results that Sema5B is present in the tectum at embryonic day 8 (Figure III-2C). Low level staining was observed across most developing tectal layers, with the most intensely stained tectal cells distributed in a specific pattern below the pial surface (Figure III-2C). Expression was also observed throughout the ventricular zone of the developing optic tectum as previously described (Adams et al, 1996). Immunohistochemical controls were conducted on serial sections with rabbit polyclonal IgG  33  Figure  III-2.  34  Figure UI-2. Immunohistochemical examination of Sema5B expression in the E8 avian tectum. Optic tecta were cut into 15um sections as indicated in the dorsal schematic representation (A). Tectal lobes (purple) were sectioned saggitally (dotted red line) along the anterior and posterior axis (arrow). Dotted line B illustrates a section taken from a more ventral region than section C. B is a montage of low magnification images (10X) and depicts an entire tectal section. The diencephalic ventricle (vent) as well as anterior (Ant) and posterior (Post) poles are indicated. Higher magnification (20x) images (C) of protein expression along the lateral wall of the tectum, indicated (box) in B, demonstrate broad, low level fluorescence across the developing tectum. Of interest were cells (arrow) located in a superficial tectal layer near the pial surface (Pi). These cells expressed high amounts of Sema5B. IgG immunohistochemical controls were conducted in parallel on adjacent sections and did not share this fluorescence pattern(C, 20X).  35  antibodies diluted to experimental antibody concentrations. IgG controls elicited little to no fluorescence in all sections examined, and confirmed the specificity of the observed Sema5B expression pattern (Figure III-2D). Molecular cues with known involvement in retinotectal guidance, typically elicit their function through a graded expression pattern within the optic tectum. To determine if sema5B was expressed as a gradient, expression along the A-P and D-V tectal axes were analyzed. As shown in Figures III-2A and III-3 immunofluorescence staining indicated that Sema5B is distributed differentially along the A-P and D-V axes. Saggital sections taken from ventral tectum (Figure III-3 A, C, E) showed broad Sema5B expression along the entire A-P axis while more dorsal sections had a prominent decreasing A-P gradient (Figure III-3 B,D,F). In these sections the anterior tectum displayed a broad expression pattern, with a large number of labelled cells (Figure III-3B, III-4A). Progression along the A-P axis resulted in a gradual reduction in the overall number of cells expressing Sema5B. In comparison to the anterior tectum, the posterior tectal pole contained only a thin layer of cells with Sema5B expression (compare Figure III-4B; 4C). These results indicated that semaphorin 5B is distributed in a decreasing A-P gradient during RGC innervation. Together these data suggest a complex pattern of expression across the avian tectum with SemaSB expression being highest in the anterior and ventral and weakest in the dorsal and posterior areas. The innervating RGC fibers of the stratum opticum are in close contact with and receive guidance cuesfrom,the superficial most tectal layer of cells (Mey and Thanos, 2000). To determine relative proximity of Sema5B expression to the SO layer, sections were double labelled with flourescence for Sema5B and the post-mitotic neuronal cell  36  Figure IH-3.  Figure 111-3. Immunoflourescence analysis of Sema5B expression in dorsomedial versus the ventrolateral tectum. Left panels are low magnification (10X montage; A) and high magnification (20X; C,E) images of a ventral section of the optic tectum (line C, Figure III2A). Low magnification (10X montage; B ) and high magnification (20X; D,F) images of a dorsal section are to located on the right. Sections from ventral areas of the tectum displayed broader Sema5B protein expression (anterior expression = arrows, posterior expression =  arrowheads) along the A-P axis when compared to sections from more dorsal regions ( B , line B Figure 111-2 A). Ant= anterior; Lat= lateral; Post= posterior walls of the tectum  as indicated.  38  Figure III-4.  B  ANTERIOR  POSTERIOR  -  '  ' •  * \  w  V  E  D  /  "Hi  F ' aSemaSB  \ a TUJ-1  a SemaSB 39  a tllJ-  Figure IQ-4. Comparison of Sema5B immunohistochemical staining in the anterior versus the posterior tectum. Anterior tectal staining is depicted in left panels (B,D,F), posterior to the right (C,E,G). Panel A is a montage of low magnification images with anterior (Ant) and posterior poles (Post) labelled for orientation. As indicated, anterior expression (arrow, B,D) was greater than that observed in posterior tectal regions (arrow, CJE).  Double labelling of tectal tissue with Sema5B (green) and the RGC marker TUJ-1  (red) are shown in F and G. TUJ-1 fluorescence indicated that innervating RGC fibers grew adjacent and superficial to strong Sema5B expression (D-G). More TUJ-1 positive fibers were observed in the anterior tectum (F) compared to the posterior (G). RGC fibers never penetrated past high expression into deeper tectal layers. Panels B-G were imaged at 20X magnification. V= ventricular surface.  40  marker TUJ-1. TUJ-1 antibodies are specific for type III p tubulin and have been used in avian studies to identify terminally differentiated RGC's (Pimentel, 2000; Sakagami et al., 2003). Sections fluorescently labelled with mouse TUJ-1 antibodies successfully distinguished a layer of fibers ainning along the superficial tectal surface (Figure IH-4F, G). In double labelled sections, high level Sema5B expression appeared in the layer below the TUJ-1 positive SO (Figure III-4F,G). Sema5B was largely excluded from the SO layer (Figure II1-4D, E) and TUJ-1 labelled fibers rarely penetrated past Semaphorin expressing cells into underlying tectal layers. TUJ-1 identified more fibers in the anterior tectum than in posterior regions, which is consistent with previous studies on RGC innervation (Cowan et al, 1968; LaVail and Cowan, 1971a; LaVail and Cowan, 197lb; Thanos and Mey, 2000).  -Temporal pattern of Sema5B expression in the embryonic tectum-  To examine whether Semaphorin 5B was an appropriate retinotectal guidance cue candidate, we assessed its temporal expression in the optic tectum. At E3, the earliest age examined, relatively weak and uniform expression was already present across the developing tectal neuroepithelium (Figure III-5A). At this early age, high level Sema 5B staining was not observed in superficial tectal cell layers. Beginning at E5, a clear gradient was evident with the appearance of intensely stained cells in superficial layers of the anterior tectum (FigureIII-5C, 6C). In subsequent stages superficial cell labelling progressed along the A-P axis into the lateral wall of the tectum. High level Sema5B expression was first detected in the superficial layers of the posterior tectum at E7 (FigureHl-6E). By E8 posterior expression was prominent; however, differential protein distribution was  41  Figure III-5.  42  Figure UI-5. Temporal examination of Sema5B expression in the anterior tectum. Panels display images of anterior tectum ranging from E3-E8 stages of development. Low level Sema5B protein expression was observed across the developing neuroepithelium from E3, the earliest age examined (A-F). Beginning at E5 increased labelling was observed by a subset of superficial tectal cells (arrow, C). As development proceeded superficial expression broadened with an increasing number of these cells displaying high level Sema5B expression (D-F). Images A-C were taken at 40X magnification, D-F at 20X magnification.  43  Figure III-6.  44  Figure TJI-6. Temporal examination of Sema5B expression in the posterior tectum. Low level expression across the developing neuroepithelium was observed beginning on E3 and continued throughout all ages examined (A-F). High level Sema5B protein expression (arrow) was first distinguished in the posterior tectum at E7 chickens (E). Posterior Sema5B expression was also observed in E8 optic tecta (F). Panels of early developmental stages (A-D) were imaged at 40X, later stages (E,F), at 10X and 20X magnification.  45  maintained as Sema5B expression in the anterior tectum had broadened to include a greater cell number (Figure III-5F). Development of the Sema5B gradient continued with anterior expression preceding that of the posterior until E l 2 when Sema5B protein expression dramatically declined (data not shown). Expression was no longer observable by El4. Together this data indicates that Sema5B is expressed from E3-E14, and in a graded decreasing A-P pattern beginning at E5.  -Retinal ganglion cell fibre response to Sema 5 B in vitro-  Semaphorin 5B protein presence in superficial tectal layers during E5-E12 stages of avian development supported a potential role in RGC axon guidance. In order to evaluate whether Sema5B functions as a RGC guidance cue, an in vitro guidance assay was employed. Explants were dissected from E7 avian neural retina and co-cultured with HEK 293 cells transfected with either HA tagged Sema 5B or control vector constructs. RGC neurite response was evaluated based on percentage contact with HEK 293 cell islands. An anti neurofilament antibody was used to identify growing neurites but as indicated in Figure III-7, also labelled the intermediate filaments of HEK 293 cells. Results from initial experiments indicated that RGC axons made significantly fewer contacts (p<0.001) with cells expressing Sema5B protein (mean ~ 20%, n=168) than with control cells (mean~85%, n=89; Figure II1-7A). Previous tracing studies have demonstrated RGC position within the retina detennines the location of fibre termination on the tectum (Goldberg, 1974; reviewed by Inatani, 2005). Furthermore, investigations of known RGC guidance cues have locations of origin within the retina (Walter et al., 1987; Walter et al., 1990; Rosentreter et  46  Figure III-7.  47  Figure III-7. In vitro analysis of Retinal Ganglion Cell response to Sema5B. Retinal explants were cultured in the presence of HEK 293 cells transfected with Sema5B-HA tagged construct or control plasmid only containing cells. Sema5B expression was visualized using anti HA antibodies (green) and RGC neurites with anti neurofilament (red).  Sema5B expressing cells appear yellow as they labelled with both anti-HA and anti-  neurofilament antibodies. Neurite behaviour was characterized as one of two possible phenotypes, contact (B) or avoidance (C, D). Quantification of neurite guidance showed that RGC neurites made significantly fewer (p<0.001) contacts with Sema5B expressing cells (mean ~ 20%, n=168) than with controls (mean~85%, n=89). Each bar in A represents the average percent of neurite contact (±SEM) for control and experimental groups. A student's t-test was used to compare mean percent contact between assays. Typical RGC neurite contact with control cell islands is illustrated in (B) where outgrowth is observed across cell islands. In contrast, RGC neurites cultured with experimental Sema5B expressing cells avoided islands (C) often dramatically changing neurite direction (arrow) to prevent contact (D). Scale bar is 40 um for B, C and 20 um for D. Isl= cell islands.  48  demonstrated the capacity of cues to elicit differential responsesfromRGC's with differing al, 1998). To investigate whether RGC's from distinct retinal regions responded differentially to Sema 5B,retinal explants were takenfromall four retinal poles at varying developmental stages and tested using the in vitro cell island assay described above. Quantification of mean percent contact demonstrated that RGC neurites from both nasal and temporal eye regions made statistically fewer contacts (p<0.001, F: 252, 206) with Sema5B expressing cells than with controls (Figure IIi-8A,B). ANOVA analysis determined that there was no significant difference in percent contact between nasal and temporal experiments (p<0.4225; Figure 8). As graded Sema5B expression had also been observed along the D-V axis of the tectum it was possible that dorsal and ventral RGC's would exhibit differential guidance responses in vitro. To test this, retinal explantsfromeither ventral or dorsal retinal regions were cultured with Sema5B expressing cells. Analysis of experimental and control cultures demonstrated that similar to originating along the N-T axis, neurites from both dorsal and ventral RGC's made significantly more contacts with control than experimental Sema 5B cell islands (p<0.001, F; 95.6; Figure II1-8C). No statistical difference in RGC neurite response to Sema 5B expression was observed across the D-V retinal axis as determined using ANOVA (p<0.9678, F: 0.002; Figure III-8C). Together these data indicate that retinal ganglion cells across the two retinal axes respond similarly, by making fewer contacts with cells expressing Semaphorin 5B. Further analysis using ANOVA determined that no significant difference in experimental contact number between individual developmental stages was present (p<0.225, F: 1.25; p< 0.7562 F:0.396). Interestingly, considerably fewer in an average 38% reduction in n values when compared to controls.  49  Figure III-8.  Nasal RGC Neurite Response  Control SemaSB  Age  Temporal RGC Neurite Response 100  90 30 70 o ro  60  C o o  50  Control SemaSB  40 30 20 10 0  E5  E6  E7  E8  Age  C  Dorsal and Ventral RGC Neurite Response  o ro  Cortrol  —  Sema5B  o  Dorsal  V  Position in Retina  50  Figure III-8. Temporal and spatial analysis of RGC response to Sema5B. Panels show bar plots depicting the mean percent contact of RGC neurites with either control or Sema5B expressing cells (A,B»C) throughout stages of development (A,B)- Error bars represent the calculated standard error of the mean. Explants derived from either temporal or nasal retinal poles were cultured with HEK 293 cells transfected with either HA tagged Sema5B construct or control plasmid only controls. ANOVA analysis of percent collapse determined that both nasal (A) and temporal (B) RGC neurites make significantly fewer contacts with Sema5B expressing cells than control cell islands (p<0.001, F:252,206). No significant difference was observed between developmental stages E5-E8 for retinal areas (nasal: p<0.225, F: 1.25; temporal: p<0.7562 F: 0.396). As indicated in C, RGC's from dorsal and ventral retinal regions also made significantly more contacts with control than experimental cells (p<0.001, F:95.6). No statistically noticeable difference was observed in response between RGC's originating in dorsal versus ventral poles of the retina (p<0.9678, F:0.002). Data from E7 and E8 dorsal versus ventral experiments were compiled for the plot shown in C. A l l investigations were conducted under double-blind conditions. All significant differences between groups were verified using Student-Neuman-Keuls post-hoc analyses.  51  explants adhered to coverslips in experimental conditions resulting  -Localization of Sema 5B in the developing retina-  Recent work has demonstrated that some neural populations sometimes also express a guidance cue that they can respond to. This is true for RGC's which respond to, but also express the guidance cue ephrin-A (Hornberger et al., 1999; Iwamasa et al., 1999; Marquardt et al., 2005). In the following experiments we were interested in deteimining whether RGC's capable of responding to Sema 5B also expressed this cue on its cell surface. To determine this we first conducted western analysis using polyclonal antibodies raised against the N-Terminal epitope of Sema 5B. Western analysis indicated that an 80 kDA Sema5B protein was present in the retina (Figure III-9A). Control blots were conducted in parallel using primary antibodies incubated with antigen prior to blotting and produced blots devoid of signal (Figure I1I-9B). Immunoprecipitation and subsequent blotting with a Sema5B antibody was unable to identify additional bands, indicating that Sema5B is primarily expressed within the retina as an 80kDa protein (data not shown). Immunoflourescence was then employed to determine the localization pattern of Sema5B expression within the retina. Low level Sema5B protein expression was observed across the entire width of developing retina beginning at embryonic day 3; however later ages displayed a specific high level of expression localized to the inner retinal layer. This pattern of expression was first apparent at E4 and continued until the E l 4, the oldest developmental stage examined.  52  53  Figure III-9. Investigation of Semaphorin 5B expression within the developing avian retina using western blot and immunoflourescence. Retinal protein was extracted from E8 avian eyes using RIPA lysis buffer and analyzed using SDS-PAGE gel electrophoresis. Gels were blotted with an N-terminal Sema5B and donkey anti rabbit horseradish peroxidase antibodies. Resultant blots identified a protein with a corresponding molecular weight of 80 kDa's (A). Homogenate from E8 chicken spinal cord served as positive control (SC) and also had an 80 kDA band. Control blots were conducted in parallel with the Sema5B antibody incubated with epitope prior to blotting (B). Blots were devoid of signal in both spinal cord and retinal lanes confirming antibody specificity. Protein localization within the retina was assessed using immunohistochemistry on saggital sections and are depicted in panels C-D. High Seamphorin5B expression (green) was observed along both the inner RGC (arrow) and ventricular layers of the retina (C). Double labelling for RGC specific TUJ-1 (red) confirmed Sema5B expression was within the RGC layer (D, E). High magnification imaging of TUJ-1 (G) and Sema5B (F) confirmed expression on RGC perikarya (H). Panel F-H are lOOx magnification images of the area outlined (box) in E. Panels C,D were imaged at 10X, E at 50X magnification.  54  To determine which cells within the retina were expressing Sema5B, sections were double labelled with the retinal ganglion cell marker TUJ-1 and Sema5B (Figure III-9C,D) Results revealed high level Sema5B protein expression co-localized with TUJ-1 staining. Observation at high magnification further confirmed that Sema5B was indeed expressed by RGC's in the developing avian retina. Interestingly, TUJ-1 positive staining of the retinal fibre layer did not co-localize with Sema5B expression (Figure III-10C,D). To help elucidate if Sema5B was differentially localized along individual RGC's, immunoflourescence was conducted on dissociated cultures. Results revealed robust Sema5B expression on RGC perikarya; however, fluorescence dropped dramatically at the axon hillock and was not observed along developing neurites (Figure 111-10). Double labelling with TUJ-1 confirmed RGC identity and visualization of developing neurites. Together this in vitro data corroborated in vivo findings that Sema5B is expressed on RGC cell bodies but not on RGC axons during RGC guidance.  55  F i g u r e  1 1 1 - 1 0  A  B  /  •  / •  a Sema5B  a TU  a Sema5B  D  a SemaSB a TUJ-1  56  f  Figure 111-10. Analysis of Sema5B protein expression on developing RGC fibers. Immunoflourescence staining was conducted on dissociated retinal cells obtained from E8 chickens. High level Sema5B expression (green) was observed on RGC cell bodies (arrowhead, A).  RGC identity was confirmed with the RGC specific marker TUJ-1 (red,  C). As indicated in panel A, Sema5B expression was not observed along developing neurites (arrows) identified by TUJ-1 in B. In vitro results were verified in vivo using immunoflourescence on 15um saggital sections. Observation at high magnification concluded that high Sema5B expression (C) was not present along axons (arrow) of the developing retinal fibre layer as identified with TUJ-1 staining (D). AU panels imaged at 100X magnification  57  C H A P T E R  I V :  D I S C U S S I O N  This study analyzed the expression pattern of a class five semaphorin, Sema5B, in the developing avian visual system. Previous research has implicated semaphorins in a number of developmental processes including axon guidance (Pasterkamp and Kolodkin, 2003; Gherardi et a., 2004; Kruger et al., 2005). Preliminary datafromour lab indicated that this novel transmembrane semaphorin, Sema5B, was expressed in the developing avian optic tectum. Based on these findings and the expression pattern of other guidance cues involved in visual system development, I hypothesized that Sema5B would be expressed in both the retina and its target, the optic tectum. Furthermore,  I predicted that expression would  localize to regions adjacent to growing retinal ganglion cell axons,  enabling Sema5B to  influence RGC axon guidance and target selection. Since the majority of semaphorins have been shown to be inhibitory in nature, I also hypothesized that Sema5B will act as a repulsive guidance molecule and repel growing R G C axons away from areas of expression.  The resultsfromthis study demonstrate that Semaphorin 5B is expressed in both the retina and the optic tectum during the stages of retinotectal map formation. In both regions expression was adjacent to growing retinal ganglion cellfibresand suggests possible Sema5B involvement in RGC axon guidance. Within the tectum, Sema5B expression was highest in superficial cell layers, adjacent to the developing stratum opticum. Furthermore, Sema5B expression was graded along both the A-P and D-V tectal axes and corresponds with the known tectal development pattern. Interestingly, Sema5B was also expressed by RGC's; however, expression was limited to perikarya and was not observed along developing fibres in vitro or in vivo. In addition, functional analyses using a co-culture guidance assay 58  deterrnined that RGC's from all four poles of the retina are repelled away from areas of Sema5B expression. Overall these results show that Sema5B is an inhibitory RGC guidance cue expressed in areas adjacent to growing RGC axons and suggest likely involvement in RGC guidance and target selection. This is the first evidence of transmembrane semaphorin involvement in RGC guidance and arborisation within the avian tectum.  -Implications of Sema 5B expression in the avian tectuin-  Preliminary in situ hybridization analyses conducted in our lab had indicated that Sema5B was expressed in the developing optic tectum. Using western analysis and immunoflourescence, I have confirmed these results and provided a further description of the Sema5B pattern of expression in the tectum. Western analysis with an anti-semaphorin 5B antibody revealed a strong 80 kDa band present in the developing tissue of the tectum. As earlier investigations conducted in our lab had confirmed Sema5B expression in the developing avian spinal cord, spinal tissue was used a positive control during western analysis. Semaphorin 5B mRNA expression in the spinal cord has also been demonstrated in the rodent (Adams et al, 1996; Sakliora et al., 1998). Gels blotted with the sema5B antibody successfully identified the 80 kDa band in both spinal and tectal tissue. Not surprisingly, analysis with the sema5B antibody indicated that this semaphorin member is expressed in an 80 kDa form in both the tectum and the spinal cord. Control gels with either secondary antibodies only or primary sema5B antibodies incubated with antigen containing peptide fragments, were devoid of signal and further confirmed the specificity of our results.  59  Previous analysis has determined that the Sema 5B protein is 1093 amino acids in length (Adams et al., 1996). Based on these results, the expected molecular weight of Sema5B is 110 kDA's. This data conflicted with our findings which indicated that in the tectum this molecule is present in a lower molecular weight, 80 kDA form. These findings indicate that Sema5B may be processed through proteolytic cleavage resulting in the generation of different Sema5B protein products. Work published by the Piischel lab has shown that a secreted semaphorin member, Sema3 is synthesized as an inactive precursor and becomes repulsive through fiirin mediated proteolytic cleavage (Adams et al., 1997). In order to determine if higher molecular weight forms were present in the tectum but expressed at lower levels, we employed immunoprecipitation prior to western analysis. Analysis of precipitated proteins identified an additional band having an estimated molecular weight of approximately 130 kDa's. This data suggests Sema 5B is expressed in the developing tectum in at least two forms, 130 and 80 kDa products, during the time of RGC fibre innervation. These findings are supported by work in our lab which has found HEK 293 cells transfected with full length Sema5B constructs also expressl30 kDA and 80 kDA products. Interestingly, the lower molecular weight, 80kDA., product has been isolated in cell culture supernatant and provides additional evidence of Sema5B proteolysis. Similar to other Semaphorin3A we hypothesize that Sema5B might confer different activities through proteolytic processing (Adams et al., 1997). In addition, research from our lab demonstrated different functions conferred by different Sema5B domains. While HEK 293 cells transfected with the sema domain repelled sensory and RGC neurites, cells expressing the TSR-1 acted as attractive substrates for neurite outgrowth (Wang, unpublished data). This substantiates our hypothesis and provides a possible mechanism whereby cells can cleave  60  Sema5B to generate diffusible molecules containing fewer TSR-1's, and therefore greater repulsive activity. Remaining transmembrane products may have other roles in neuronal development such as adhesion and synaptogenesis. Although putative cleavage sites have been identified, further investigation is needed to elucidate the mechanisms underlying Sema5B cleavage. Immunohistochemical analysis of the spatial distribution of Sema5B in the developing tectum confirmed the western blot results. Low level expression was observed across the entire width of the developing tectum at all ages examined, E3- E12. This expression corresponded with previous data showing class five semaphorin members localized to the neuroepithelium of CNS structures during neural development (Adams et al., 1996). The function of Sema5B in these areas has yet to be determined; however proposed hypotheses suggest Sema5B may direct the emigration of postmitotic neuroblasts from the ventricular zone or prevent penetration of misdirected axons into areas of expression (Adams et al., 1996). Beginning at E5, increased Sema5B expression appeared in the superficial most cell layers in the anterior and ventral regions of the tectum. Interestingly, this pattern of expression corresponds with existing data on tectal development. Developmental processes including cell proliferation, migration and stratification occur asymmetrically across the avian tectum (La Vail and Cowan, 1971a; Cowan et al., 1975; Mey and Thanos, 2000). Ventro-lateral and anterior tectal development precedes that occurring in more dorso-medial and posterior areas (La Vail and Cowan, 1971a; Cowan et al., 1975; Mey and Thanos, 2000). Therefore, Sema5B expression is observed first in the most advanced areas of the tectum and corresponds with the spatial pattern of tectal development. As migration is occurring in the  61  tectum at this time, it is likely that Sema5B appearance corresponds with the arrival of migrating neurons at the superficial tectal cell surface. Between E4-E8 tectal cells are migrating to form two distinct laminae. Cells migrating to the inner tectal surface form the stratum griseum centrale (SGC) while cells reaching the superficial surface will eventually form the SGFS layers a-g (reviewed by Mey and Thanos, 2000). Before lamination is completed around El6, the superficial cellular layer is denoted as layer IV (Sugiyama and Nakamura, 2003). Therefore Sema5B is expressed by cells of the transient layer IV which will eventually form the SGFS in later developmental stages. Temporal analysis revealed that in later stages, E6-E8, superficial Sema5B expression continues to extend further along the A-P and D-V axes. Expression was first observed in the superficial cell layers of the posterior tectal pole around E7. This timeline corresponds with the generation of layer IV in the posterior tectum. As semaphorin expression appears in more dorsal and posterior regions, the existing expression in the anterior tectum broadens. This increased anterior expression maintains the A-P gradient, as anterior expression remains greater that that observed in posterior regions. Since migration is continuing in the anterior tectum these results indicate that newly arriving cells, which will later form the SGFS layers e-a also express Sema5B. Altogether this data indicates that Sema5B is expressed as an apparent gradient along the A-P and D-V axes. Furthermore this expression localizes to cells of the presumptive SGFS lamina and the appearance of Sema5B protein likely corresponds with the arrival of SGFS cells. To confirm that Sema5B expression indeed localized to the superficial layers of the tectum, the postmitotic neural cell marker TUJ-1 was used to verify the presence of RGC axons. Previous studies have confirmed that TUJ-1 is specific for RGC's at this time and  62  was therefore used to visualize the developing stratum opticum (Pimentel, 2000; Sakagami et al., 2003). Double labelling indicated that high level Sema5B expression localized directly below the TUJ-1 positive SO. These results indicate that Sema5B is expressed in regions of the tectum adjacent to innervating RGC axons. Interestingly, other guidance cues involved in retinotectal map formation are also expressed in SGFS layers during RGC innervation (Rodger et al., 2000; Sakurai et al, 2002). For example, the repellent cue ephrinA2 is expressed in the SGFS of the goldfish tectum and has been shown to be involved in retinotectal map reformation following optic nerve injury (Rodger et al., 2000). Therefore these results indicate Sema5B expression within the tectum localizes to an area adjacent to growing RGC axons and is likely to influence their pathfinding and target selection. Higher magnification images further revealed that Sema5B was not expressed in the SO and that TUJ-1 labelled axons rarely penetrated past Sema5B. These results corroborate the majority of findings that RGC fibres do not penetrate into tectal cell layers until E10 (Crossland et al., 1974; Thanos and Mey, 2000). Penetration of RGC axons into underlying lamina is followed by synapse formation within SGFS layers. Studies have shown the involvement of repellent molecules in the restriction of RGC fibre arborisation during initial innervation (Sakurai et al., 2002; McLaughlin et al, 2003). One such cue is ephrinA5, which is expressed as a gradient within the avian optic tectum during SO formation (Cheng et al., 1995; Sakurai et al., 2002). Chromophore assisted laser inactivation of ephrinA5 function in the avian tectum resulted in increased RGC axon arborisation into underlying SGFS layers (Sakurai et al., 2002). Therefore it is possible that Sema5B may provide a repellent activity directly below RGC axons to prevent their premature arborisation. This hypothesis is further  63  supported by our findings that Sema5B expression in SGFS layers has decreased significantly by El2, when RGC axons are penetrating into underlying tectal cell layers. -Implications of Sema5B in topographic mapping across the tectum-  The Sema5B gradient appears across the tectum beginning on E5 and continues until E l 1. Initial expression in the superficial tectal layers is observed at E5 and is limited to the anterior and ventral poles at this time. This differential expression establishes a graded distribution along both the A-P and D-V axes. To date guidance cues have been implicated in topographic mapping of RGC fibres along these two tectal axes. The repellent ephrinA2 and eprhinAS's are expressed differentially along the A-P axes and limit temporal axons to the anterior region of the tectum (Cheng et al., 1995; Nakamoto et al., 1996). In contrast, ventral RGC axons are directed away from ventral tectal areas by semaphorin 3D expression (Liu et al., 2004). Simultaneously, ephrin B expression in the dorsal tectum attracts these fibers into the dorso-lateral regions (Mann et al., 2002; McLaughlin et al., 2003). As gradients of both semaphorin and ephrin guidance cues have been shown to direct retinotectal map formation it is possible from our data that Sema5B could also be involved in this process. As early tracing studies have determined that the first RGC's fibres reach the anterior pole of the tectum at E6, Sema5B may selectively repel nasal axons into the posterior tectum. Likewise, high Sema5B expression in the ventro-lateral tectum may preferentially direct ventral RGC axons to the dorso-medial tectum. Our data is similar to the temporal expression pattern of other membrane associated inhibitory molecules such as ephrinA5, which is expressed in the avian tectum beginning on E2 (Cheng et al., 1995). Furthermore, an apparent gradient of this guidance cue was evident along the A-P axisfromstages E4-E8  64  Figure IV-1  ^  SO SGFS  Tectum  Retina  Figure IV-1. Schematic representation of Sema5B expression in the developing avian retina and tectum. In the retina Sema5B (green circles, Retina) is expressed by RGC perikarya located within the GCL, but are not by axons in the fibre layer. As our in vitro results demonstrate that Sema5B repels RGC axons, Sema5B may act as an inhibitory boundary preventing growing neurites in the fibre layer from penetrating into underlying retinal laminae. Sema5B expression in the tectum may serve a similar function in the tectum where the protein is expressed by cells in the putative SGFS layer. Here Sema5B expression (green circles, Tectum) would prevent RGC axons, projecting from the retina,frompenetrating into underlying tectal layers prematurely.  SO= stratum opticum, SGFS= stratum griseum  fibrosum.  65  (Cheng et al., 1995). As this inhibitory cue has been implicated in the topographic map formation of RGC axons as well as in restricting arborisation during RGC innervation, it is possible that Sema5B may also function in a similar manner (Cheng et al., 1995; Nakamoto et al., 1996; Yates et al., 2001; Sakurai et al., 2002). Further in vivo and in vitro investigations will be needed to determine this.  -Implications of Sema5B repulsion of RGC neurites in vitro-  To date the majority of semaphorins have been shown to influence axon guidance through their inhibitory function (Fujisawa et al., 1998; Kolodkin, 1998; Goshima et al. 2000; Nakamura et al., 2000; Dent et al., 2004; Wolman et al., 2004; Huber et al., 2005). For example, in mice Sema3B has been shown to prevent posterior pars fibres from deviating off their normal trajectory, enabling the proper formation of the anterior commisure (Julien et al., 2005). In addition, semaphorin 4E acts as a repellent to facial and gill motor axons, preventing extending fibres from entering into epithelial tissue (Xiao et al., 2003). Many semaphorins are expressed in the developing avian nervous system and have appear to have redundant roles to ensure proper pathfinding (Luo et al., 1993; Luo et al., 1995; Feiner et al., 1997; Shepard et al., 1997;Steinbach et al., 2002; Steffensky et al., 2006). Our observations of expression in the avian optic tectum indicated that Sema5B may act as a cue to direct axon growth and target selection. As another transmembrane class five semaphorin, Sema5A, has been shown to repel RGC axons in vitro we hypothesized that Sema5B would elicit a similar response (Goldberg et al, 2004). In order to test this, RGC explants were co-cultured with HEK 293 cells transfected with Sema5B-HA tagged constructs. Based on our hypothesis we expected to observe RGC neurite avoidance of cell islands expressing Sema5B. As expected,  66  RGC neurites made significantly fewer contacts with cells expressing Sema5B than with control, plasmid only containing cells. Together this data indicated that Sema5B is an inhibitory RGC guidance cue. As our observations indicated that Sema5B was expressed as a gradient across the tectum, it was likely that RGC's from discrete retinal regions would respond differently to Sema5B. To test this, retinal explants from all four poles of the eye, nasal, temporal dorsal and ventral, were cultured with Sema5B expressing cells. Surprisingly, no significant difference in response to Sema5B was observed across the retinal axes. Explants from all four retinal poles responded similarly by showing statistically fewer contacts with Sema5B expressing cells. These findings contradict in vitro characterizations of other guidance molecules. For example, temporal and not nasal RGC axons express the ephrinA-5 and ephrinA-2 receptor EphA, enabling the preferential repulsion of temporal axons out of posterior tectal regions (Cheng et al., 1995; Nakamoto et al., 1996). Similarly, the differential distribution of the ephrinB receptor, EphB, enables the directed growth of ventral retinal axons toward the attractive ephrinB cue in dorsal regions of the tectum (Mann et al., 2002; McLaughlin et al., 2003). Our results are the first to document an inhibitory guidance cue expressed differentially across the tectum that elicits a uniform response from all RGC populations. These observations suggest that all RGC axons express the putative Sema5B receptor and indicate that tectal Sema5B may function primarily as an inhibitory barrier to all RGC axons. Such a structure would prevent penetration of growing RGC fibres into underlying layers until the appropriate stage of development. Another possible hypothesis is that robust Sema5B expression in the first areas of the tectum to receive RGC axons may act to slow fibres as they enter the optic tectum, enabling more accurate target selection.  67  In order to ensure that spatial differences in RGC response didn't arise earlier in development, explantsfromE4-E9 avian retinas were co-cultured with Sema5B expressing HEK 293 cells. Again, we were surprised by our results as no significant differences in response to Sema5B were noticeable across the D-V and N-T axes of the retina. Altogether our data indicates that RGC's from all retinal regions during E4-E9, the stages of retinotectal innervation, are repelled by Sema5B. It should be noted that explantsfromE4 and E9 retinas were difficult to adhere to coverslips and were therefore not analyzed in depth. In addition it was apparent that in all cell experiments fewer explants adhered to coverslips containing Sema5B expressing cells, indicating that Sema5B may prevent neuronal-substrate adhesion interactions.  -Implications of Sema5B in the developing avian retina-  Based on the observed inhibitory response of RGC axons to Sema5B in vitro, we wondered whether Sem5B could be involved in directing RGC axon guidance within the retina. To determine this, Sema5B expression within the developing retina was investigated using western analysis and immunohistochemistry. Western analysis with Sema5B antibodies detected an 80 kDA band in retinal tissue extracts, similar to that observed in both tectal and spinal cord tissues. Surprisingly, immunoprecipitation was unable to identify a 130 kDa form similar to that found in the optic tectum. These results indicate that Sema5B protein is found predominately in a lower molecular weight 80 kDa form within the avian retina. Immunohistochemical analysis also confirmed Sema5B expression within the retina Similar to the tectum, Sema5B expression was observed along the entire retinal  68  neuroepimelium. Again supporting previous work on class five semaphorins, Sema5B expression was high along the ventricular surface of the retina (Adams et al., 1996). These findings support earlier hypotheses of class five semaphorin involvement in promoting emigration of neural cells out of the ventricular layer of nervous tissue. Of particular interest was high level Sema5B expression observed along the inner, vitreal surface of the retina. To determine which retinal lamina Sema5B localized to, we double labelled sections with Sema5B and the post-mitotic neural cell marker TUJ-1, specific for RGC's at this time (Pimentel, 2000; Sakagami et al., 2003). Results revealed that Sema5B was expressed in the GCL, adjacent and just deep to the developing inner fibre layer (IFL). Interestingly, analysis at higher magnification detennined that Sema5B and TUJ-1 expression overlapped, confirming that Sema5B is expressed by RGC cells. However, Sema5B expression was not observed in the IFL indicating that Sema5B may not be expressed on RGC axons during axonogenesis. These findings corroborated with our earlier results that Sema5B was not present in the developing RGC fibre layer (SO) of the optic tectum. To investigate the localization of Sema5B on RGC's, immunocytochemical analysis was conducted on dissociated retinal cultures. Retinal ganglion cells were identified as TUJ1 positive cells with long processes. As expected, Sema5B expression was high on RGC perikarya but was not observed along axons. These findings substantiate earlier results that Sema5B is expressed on RGC perikarya located in the GCL of the retina. This expression is adjacent to developing RGC axons, which do not express Sema5B. This pattern of expression suggests Sema5B may function to prevent penetration of RGC axons into underlying layers of the retina, enabling RGC axon guidance to the optic disc. Further  69  research will need to be carried out to determine the exact function of Sema5B protein in vivo. Although guidance cues have been shown to be preferentially expressed by specific neuronal populations, this is the first known example of the cellular compartmentalization of a molecule influencing axon guidance (Redies et al., 1992; Cheng et al., 1995; Nakamoto et al., 1996; Eberhart et al., 2002). Therefore, we find the mechanism underlying the cue's spatial restriction intriguing. As we expect this process to likely be integral in its function, future experiments will also focus on elucidating how RGC's target Sema5B specifically to their cell bodies and not their growing axons.  -Final RemarksThis study has provided the first characterization of Sema5B in the developing visual system. Sema5B was found to be present in both the developing avian retina and optic tectum in regions adjacent to growing RGC axons. In vitro analysis confirmed speculation that Sema5B was an inhibitory guidance molecule and indicates that Sema5B in both the retina and tectum likely functions by creating an inhibitory barrier to prevent retinal fibres from straying into underlying layers. Further studies will focus on testing Sema5B function in vivo through ectopic expression of Sema5B in areas of RGC growth including the SO or the posterior or dorsal regions of tectum. In addition, loss of function experiments will be conducted using either RNAi or morpholino induced knockdown of Sema5B in the anterior and lateral regions of the tectum. If Sema5B is involved in keeping RGC axons within the IFL and SO we would expect to see increased arborisation in Sema5B's absence.  70  R E F E R E N C E S  Adams RH, Betz H, Puschel AW. 1996. 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