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Neurodevelopmental characterization of semaphorin 5B in C57Black mice Lett, Robyn Lynn Mwuese 2003

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N E U R O D E V E L O P M E N T A L CHARACTERIZATION OF SEMAPHORIN 5B IN C57BLACK MICE by R O B Y N L Y N N MWUESE LETT B.Sc. Carleton University, 2001 A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Anatomy and Cell Biology; Programme in Neurosciences) We accept this thesis as conforming /to the required standard/7 THE UNIVERSITY OF BRITISH C O L U M B I A October 2003 © Robyn Lynn Mwuese Lett, 2003 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date Abstract The mammalian nervous system develops through five recognized stages: induction, differentiation, migration/axon guidance, synaptogenesis and pruning/apoptosis. Guidance cues, generally thought to be responsible for guiding axons through the developing organism, have been identified as playing additional roles in differentiation, directed cell migration, synaptogenesis, as well as pruning and apoptosis. Semaphorin 5B (Sema5B) is a member of the semaphorin family of guidance molecules; therefore, it is hypothesized that it is expressed in the developing nervous system of mice. This thesis has investigated the developmental expression pattern of Sema5B in C57Black mice by in situ hybridization, which demonstrated expression of Sema5B m R N A in four specific regions of focus: the spinal cord and associated dorsal root ganglia, the eye, the olfactory bulb and epithelium and the dorsal forebrain. In the spinal cord, Sema5B undergoes dynamic changes between E12 and E l 8 that are consistent with a potential role in the formation of laminar-specific targeting of sensory neurons within the dorsal horn. In the eye, expression of Sema5B is specific to the lens epithelium and the retinal ganglion cell (RGC) layer, which implies a role in either R G C differentiation, migration, or in the guidance of R G C axons to the optic disk. Expression of Sema5B by olfactory receptor neurons at E l 8 implies that expression may continue postnatally, thus indicating a potential role in the maintenance of the regenerative nature of the adult olfactory epithelium. Finally, expression of Sema5B in the ventricular zone and the cortical plate as it develops denotes a possible function of Sema5B in the differentiation and/or migration of neurons in the developing mouse forebrain. i i i Table of Contents Abstract i i Table of Contents i i i List of Figures v Acknowledgements v i Chapter 1: Introduction and Background 1 Nervous system development and the establishment of connectivity 1 The growth cone 2 Theories of axon guidance mechanisms 3 Guidance cues 6 Functional properties of guidance cues 8 The semaphorin family of guidance cues 9 Invertebrate semaphorins 13 Secreted vertebrate semaphorins 14 Transmembrane vertebrate semaphorins 15 Semaphorin receptors 17 Class 5 semaphorins 18 Objectives: Describing semaphorin 5B expression in mice 20 Chapter 2: Methodology 21 Animals and Care 21 Isolation of Embryonic Tissue and its Preparation 21 Non-Isotopic In Situ Hybridization 22 Prehybridization 22 Digoxygenin-labelled R N A Probe Synthesis 22 Dot Blot 23 Hybridization and Post-hybridization 24 Immunological Detection of Digoxygenin-UTP 24 Niss l Staining of Tissue Sections 25 Data Collection 25 iv Chapter 3: Results 26 Sequence and Structure of Murine Semaphorin 5B 26 Location of Probe Hybridization 26 Results of Dot Blots and Gels 26 Expression pattern of semaphorin 5B m R N A 29 Semaphorin 5B expression within the mouse spinal cord and dorsal root ganglion 30 Semaphorin 5B expression within the mouse eye 36 Semaphorin 5B expression within the mouse olfactory system 41 Semaphorin 5B expression within the mouse forebrain 46 Chapter 4: Discussion 51 Functional implications of semaphorin 5B structural domains 52 Guidance cues are associated with diverse developmental processes 53 Semaphorin 5B functions as an axon guidance cue in the spinal cord and retina of the chick 54 Potential role of semaphorin 5B in axon guidance within the mouse spinal cord 55 Potential roles for semaphorin 5B in axon guidance;within the developing mouse eye 58 Semaphorin 5B may serve an important role in cortical neuron migration 61 Semaphorin 5B may play a role in the guidance of mouse olfactory bulb axons 64 Potential role for semaphorin 5B in the mouse olfactory epithelium 65 Chapter 5: Summary 66 References 67 Appendix A 81 List of Figures Figure 1: The semaphorins are a family of guidance molecules 11 Figure 2: Semaphorin 5B probe alignment and probe analysis 27 Figure 3: In situ hybridization of semaphorin 5B in the mouse spinal cord and dorsal root ganglion 31 Figure 4: Comparing Sema5B labelling and Niss l staining of the E l 8 ventral spinal cord 33 Figure 5: In situ hybridization semaphorin 5B in the retina and lens o f the mouse eye... 37 Figure 6: Niss l staining and Sema5B expression in the E l 8 mouse retina 39 Figure 7: In situ hybridization of semaphorin 5B in the mouse olfactory bulb and epithelium 42 Figure 8: Comparing of mSema5B labelling with Nissl staining of E l 8 O B and O E 44 Figure 9: In situ hybridization of semaphorin 5B in the developing mouse cortex 47 Figure 10: Comparison of E l 8 cortex labelled for mSema5B with Nissl-stained E l 8 cortex 49 VI Acknowledgements I would first and foremost like to thank Dr. T im O'Connor for inviting me into his lab. I sincerely appreciate the patience you have shown, the beer you have bought and the conversations we have had over the last two years. I greatly anticipate my future work in your lab under your guidance. I would like to thank my committee (Dr. Jane Roskams, Dr. Vanessa Au ld , Dr. W o l f Tetzlaff and Dr. T i m O'Connor) for the time they have put into my degree progress, and for being wil l ing to bend a few rules to keep me on track. I would especially like to thank Dr. W o l f Tetzlaff for all his time and effort spent teaching me many of the techniques I needed or w i l l need, and allowing me the use of his lab space. I also would like to thank the members of his lab for putting up with me, especially Jei for helping me with my surgeries, Clarrie for caring for my mice and everything else, and Egidio for doing for me what I couldn't bear to do myself. Thank you to Dr. Victor Viau , though not on my committee, has been a source of good advice and along with his technician Patricia, taught me how to perform radioactive in situ hybridizations, which unfortunately did not make it into this thesis. Thanks to everyone in the O'Connor lab; your friendships, support, advice and tolerance have helped this become my new home, and are a large part of what made me want to stick around for a few more years. Finally I want to thank my family. I love you all and I wouldn't be here without you. 1 Chapter 1: Introduction and Background The mammalian nervous system constitutes a remarkably complex network of connections amongst neurons, and between neurons and other cell types, established during development with relatively few errors. B y describing and investigating the rules governing development, this knowledge can be applied to scenarios where nervous system development does not proceed properly resulting in such neurological disorders as epilepsy or schizophrenia (Malas et al. 2003; Miyoshi et al. 2003; Sheen and Walsh 2003), or when damage occurs as in the case of spinal cord injury, neurodegenerative disorders and stroke (Bothwell and Giniger 2003; Dawbarn and Allen 2003), or to simply improve our understanding for such processes as learning and memory (Craver 2003; Klass et al. 2003). This thesis describes the spatial and temporal expression of semaphorin 5B in the developing nervous system of C57Black mice. Based on this expression pattern, I propose that semaphorin 5B is involved in the differentiation, migration and axon guidance of neurons in the developing nervous system. Nervous System Development and the Establishment of Connectivity The development of the nervous system can be described in five defined stages: induction, differentiation, cell migration and axon guidance, synaptogenesis, and pruning/apoptosis. Induction and differentiation are necessary for the production of the cells that compose the nervous system. A x o n guidance, synaptogenesis, and pruning/apoptosis are crucial steps leading to the ultimate functional outcome, as they are the processes governing wiring of the nervous system (Frank and Wenner 1993). 2 The viability of an organism necessitates a properly functioning nervous system that produces appropriate behaviours in response to various stimuli. Therefore, establishment of neural connections during development must be consistently precise and accurate. Axons often traverse long distances in the developing organism, past many potential though erroneous targets in order to reach their final correct target. Santiago Ramon y Cajal, the 19 t h century Spanish anatomist, was the first to propose that neurons reach targets and form functional connections by sensing the environment with a structure at the tips of their axons, which he called the growth cone. The Growth Cone A n important step of neuronal differentiation is the generation of an axon. The axon is a membranous process that is structurally supported by microtubules and intermediate filaments. The tip of the growing axon (or neurite, as it is referred to when axonal or dendritic identity has not yet been determined) terminates with a specialized structure called the growth cone, an expanded portion of the neurite rich in filamentous (F) actin. The growth cone is characterized by long thin individual protrusions called filopodia^ which rapidly extend and retract (Jay 2000), and are bridged by wide flat ruffle-like membrane extensions called lamellipodia (Condeelis 1993). Two forms of actin cytoskeletal architecture underlie these two structures: filopodia consist of relatively long F-actin filaments and lamellipodia are formed by a dense meshwork of shorter actin filaments (Lewis and Bridgman 1992). At the core of the growth cone are bundles of tightly packed microtubules that w i l l occasionally project into the filopodia (Cheng and Reese 1985). 3 The cytoskeletal framework of microtubules and actin in particular, underlies the intrinsic motility of the growth cone (Forscher and Smith 1988; L i n et al. 1994). Highly concentrated receptor proteins enable the growth cone to sample the surrounding environment with its rapidly extending and retracting filopodia (Condeelis 1993). A wide variety of cues are available in the environment to interact with the receptors on the growth cone, which transduce information into signals that rearrange the cytoskeleton, ultimately guiding the direction of outgrowth. Environmental cues can be grouped into those that are attractive to the growth cone and stimulate axon extension (Tessier-Lavigne et al. 1988), and those repel the growth cone causing collapse of the growth cone's internal cytoskeletal architecture (Fan et al. 1993), retraction, or partial collapse that causes the growth cone to split or turn (Kal i l et al. 2000). The growth cone thus guides the growing axon to its final target (Lander 1990; Doe and Sanes 2000; Keynes and Cook 2001; Dickson 2003; Lett and O'Connor 2003) with which it w i l l establish permanent, functional connections, called synapses. Theories of Axon Guidance Mechanisms It was first believed that the final pattern of connections within the nervous system was entirely based upon functional properties; connections were made at random with any tissue or cell encountered by the outgrowing axon (Weiss 1939). It was proposed that the massive arbourization this would entail would be later pruned by function, such that only neurons and targets with matching resonant properties - where "resonance" was determined by the type of action potential produced and conducted by a given neuron - would maintain a connection, and all others would be degraded (Weiss 1935,1936). In the 1950s and 60s, Roger Sperry countered the resonance theory with the proposal that the nervous system did not establish connections in a stochastic haphazard fashion. He found that axons of the retina made their way to the contralateral tectum in an organized and precise manner, bypassing other potential yet erroneous targets (Sperry 1943, 1963). This was demonstrated through regeneration experiments in the retinal-tectal systems of amphibians and fish. In these experiments, the entire optic nerve was removed, cells of specific portion of the retina were destroyed, and axons of the remaining retinal neurons were allowed to regenerate. The pattern of re-irrnervation in the tectum was found to be a complete topographical representation of the retina (retinotopic map) within the contralateral tectum (Sperry 1963). In addition, this topographically dependent pattern of innervation was maintained subsequent to the severing of the optic nerve and 180 b rotation of the eyeball (Sperry 1943). From these two very basic but elegant experiments, Sperry concluded that neural connections were not made at random, but that a co-ordinate system of gradients, widely available in the extracellular space, directed growth cones to their appropriate locations. He also proposed that each neuron and its target had identical co-ordinates imposed by these gradients, thus enabling axons to properly identify the target neuron(s) with which they were supposed to form connections. Although identification of the final correct target is crucial for the development of neural specificity, other processes must exist to guide growth cones to the correct pathway leading to that target. Examinations of appropriate pathway selection by neurons in developing insects lead to the proposal of the labelled pathways hypothesis (Goodman et al. 1982). This hypothesis states that growth cones choose amongst, and follow along, a given pre-existing axon tract based upon specific differentially expressed surface tags. The grasshopper Schistocerca americana was used to illustrate this principle. In these experiments they showed that a cell that differentiates from the ectoderm to become a neuronal precursor divides to form two sister neurons. The projecting axons of these neurons then choose to jo in different tracts along the ventral midline. The authors proposed that the choice was based upon differing labels upon the tracts and some type of system of recognition that had been differentially distributed during the division of the two neurons. Thus, specificity of neural connectivity and development appears to be determined by how a growth cone chooses the appropriate pathway (Raper et al. 1983). Labeled pathways were thus assumed to guide the direction of axon outgrowth by creating permissive routes (Goodman et al. 1982); however, there must be a mechanism that inhibits axon growth into undesirable regions, driving it instead towards the permissive regions/pathways. One of the first examples of growth cone inhibition occurred when Kapfhammer and Raper demonstrated that sympathetic and retinal neurites could maintain exclusive territories through mutual inhibition (Kapfhammer et al. 1986). This form of inhibition was called collapse, as it involved a complete loss of the internal cytoskeletal structure of the growth cone. They showed that collapse did not occur due to modulation o f growth cone/substrate adhesion, rather by the active retraction of the growth cone away from the contacted inhibitory substrate, in this case another type of neurite (Kapfhammer and Raper 1987a). These activities were proposed to be involved with segregating central from peripheral nervous systems, and made arguments in support of Goodman's labelled lines hypothesis (Kapfhammer and Raper 1987b). This was one of the first indications that labels could be non-permissive as wel l as permissive. 6 Although the labels along pre-existing tracts are helpful for axons that are generated later during the development of a particular pathway, guidance must also occur in the absence of these labels. Pioneer axons are the first axons to extend into a given area of the embryonic environment (Bate, 1974) and must pathfmd without the benefit of previously existing labeled tracts. It has been proposed that complex axon pathfmding must be accomplished in a step-by-step process called segmental pathfinding; each segment of the pathway is dealt with individually as a single targeting event. An example of segmental pathfinding in vertebrates occurs in the establishment of the spinothalamic tract by commissural axons in the developing spinal cord (Lett and O'Connor 2003). The cell bodies of these neurons reside in the dorsal lateral portion of the spinal cord and they project an axon ventrally within the spinal cord grey matter toward the floorplate at the ventral midline, their first intermediate target. From there, they must cross the floorplate and then contact and fasciculate with the appropriate ascending axon tract, their second intermediate target. Once this contact is made, these axons extend along the spinothalamic tract until they reach the thalamus, their final target where synaptic contacts are formed. The intermediate targets along this trajectory are referred to as guideposts, and represent points where growth cones must "decide" through sampling of the environment to continue their journey to the final target (O'Connor, 1999). Guidance Cues Theories and observations of axon guidance made over the last century have attempted to explain how the intricate connections of the nervous system form. Each theory and/or observation has been explained by the existence of molecular guidance cues that are responsible for guiding 7 growth cones and thus the growth trajectory of extending axons ((Kennedy et al. 1994; Frisen et al. 1998; Isbister and O'Connor 2000; Zou et al. 2000; Nguyen et al. 2002)). Guidance cues are secreted or membrane-associated proteins, generated by many different cell types (included those o f non-neuronal origin). The action of guidance cues is mediated by their specific receptors located on growth cones which stimulate axon behaviours such as attraction, repulsion, branching, turning and collapse (Kal i l et al. 2000). These guidance molecules are regulated in their expression by specified tissues throughout development, and often into adulthood as well , and thus exert intricate control of axon growth in both a spatial and a temporal fashion (Yu and Bargmann 2001), as well as controlling synaptogenesis, plasticity, regeneration and disease states in adult organisms (Kageyama and Robertson 1993; Bo lz et al. 1996; Super et al. 1998; Gavazzi 2001; Pasterkamp and Verhaagen 2001; Yamamoto et al. 2002; Bagri et al. 2003; Bothwell and Giniger 2003; Dawbarn and Al l en 2003; Malas et al. 2003; Miyoshi et al. 2003). In recent years the numerous axon guidance cues have been identified, some of which belong to extremely large protein families. O f the all the different families, there, are four which are most prevalent: ephrins, netrins, slits, and semaphorins. The ephrins, in conjunction with their receptors the EPHs, are responsible for the retinotopic layout in the tectum of amphibians described by Roger Sperry (Frisen et al. 1998; Kullander and K l e i n 2002). Therefore the chemoaffinity of specific retinal axons for specific portions of the tectum can be explained in part by the differential expression of the EPFf/ephrins within that system. Netrin is involved with segmental pathfinding displayed by commissural axons of the spinal cord. It was found to be 8 secreted from the floor plate cells in the developing spinal cord (Kennedy et al. 1994) and was shown to be attractive to outgrowing commissural axons that had yet to cross the midline. There are also inhibitory cues, slits and semaphorins, produced by the floor plate and surrounding grey matter to which the commissural axons acquire responsiveness, that are responsible for forcing these growth cones off the floorplate and towards the correct white matter tract (Zou et al. 2000; Dickson 2003). Functional Properties of Guidance Cues Guidance cues are often described as attractive and inhibitory, though many which were originally thought to be repulsive molecules have in some cases been shown to exert attractive effects upon growth cones. The exerted effects of netrin and semaphorins have been shown to be reversed based upon intracellular levels of cyclic nucleotides that mediate the downstream effects within the growth cone (Song et al. 1998; Polleux et al. 2000). It is therefore perhaps presumptuous to definitively label any cue as either attractive or repulsive; it is more appropriate to define a cue as inhibitory or attractive to a particular growth cone in a given scenario. Guidance cues can be further defined by their range of action. Cues may be secreted, or membrane-bound, as stated earlier, which determines whether they may exert short range (discrete) or long range (diffuse) effects. Secreted factors may be important for establishing gradients of guidance information. Isbister et al (2002) have proposed that the concentration of a secreted factor at any given point may affect the size of the growth cone, but the magnitude at any given point along the gradient is irrelevant for guidance. They propose that the slope of the gradient or the fractional change in concentration, rather than absolute change, is potentially 9 important for determining the cue's guidance effects. Thus, the effects of a secreted factor are "long" range, as it diffuses away from the site of secretion into regions where it is not expressed. Membrane-bound cues are by nature of short range action. They are restricted to movement within the plane of the plasma membrane of the cell by which they are expressed. In order for effects to be exerted by short-range cues, there must be contact occurring between the exploring filopodia of a growth cone and the surface of the expressing cell (Kapfhammer and Raper 1987a, b). The Semaphorin Family of Guidance Cues The family of semaphorin guidance molecules is delineated by a highly conserved 500 amino acid "semaphorin" domain that contains 14 (vertebrate) to 16 (invertebrate) conserved cysteine residues (Kolodkin et al. 1993). The semaphorin family is comprised of 8 classes, and over 20 members have been identified to date (Fig. 1); invertebrates exclusively express classes 1 and 2, as well as a novel class 5 member (Khare et al. 2000; Bahri et al. 2001), 3-7 are vertebrate-specific, and class 8 is viral (Semaphorin-Nomenclature-Committee 1999). Classes 2, 3 and V are secreted, whereas class 1 and 4-6 are transmembrane, and class 7 is membrane-associated through a GPI-linkage ( X u 1998; Yamada 1999).1 ' As per the Semaphorin Nomenclature Cornmittee (1999), all rodent semaphorins are abbreviated as Sema, followed by the class number and capitalized letter of the subclass (e.g. Sema3C). Semaphorins of other vertebrate origin, including humans, are abbreviated as SEMA, followed by the class number and capitalized subclass letter (e.g. SEMA3C). Invertebrate semaphorins are abbreviated as sema, followed by class number and lowercase subclass letter (e.g. semala). Often the organism of origin is denoted in a one letter prefix to the specific semaphorin name (e.g. m-Sema3C, for mouse, or g-semala for grasshopper). This format will be followed throughout this thesis. 10 Figure 1: The semaphorins are a family of guidance molecules. The semaphorin family is distinguished by a -500 amino acid "semaphorin" domain. Members are grouped into classes based upon organism of expression and domain specializations, and are secreted, GPI-linked or transmembrane. Class 1 and 2 members are exclusively expressed in invertebrates. Class 1 semaphorins are transmembrane, and class 2 semaphorins are secreted, and contain an immunoglobulin (Ig) domain C terminal to the semaphorin domain. Classes 3-7 are expressed in vertebrates, with one exception. Class 3 members are secreted, and contain a C terminal Ig domain and terminate in a basic tail. Class 4 molecules also contain an Ig domain, and are transmembrane. Class 5 members contain a unique domain among semaphorins, bearing seven-thrombospondin-type-l-like repeats, and is also transmembrane. Class 6 semaphorins are transmembrane with many intracellular binding domains. Class 7 semaphorins are GPI-linked and class V semaphorins are virally expressed and are secreted. 11 1 2 3 4 5 6 7 V £ Immunoglobulin Domain • Thrombospondin Type-1-Like Repeat Hi Plexin-Semaphorin-lntegrin (PSI) Domain Intervening Sequences and Minor Domains Semaphorin Domain Signal Sequence Basic Domain 12 Semaphorins are members of a superfamily of molecules, which consists also of the semaphorin receptors, the plexins, and scatter factor receptors (Maestrini et al. 1996; Artigiani et al. 1999; Springer 1999). Interestingly, scatter factors receptors - the ligands o f which are the M E T proto-oncogene family - are also involved in cell migration/guidance, mediating cell-cell dissociation, and in pathological cases, metastasis (Artigiani et al. 1999). Each of these families contains a semaphorin domain as well as a MET-related cysteine-rich sequence located C terminal to the sema domain 2, which is now referred to as the PSI domain (for "plexin, semaphorin and integrin"; (Kolodkin and Ginty 1997)). A n example of its function, the PSI domain in the a(32 integrin appears to be integral to inactivation of its ligand binding capabilities (Zang and Springer 2001), and may serve a similar function among plexins and semaphorins. The semaphorin domain has been shown to confer specific biological activity and binding patterns for each subclass, whereas the C terminal domains are thought to enhance this activity rather than directly contribute to it ((Koppel et al. 1997) among others). It is the collapsing activity of semaphorins which is thought to be a component of the molecular inhibition of central nervous regeneration, particularly subsequent to spinal cord injury (Pasterkamp et al. 1998; Pasterkamp et al. 1999; Goshima et al. 2000; Gavazzi 2001; Pasterkamp et al. 2001; Pasterkamp and Verhaagen 2001). However, what differentiates each class o f semaphorins, other than organism of origin, is the region C terminal to the conserved semaphorin domain. It may be these regions of individuality that permit functions differing from that of collapse and/or defasciculation (a more attenuated form of repulsion) originally proposed by Kolodkin et al in 1993. 2 One exception to this are the two class V semaphorins, which in addition to containing truncated semaphorin domains, also lack the PSI domain typical of the abovementioned molecular families Springer T. A. (1999) Domains in plexins: links to integrins and transcription factors. Trends in Biological Science 24, 261-263.. 13 Recent evidence suggests that the C terminus of semaphorins may confer activity. This is an especially relevant concept in terms of transmembrane semaphorins; class 5 semaphorins bear thrombospondin type-1 like repeats, and class 4 and 6 semaphorins are endowed with larger cytoplasmic domains that would allow these molecules to have inverse signalling capabilities that the secreted class 3 semaphorins do not possess. Various members of the semaphorin family have been shown or proposed to be involved in processes such as tumour suppression (Sekido et al. 1996; Xiang et al. 1996; Takahashi et al. 1997), immunological functions such as T cell proliferation and activation (Mine 2000; Giordano et al. 2002; Kumanogoh et al. 2002), as well as apoptosis or programmed cell death (Gagliardini and Fankhauser 1999; Shirvan et al. 1999). Invertebrate Semaphorins Class 1 and 2 semaphorins have been characterized in the nematode worm, Caenorhabditis elegans, the fruit fly, Drosophila melanogaster, and are particularly wel l defined in the limb of the grasshopper, Schistocerca americana. It was in the grasshopper that fasciclin IV, the prototypical semaphorin, was discovered (Kolodkin et al. 1992). It was originally grouped as a member of the fasciclin family of cell adhesion molecules. In the grasshopper, peripheral neurons are born peripherally and project centrally. The pair of T i l neurons, so called because they are the first neurons born from the tibial portion of the limb bud, extend axons proximally towards the central nervous system due to a gradient of a secreted chemorepellent, g-sema2a, concentrated distally and dorsally (Isbister et al. 1999). T i l neurons encounter a band of cells expressing the transmembrane g-semala at the trochanter, where they turn and follow this band ventrally until they reach an intermediate target, the C x i guidepost 14 cells, that allows them to turn and continue proximally towards the central nervous system (Bonner and O'Connor 2000). It was determined that semala had chemoattractive properties, differing from the general conception of repulsion as semaphorin function (Kolodkin et al. 1993; Isbister et al. 1999; Wong et al. 1999). Vertebrate Secreted Semaphorins Nearly all of what is known and understood about semaphorins comes from work within the class 3 family members. This class of semaphorins is secreted and has an immunoglobulin domain followed by a basic tail C terminal to the semaphorin domain. The prototypic vertebrate semaphorin was originally called collapsin, found in the developing chick nervous system. It was demonstrated to inhibit growth cones and cause total collapse of their internal structure (Luo et al. 1993)). After initial isolation of collapsin, further investigation yielded an entire family of related molecules, all o f which were seemingly specific to the developing nervous system (Kolodkin et al. 1993; Messersmith et al. 1995; Puschel et al. 1995). Secreted semaphorins are synthesized as pro-proteins, and require cleavage by the proteolytic enzyme furin (Adams et al. 1997) and dimerization in order to be functional (Klostermann et al. 1998; Koppel and Raper 1998) . Sema 3 A (the mammalian homologue of collapsin-1, formerly SemD) and chick S E M A 3 A (as collapsin-1 is now called) were found to be short-range guidance cues integral for appropriate central and peripheral pathway projections and terminations formed by nociceptive (TrkA-positive, NGF-responsive) dorsal root ganglion cells (Messersmith et al. 1995; Puschel et al. 1995; Wright et al. 1995; Puschel et al. 1996; Shepherd et al. 1996; Keynes et al. 1997; 15 Kitsukawa et al. 1997; Fu et al. 2000; Dontchev and Letourneau 2002; Pond et al. 2002). However, although Sema3A is important for maintaining the ventral spinal cord as an inhibitory domain, evidence from the knockout (Behar et al. 1996) shows that it is not acting alone and other factors must be important in ensuring that each set of sensory afferents connects appropriately within a given lamina of the dorsal spinal cord (Behar et al. 1996; Catalano et al. 1998; Sanes and Yamagata 1999). Secreted semaphorins are involved with the guidance of many central nervous system pathways. Sema 3 A , 3C, 3E and 3F guide the development of the hippocampal formation (Steup et al. 2000; Pozas et al. 2001). Sema3A is integral for the correct orientation o f cortical neurons, exerting attraction from apical dendrites and repulsion from axons (Polleux et al. 2000). In concert with Sema3C and 3F, Sema3A is also implicated in the establishment cortical projections (Bagnard 1998; Bagnard 2000; Bagnard 2001) and cortical neuron migration (Tamamaki et al. 2003). Additionally, Sema3A in involved in the guidance of migrating olfactory receptor neurons (Williams-Hogarth et al. 2000) and their projecting axons (Schwarting et al. 2000), while Sema3B and 3F are respectively attractive and repulsive toward olfactory bulb axons (de Castro et al. 1999). Although secreted semaphorins are typically thought to inhibit axon growth, there are occasions where they may serve as chemoattractants to specific subsets of neurons (Bagnard et al. 1998; Bagnard et al. 2000; Polleux et al. 2000; Castellani et al. 2002). Transmembrane Vertebrate Semaphorins Only recently has attention been given to vertebrate transmembrane semaphorins, in particular classes 4 and 6. Members of the 4 and 6 classes have also been found to be repulsive to select 16 groups of neurons though the roles of transmembrane semaphorins appear to be diverse. There are several reports of intracellular interactions of transmembrane class 4 and 6 semaphorins with anchoring proteins, and arguments for the physiological relevance o f these interactions are repeatedly made for retrograde signaling or synaptogenesis (Eckhardt et al. 1997; Encinas et al. 1999; Kikuchi et al. 1999; Wang et al. 1999; Klostermann et al. 2000; Schultze et al. 2001). Class 4 semaphorins are characterized by an extracellular immunoglobulin domain that lies between the semaphorin domain and the transmembrane domain, as wel l as a variety of proline-rich intracellular domains. The cytoplasmic domain of Sema4C (first described in (Inagaki et al. 1995) as m-SemF) contains a consensus sequence for interaction with SH3 domains and cytoskeletal proteins. In the nervous system it has been found to interact with a class I P D Z protein called M-SemF cytoplasmic domain-associated protein ( S E M C A P - 1 ) . S E M C A P is thought to regulate subcellular localization of Sema4C by binding and forming detergent-insoluble aggregates (Wang et al. 1999). Sema4D is also known as C D 100, and though it is present in nervous tissue at low levels, it is expressed predominantly on lymphocytes and in lymphoid tissue of the immune system (Furuyama et al. 1996). Intracellularly, Sema4F has been found to co-localize with synapsin-1, and with SAP90/PSD-95 in dendrites, both of which occur through a conserved C terminal PDZ-binding motif (Encinas et al. 1999; Schultze et al. 2001). Class 6 semaphorins have been described less than members of class 4. N o additional domains or motifs in the region C-terminal to the semaphorin domain characterize them. They have much longer intracellular domains than other transmembrane semaphorins, and it has been postulated that these semaphorins engage in retrograde signaling, and might therefore be important in cell-17 cell signalling (Eckhardt et al. 1997; Zhou et al. 1997). Sema6A has been found to repel sympathetic but not sensory neurites in vitro, and in vivo to be greatly involved in maintenance of a fasciculated sympathetic chain between ganglia and white rami exit points (Xu et al. 2000). Sema6A-l is 59% homologous to Sema6A extracellularly, but greatly diverges with a zyxin-like intracellular domain found to interact with the enabled/vasodilator-stimulated phosphoprotein-like protein ( E V L ) . E V L contains consensus sequences for binding the G-actin-associated protein profilin, focal adhesion proteins, as well as SH3 domains of Src kinase and Abelson kinase (Klostermann et al. 2000). Sema 6B dimerizes and binds SH3 domains of the proto-oncogene, c-Src (Eckhardt et al. 1997). Sema6C has two splice alternates and shares an intracellular short consensus sequence with Sema6A. Sema6C is widely expressed in both embryonic and adult rat C N S , and is capable of collapsing D R G growth cones in vitro (Kikuchi. etal. 1999). Semaphorin Receptors There are five known types of receptors involved with transduction of semaphorin signaling; the plexin family are the predominant receptors, while neuropilin (Chen et al. 1998; Rohm et al. 2000; Gavazzi 2001), L I (Castellani et al. 2002), offtrack (Winberg et al. 2001), and the M E T (Giordano et al. 2002) receptor also act as semaphorin co-receptors in specific contexts and in conjunction with plexins. Only two forms of the vertebrate-exclusive neuropilin (abbreviated NP-1 and NP-2) exist (Takagi et al. 1991; Takagi et al. 1995; Feiner et al. 1997; He and Tessier-Lavigne 1997; Kolodkin et al. 1997; Chen et al. 1998; Giger et al. 1998; Renzi et al. 1999). Neuropilins do not transduce the semaphorin signal directly; they serve as the receptor, and through their interaction with plexin permit transduction of the signal (Rohm et al. 2000). In 18 addition, neuropilins are exclusively bound by members of the semaphorin class 3 (Feiner et al. 1997; Chen et al. 1998), whereas the plexins appear to serve as the predominant receptor for all semaphorins. There is great diversity of plexins in both vertebrates and invertebrates (Winberg et al. 1998; Tamagnone et al. 1999; Cheng et al. 2001; Fujii et al. 2002; Suto et al. 2003). The plexin family is thought to be evolutionarily older than, and perhaps the point of origin for, the semaphorin family (Winberg et al. 1998). Plexins were discovered in 1987 (Takagi et al. 1987); however, not until a protein distantly related to the plexins (virus-encoded semaphorin protein receptor -V E S P R ) was determined to be the receptor for the viral semaphorins (Comeau et al. 1998) were plexins thought to have a role as receptors for other semaphorins. Drosophila plexinA is predominantly expressed in the nervous system, and when knocked out displayed the same loss-of-function phenotype as Drosophila-semaphorm l a knockouts. Further investigation determined that plexinA binds specifically to d-semala, thus providing the first conclusive evidence that plexins were semaphorin receptors (Winberg et al. 1998). Since this initial study, it has been found that a variety of plexins function as the receptors for class 4, 7, and viral semaphorins (Comeau et al, 1998; Tamagnone et al, 1999). The exact receptor type for some of the semaphorins - class 5 and 6 specifically - is presently unknown. Class 5 Semaphorins Interestingly, there has been little investigation of transmembrane class 5 semaphorins, which are differentiated from other semaphorins by a unique domain of seven thrombospondin type-1-like repeats (partially conserved to the thrombospondin type-1 repeats or TSRs; C S X X C G instead of 19 C S V T C G ) between the semaphorin domain and the transmembrane domain. This is an intriguing feature, as thrombospondin molecules are growth factors that enhance cell-survival and create a permissive environment for neuritic extension (Adams and Tucker 2000). The Piischel group cloned the semaphorins 5 A and 5B in mouse (Adams et al. 1996), and have briefly examined the embryonic expression patterns in mice and rats in relation to the expression of the secreted semaphorins. The semaphorin domains of the two family members are 64% identical, and when compared to other semaphorin classes are most highly homologous (60-66%) to the invertebrate semal group (Adams et al. 1996). Sema5A is expressed by retinal ganglion cell axons along the optic nerve and optic disk, and has been shown that its disruption by antibodies results in defasciculation of the retinal axon bundle and errant pathfinding (Oster et al. 2003). Recently, a transmembrane semaphorin containing thrombospondin and thrombospondin-like repeats has been discovered in the Drosophila genome, and as such was called sema5c (Khare et al. 2000; Bahri et al. 2001). N o specific function of d-sema5c has been shown, but mutant analysis thus far demonstrates that it is not essential for fly development (Bahri et al. 2001). 20 Objectives: Describing Semaphorin 5B Expression in Mice Semaphorin 5B is relatively unknown in terms of its developmental expression pattern, and no attempt has been made to assess its potential functions in the establishment of the nervous system of mice. This thesis w i l l describe in detail the spatial and temporal expression pattern of murine semaphorin 5B in four specific regions of the nervous system: spinal cord and dorsal root ganglia, eye, olfactory system and forebrain. This thesis w i l l demonstrate that semaphorin 5B m R N A expression correlates spatially and temporally with the penetration of sensory axons into the developing spinal cord, thereby potentially serving a complementary role to the ventral inhibition provided by Sema3A by controlling the establishment of lamina-specific connections within the dorsal horn. The expression of Sema5B within the developing cortex correlates with the advent of nervous cell proliferation, differentiation and migration that establish the inside-out patterning of the cortical plate and therefore the ultimate determination of specific neuronal identities within the neocortex. Sema5B is also expressed in discrete regions of retina and lens within the developing eye and w i l l be discussed as having a potential role in the differentiation, migration and axon guidance of retinal ganglion cells. 21 Chapter 2: Methods Animals and Care A l l mice were strain C57Black, obtained from Charles River Laboratories. M i c e were housed in a 12-hour dark-light cycle with ab libidum access to food and water. Pregnant mice were caged in groups of a maximum of four. Isolation of Embryonic Tissue and its Preparation Pregnant mice were injected intraperitoneally with O. lmL 2.5% chlorohydrate. Once anesthesia was confirmed by absent response to toe or tail pinch, the chest wall was opened and a cut was made in the right atrium. To perfuse, the left ventricle was injected with lOmL of 20mM P B S , followed by 20mL 4% paraformaldehyde in P B S . After perfusion, the abdominal wall was opened and the uterus was completely exposed. Embryos were singly removed from their individual amnions and the umbilical cords cut. Once isolated, embryos were washed in 2 0 m M P B S and placed in 4% paraformaldehyde overnight at 4C for post-fixation. A t stages E l 4 or E l 8 the nervous system and its boney encasements were dissected out to ensure better post-fixation, whereas stage E l 2 embryos were not dissected prior to post-fixation. For sectioning, the head was separated from the spinal column. The spinal column was cleaned of skin and muscle and the cervical and thoracic regions were removed. Following post-fixation, all tissue samples were placed in 30% sucrose to cryoprotect the tissues, and stored at 4 C until sectioning. A l l sectioning of the head was performed in the coronal orientation, and all spinal column sections were made in the transverse direction. Samples cooled to -20 C, mounted on sectioning blocks using O C T embedding material (TissueTek) and cut into 40um sections, which were placed 12-15 per slide on Perma-frost ( V W R ) slides. Sections were dried at room temperature. Subsequent 22 processing differed depending upon the procedure to be used. Sections to be used for non-isotopic in situ hybridization were doubly sealed, baked at 40 C for at least 1 hour and stored at -80 until used. Sections were equilibrated to room temperature for one hour prior to in situ hybridization. Non-Isotopic In Situ Hybridization A l l steps took place at room temperature unless otherwise stated. A l l prehybridization solutions were prepared with diethylpyrocarbonate-treated water ( D E P C , 0.1% - Sigma). Sections stored at -80 were warmed to room temperature for one hour and the sections circled with a wax P A P pen. Prehybridization Sections were incubated in two changes of l x P B S - D E P C for 5 minutes each, followed by two 5 minute incubations in lOOmM glycine in l x P B S - D E P C (NaCl , KC1 , N a 2 H P 0 4 , K H 2 P 0 4 ) and one 15 minute wash in l x P B T ( l x P B S - D E P C , 0.3% Triton X-100). Sections were washed 2x5 minutes in l x P B S - D E P C , permeabilized for 10-12 minutes with 5pg/mL proteinase K (Sigma) in T E buffer ( lOOmM Tris, 5 0 m M E D T A , p H 8.0) in a 37 C shaking water bath. Sections were fixed for 5 minutes with 4% paraformaldehyde in l x P B S - D E P C (pH 7.0) at 4 C and washed 2x5 minutes in l x P B S - D E P C . Sections were acetylated 2x5 minutes in lOOmM triethanolamine (TEA) containing 0.25% v/v acetic anhydride (Fisher Scientific). Digoxygenin-labeled RNA Probe Synthesis The plasmid p B K - C M V containing murine Sema5B sequence was kindly provided by Dr. A . Puschel. P C R primers (Invitrogen) were designed to the last 390 nucleotides, and the P C R product was subcloned into the p - G E M - T vector (Chemicon), all o f which was performed by Dr. W. Wang. The subcloned fragment was sequenced by N A P S ( U B C ) and verified using Blastn and Vector N T I Suite 7. Miniprep and maxiprep D N A isolations were performed using kits from Qiagen, and all plasmids were amplified using D H 5 a transformation-competent E.coli bacteria. The concentration o f the final maxiprep of p - G E M - T N-mSema5B was 0.635[xg/uL. Two R N A probes were used for in situ hybridizations: the sense and antisense transcripts of the C terminal fragment of mSema5B that was subcloned into p - G E M - T (Promega). The plasmid was linearized with restriction enzymes from New England Biolabs (BamHI for antisense, NotI for sense). Restriction enzymes were used at 3u/10uL of plasmid. Digoxygenin-labeled probes were made using the D I G - R N A labeling kit (Roche Biochemical), and the final probes were cleaned using the R N A clean-up protocol from the RNeasy kit (Qiagen). A l l probes were eluted with autoclaved ddH20 . Probe concentrations were verified by photospectrometer (Eppendorf), and size was estimated by agarose gel (1%). Dot Blot Labeling efficiency was verified by dot blot. R N A probes and R N A controls (obtained from probe synthesis kit) were diluted at 1:100, 1:1000 and 1:10,000 in a mixture of formaldehyde, 20x standard sodium citrate (SSC) and distilled water (volume ratio 2:3:5). l u L of each dilution was dotted on a nitrocellulose membrane ( E C L HyBond) and crosslinked by U V light on a transilluminator for 30 minutes. Blots were developed using the DIG-luminescence detection kit (Roche Biochemical). R N A probes were stored at -80°C. 24 Hybridization and Post-Hybridization Sections were prehybridized at 55°C for 30 minutes with hybridization buffer (DIG-EasyHyb from Roche Biochemical, or 40% deionized formamide, 10% dextran sulfate, l x Denhardt's solution, 4x SSC, l O m M D T T , lOmg/mL RNase free bovine serum albumin - Fisher Scientific) plus lmg/mL yeast t R N A (Roche Biochemical) and l m g / m L salmon sperm D N A (Sigma; denatured for 20 minutes at 65°C and sheared with a 27 gauge needle). The prehybridization solution was replaced with a mixture of the same solution containing l p g / m L of DIG-labeled riboprobe. Probes were boiled for 10 minutes at 100°C and cooled on ice for 1 minute prior to their addition to the hybridization solution. Slides were covered with parafilm and hybridization proceeded in a humid chamber (maintained with 5x SSC) at 55°C overnight. Slides were placed in 2x SSC at 50-55°C until parafilm floated off. They were washed at 50-55°C for 2x15 minutes in 2x S S X and 2x15 minutes in l x SSC. Immunological Detection of Digoxygenin-UTP Sections were washed 2x10 minutes in buffer 1 ( lOOmM Tris, 150mM N a C l , p H 7.5), then blocked for 30 minutes with buffer 1 plus 0.1% Triton X-100 and 2% normal sheep serum (Jackson Laboratories). The blocking solution was decanted and sections were incubated overnight at 4.C in antibody solution (buffer 1, 0.1% Triton X-100, 1% normal sheep serum, 1:5000 sheep anti-DIG-alkaline phosphatase (Fab fragments; Roche Biochemical). Sections were then washed twice for 10 minutes in buffer 1 and once for 10 minutes in buffer 2 ( lOOmM Tris, lOOmM N a C l , 5 0 m M M g C h , p H 9.5). m R N A hybridization was then detected by covering sections for 2 hours with a colourizing solution of buffer 2 containing 337.5 pg/ml nitroblue tetrazolium ( N B T - BioRad), 175 pg/ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP -25 BioRad), and l m M levamisole. The colourization reaction was stopped by briefly incubating sections in buffer 3 ( l O m M Tris, l m M E D T A , p H 8.1), and then dipping them in distilled water. Sections were allowed to air dry, and were mounted with Cytoseal mounting medium (Stephens Scientific) and 24x50mm glass coverslips (Fisher Scientific). Images were viewed under inverted light transmission (Nikon) and photographed with a Nikon FE2 camera on Fujichrome Sensia 400RH135 film. Nissl staining of tissue sections Following mounting on permafrost coverslips, tissue sections were allowed to dry at room temperature. Sections were placed for 3 minutes in Cresyl Violet solution, then dipped 10 times in distilled water, dipped 10 times in 70% ethanol, and then i f staining was adequate, sections were dehydrated in two changes of 95% ethanol for two minutes. Data Collection The total number of mice whose results were used to support the data presented in this thesis were: E12; n = 6, E14; n = 21, E18; n = 19. 26 C h a p t e r 3: Results Sequence and structure of murine Semaphorin 5B Murine semaphorin 5B was originally cloned by A . Piischel and colleagues (Adams et al. 1996). It is a multi-glycosylated transmembrane protein of 1093 amino acids (a.a.), containing a 485 a.a. semaphorin domain that is 64% identical to that of semaphorin 5 A and 41% identical to that of Drosophila semaphorin 5C (Khare et al. 2000). Location of probe hybridization The antisense probe constructed for use in the experiments described herein was designed by Dr. W. Wang (O'Connor laboratory) to hybridize to 321 nucleotides of mSema5B in the 3' region, which correspond to 107 a.a. in the C-terminal region of the mature protein. This region displays the greatest divergence between the highly similar Sema5A and 5B (51.6% identity over 334 nucleotides in the Sema5 A sequence - Fig. 2A) , and therefore represents the greatest possible specificity in terms of in situ hybridization. Results of dot blots and gels The plasmid p - G E M T - T containing the C-terminal fragment of mSema5B was linearized with Bam-HI for antisense probe synthesis and NotI for sense probe synthesis. Subsequent to the synthesis of DIG-labelled R N A and its isolation from unincorporated nucleotides and salt, a sample of each probe was removed and run on a 1% agarose gel with a mass ladder and a lkb ladder. Both antisense and sense probes were approximately 400 nucleotides in length, when compared with the ladder standards (Fig. 2B). 27 Figure 2 : Semaphorin 5 B probe alignment and probe analysis. A . Alignment of probe sequence to sema5B and sema5A. Only the regions of alignment are included. Identity between the probe and sema5B was 100%, identity between the probe and sema5A was 51.6%. B . Agarose gel (1%) comparing a mass ladder and a kilobase ladder to the sense and antisense probes following DIG-labelled R N A synthesis. Probes were determined to be approximately 400bp in length, compared to standards. C. Dot blot comparing D I G incorporation into antisense and sense probes, shown in dilutions of 1:100, 1:1000 and 1:10,000, to a lpg/pL control DIG-labelled R N A . 2 8 (3010) mSema5A (2973) mSema5B (2983) mSema5B probe (1) Consensus(3010) (3061) mSema5A (3024) mSema5B (3034) mSema5B probe (15) Consensus(3061) (3112) mSema5A (3075) mSema5B (3082) mSema5B probe (63) Consensus(3112) (3163) mSema5A (3126) mSema5B (3130) mSema5B probe (111) Consensus(3163) 3010 3020 3030 3040 3050 3060 | C A C T G C T T G T C T A C A C C T A C T G C C A G A G G T A C C A G C A G C T T G C C T T G G C A G T G T A C C T G T C T T G C C A G C A C T G C C A G C G C T G G T C T G G A O T C T G G T T A C T T G C C A G T C C A G C C C A G T C T C A G G A G T Section 61 3061 3070 3080 3090 3100 3111 C A A C T G T C A T C C A C C C T G T C T C T C C T G C C G C C C T C A A C A G C A G C A T A A C T A C C A C G C T T G T C C A T C C T G C C A C A C C T A A C C A C T T G C A C T A C A A G G G T G C G C T G T C A A A A C A T G C T A A G G G G C C A C G C T T G T C C A T C C T G C C A C A C C T A A C C A C T T G C A C T A C A A G G G T G Section 62 3112 3120 3130 3140 3150 3162 A C C A . . A T C A A C A A A C T G G A C A A A T A T G A T T C T G T G G A G G C C A T C A A G G C A T G G G G . : A C C C C C A A G A A T G A G A A G T A C A C C C C T A T G G A A T — T C A A G A C A C G G G G C C C G A A T G G C A C C C A A T T C A A G A C G G G G C A C C C C C A A G A A T G A G A A G T A C A C C C C T A T G G A A T T C A A G A C A C Section 63 3163 3170 3180 3190 3200 3213 T T A A C A A A A A C A A C T T G A T C C T A G A G G A G A G A A A C A A A T A C T T C A A C C C A C T G A A C A A G A A C A A C T T A A T C C C T G A T G A C A G A G C C A A C T T C T A C C C A C T G A A C A A G A A C A A C T T A A T C C C T G A T G A C A G A G C C A A C T T C T A C C C A C T G A A C A A G A A C A A C T T A A T C C C T G A T G A C A G A G C C A A C T T C T A C C C A C (3214) m S e m a 5 A (3177) m S e m a 5 B (3178) m S e m a 5 B p r o b e (159) C o n s e n s u s ( 3 2 1 4 ) (3265) m S e m a 5 A (3225) m S e m a 5 B (3229) m S e m a 5 B p r o b e (210) C o n s e n s u s ( 3 2 6 5 ) (3316) m S e m a 5 A (3274) m S e m a 5 B (3280) m S e m a 5 B p r o b e (261) C o n s e n s u s ( 3 3 1 6 ) (3367) m S e m a 5 A (3325) m S e m a 5 B (3328) m S e m a 5 B p r o b e (309) C o n s e n s u s ( 3 3 6 7 ) S e c t i o n 6 4 3214 3 2 2 0 3 2 3 0 3 2 4 0 3 2 5 0 3 2 6 4 1 A T ' " T C A C T G G G A A G A C C T A Y T C C A A C G C C T A C T T T A C - A G A T C T C A A C A [ T G A G C A G A C C i T G T G T A T A C A A C C A C G T A C T A C C C C A G C C C A C T G A A C A T G A G C A G A C C . . T G T G F A T A C A A C C A C G T A C T A C C C C A G C C C A C T G A A C A T G C A G C A G A C C A A T G T G T A T A C A A C C A C G T A C T A C C C C A G C C C A C T G A A C A S e c t i o n 6 5 3 2 6 5 3 2 7 0 3 2 8 0 3 2 9 0 3 3 0 0 3 3 1 5 G T A A A C T C C C C C R C A C C C C A C A A T T — A T G A T G A A T A C T A G C A G C T C T T A T A C T T T G G G C T C G T • G C C C / . G C T C C G G C G A G G C A C C ' G G A C A C C C T G T T G C C C G C T C C G G C G A G G C A C C G G A C A G C G C T S T T A G C C C A G C T T C C G G C C T G A G G C C T C A C C T G G A C A G C G C T G T T T C C C C A A C A S e c t i o n 6 6 3 3 1 6 3 3 3 0 3 3 4 0 3 3 5 0 3 3 6 6 G C T G C T C C T C A A A C C G T G C T C C A T G G C T G C C C C A T A T T T C T G A G G C T T C A G G C T G A T C C C A C C C T C C T G G G A C T T G G G C T C C T T G C C T T C C C G A G G C A G G C T G A T C C C A C C C T C T G G G A C T T G C G C T T T G C C T T C C C G A G G C A G G C T G A T C C C A C C C T C C T G G G A C T T G G G C T C C T T G C C T T C C C G A G G C A G | S e c t i o n 6 7 3 3 6 7 3 3 8 0 3 3 9 0 3 4 O 0 3 4 1 7 1 A G A T J A C G T G — T G G A A C C A T T T C A A G T G C A T T T C A A A C C A G G A C T T T C C C [ A G A A G A G T G : A A T G G G A C C G T A G A A C C A C T T T G G T T T A C C C T C T G C A C | A G T G A A A G A A G A G T G G A A T G G A C C T A C A T T Control Antisense 1Kb A S Mass Sense 29 Dot blots were used to assess the adequacy of D I G - U T P incorporation into the sense and antisense probes and demonstrate that 1 pg of probe R N A labels at the same levels as does 0.01 pg of control labelled R N A (Fig. 2C). Expression pattern of Semaphorin 5B mRNA In situ hybridizations were performed using complimentary sense and antisense probes developed to specifically recognize the 3' region of murine semaphorin 5B m R N A . The antisense probe labelled many structures within the nervous system of mice as they develop; however, the areas of focus were chosen based upon the observed patterns of expression that were most indicative of potential developmental roles played by this guidance cue. In addition, evidence from studies of S E M A 5 B in the developing chick nervous system, demonstrates the strongest and most intricate expression patterns in the spinal cord and dorsal root ganglia, the retina, the olfactory epithelium and the forebrain. Therefore, this thesis focuses on the m R N A expression of mSema5B in the four abovementioned regions. Generally, the expression of mSema5B does not appear to be greatly dynamic, rather it only appears to change as the structure of nervous system itself changes through development. There is consistent expression throughput the ventricular zones. Although tissue from E l 2 , E14 and E18 was examined for Sema5B expression, the E12 material (except spinal cord) was not included because the sense controls were so strongly labelled that no plausible conclusions could be drawn. This was not the case for E14 and E l 8 tissue, where distinct differences were observed between sense and antisense hybridizations; however, there are some exceptions to this, where sense and antisense probes labelling is similar, and these w i l l be discussed. 30 Semaphorin 5B expression within the Mouse Spinal Cord and Dorsal Root Ganglia As the age of mice increases, the expression of mSema5B m R N A throughout the spinal cord appears to increase as wel l (Fig. 3). A t E12 Sema5B expression is strongest within the proliferative ventricular zone that lines that central canal. In the grey matter, the strongest staining is restricted to cells within the dorsal third of the spinal cord grey matter (Fig. 3 A , asterisks), which includes the alar plate, indicated by the darkest staining directly under the asterisks, and a small number of cells in the ventral motor columns (Fig. 3 A , arrowhead). A t E14 there is staining throughout the spinal cord grey matter (Fig. 3D). The dorsal expression observed at E l 2 has decreased to a fewer number of more strongly labeled cells, and though the ventricular zone staining is retained, it is reduced from E l 2. The strength of ventral staining has increased markedly from E12 and has expanded to include densely packed groups o f cells in both lateromedial and ventromedial tiers of motor columns (Fig. 3D). At E l 8 there appears to be greater numbers of cells in dorsal horns that are labeled for mSema5B m R N A (Fig. 3G), although this finding is not supported by Piischel et al. who found that Sema5B was expressed in a manner paralleling the expression of glial-specific m R N A s , such as the glycine transporter and brain lipid-binding protein (Puschel et al. 1996), the expression patterns of which are apparently localized to the central canal and ventral grey matter at E l 5.5. This would appear to resemble Sema5B expression at E l 8 i f it were not for the dorsal staining; the lining of the central canal continues to be labeled, as are the motor columns, the staining of which has taken on a more defined appearance (red arrows in Fig . 3, white arrows in Fig. 4) reflecting advanced stages of differentiation (Altaian and Bayer, 1984) 31 Figure 3: In situ hybridization of semaphorin 5B in the mouse spinal cord and dorsal root ganglion. E12 (A-C; n = 6), E14 (D-F; n = 21) and E18 (G-I; n = 19) are spinal cord cross-sections. A, D, G: antisense probe hybridizations of spinal cord cross-sections at low magnification. B, E, H: high power magnification of antisense probed dorsal root ganglia. C, F, I: sense probe hybridizations at low magnification. DRG: dorsal root ganglia, DH: dorsal horn, VH: ventral horn, LH: lateral horn, CC: central canal. Asterisks; dorsal horn staining at E12, specifically the alar plate. Arrowheads; ventromedial motor columns at E12. Black arrows; dorsal root. Red arrows; dorsolateral and ventrolateral motor columns. Scale bar in A represents 400pm for A, C, D, F, G, I and 200pm in B, E, H. 32 33 Figure 4: Comparing Sema5B labelling and Nissl staining of the E18 ventral spinal cord For each image, medial is to the left, and dorsal is up. A , Hemisection of spinal cord, probed with antisense to Sema5B, shown at 20x. B , Hemisection of spinal cord, stained with Cresyl Violet, shown at 20x. C, Ventral horn in A , shown at 40x. D , Ventral horn in B , shown at 40x. V M ; ventromedial motor columns, V E ; ventral motor column, V L ; ventrolateral motor column, D L ; dorsolateral motor column, W M ; white matter. Scale bar in A represents 200um. Scale bar in C represents 200um. 35 According to Puschel et al., Sema5B m R N A in the developing mouse spinal cord is expressed primarily in the ventricular zone of the from E9.5 (Adams et al. 1996), with some expression developing in the marginal zone around E12.5 (Puschel et al. 1996). In comparison, they also found that Sema5 A was only expressed weakly in the spinal cord lateral marginal zone between E l 1.5 and 12.5, and was found again later at E l 5.5 in the superficial regions of the dorsal horn. Importantly, they did not find expression in the dorsal root ganglia (Adams et al. 1996; Puschel etal. 1996). mSema5B expression is also evident in the dorsal root ganglia (DRG) at all examined ages. At E12 this expression is as strong as that of the dorsal horn, but remains diffuse in appearance (Fig. 3B). A t E14 D R G cells display greater strength and definition of staining, though the overall appearance of the ganglion remains slightly disorganized. B y E l 8 this staining is even further defined, and the D R G has lost the disorganized appearance (Fig 3H). A t E12 and E14 the dorsal root leading from the D R G to the spinal cord is strongly stained, lined with labelled cells between the D R G to the outer limit of the dorsal root entry zone (Fig. 3 A , D). There are also a small number of labelled cells at the ventral root entry zone at E l 4 (Fig. 3D). These cells might reflect the presence of neural crest-derived boundary cap cells at the dorsal and ventral root entry zones that prevent the passage of axons into or out of the spinal cord grey matter prior to the appropriate developmental moment (Altman and Bayer 1984; Vermeren et al. 2003) Specifically, at roughly El2-13.5 in the mouse, the dorsal boundary cap is being pulled apart as the dorsal root lengthens (Altman and Bayer 1984), which explains the presence of cells along the dorsal root at E12 and E14, and their expression of Sema5B may reflect their barrier role toward sensory and motor axons. 36 Semaphorin 5B Expression within the Mouse Eye Semaphorin 5B m R N A was detected in the retina and lens of mice at all examined stages of development. A t E l 4 there are strongly labelled cells scattered within the retinal ganglion cell layer (Fig. 5A, B , double arrowhead), which is a staining pattern that closely resembles slit and robo expression (Niclou et al. 2000). This labelling increases in strength by E l 8 , and also becomes more tightly localized within the retinal ganglion cell layer (Fig. 5G, H , double arrowhead), where the E14 labelling appeared less organized. There is also some intermittent staining throughout the rest of the retina that may be non-specific, or could demonstrate labelling of other cell types (Fig. 5B, E ; arrows). Comparison with Niss l staining of the E l 8 retina indicates this staining is taking place in the retinal ganglion cells or amacrine cells within R G C layer (Fig. 6A, B) . The lens also expresses Sema5B. At E14 the numbers of labelled cells in the lens are reduced (Fig. 5A, arrow) as the fibrous portion grows and by E l 8 this staining is even further reduced (Fig. 5D, arrow). A t all examined ages, D I G - labelling was not detected anywhere in the optic disk or optic nerve (Fig. 5 A , arrowhead), which is the sole location of Sema5A expression in the retina (Oster et al. 2003). 37 Figure 5: In situ hybridization semaphorin 5B in the retina and lens of the mouse eye. E14 (A-C; n = 21) and E18 (D-F; n = 19) coronal cross-sections of the mouse eye. A, D: low magnification of antisense probe hybridizations. B, E: high magnifications of the antisense probed retina. C, F: sense probe hybridizations of the eye at low magnification. RGC: retinal ganglion cell layer, RPE: retinal pigmented epithelium. Arrowhead in D; optic nerve head/optic disk. Arrows in A, D and G; cells staining in the lens. Arrows in E, H; examples of possible cellular staining in deeper retinal layers. Double arrowheads in E, H; stong staining at the RGC layer. Scale bar represents 400um, except for B, E, H, where is represents lOOum. 3 8 39 Figure 6: Nissl staining and Sema5B expression in the E18 mouse retina A , E18 retina, cut in coronal orientation, probed with antisense to Sema5B, shown at 40x. B , E18 retina, cut coronally, stained with Cresyl Violet, shown at 40x. Arrows are to highlight R C G layer staining. O N F L ; optic nerve fiber layer, R G C ; retinal ganglion cell layer, H?L; inner plexiform layer, I N L ; inner nuclear layer, O N L ; outer nuclear layer, R P E ; retinal pigmented epithelium. Scale bar = lOOum. 41 Semaphorin 5B Expression within the Mouse Olfactory System Two structures of the olfactory system were examined: the olfactory olfactory bulb (OB) and the epithelium (OE). A t E14 the O B is distinctly labelled at or within the beginning of the lateral olfactory tract, and within the layer of the developing glomeruli, with some minor staining also seen in the layer of mitral/tufted cells (Fig. 7 A ) . A t 18 the low level staining of the O B continues in the layer of glomeruli and mitral/tufted cells (Fig. 7D). The granule cell layer ( G C L ) on the other hand is never labelled (Fig. 7A, D). Figure 8 A and C are low and medium magnifications of the E l 8 O B , showing the G C L to appear empty. Figure 8B and D are Nissl stained E l 8 O B , demonstrating that there are a large number of cells within the G C L that do not express Sema5B. Both E14 and E l 8 olfactory bulbs label strongly in the layer of developing glomeruli (Fig. 7A, D, 8 A , B) , however the antisense hybridization is very similar to that of the sense probe (Fig. 7C, F). Therefore, we cannot be confident that the staining in the glomerular layer shown in figure 7 A and C is actual labelling for Sema5B. In the E14 mouse, groups of olfactory receptor neurons (ORN) throughout the O E are strongly labelled (Fig. 7B, E). The sustentacular cell layer (SCL) that lies closest to the nasal cavity just within the dendritic layer of the ORNs is mostly unlabelled (Fig. 7D). In the E l 8 O E , ORNs continue to be labelled between the S C L and the lamina propria underlying the O E ; however, these cells are fewer and are more evenly scattered. There is no labelling of the lamina propria at any age, which labels for Sema5A m R N A (Oster et al. 2003), although at E14 and beyond there is strong labelling of the O R N dendritic layer. Figure 8G, Niss l staining of the O E , demonstrates that the non-labelling lamina propria is also full o f cells. 42 Figure 7: In situ hybridization of semaphorin 5B in the mouse olfactory bulb and epithelium. E14 (A-C, n = 21) and E18 (D-F, n = 19) coronal sections showing the olfactory bulb above and the olfactory epithelium below. A, D: antisense probe hybridization at low magnification. B, E: high magnification of OE in antisense probe hybridization. C, F: sense probe controls at low magnification. NC; nasal cavity, SCL; sustentacular cell layer, OE; olfactory epithelium, LP; lamina propria, BL; basal lamina, C; cartilage, OB; olfactory bulb. Scale Bars represent 400um for A, C and lOOum for B, D. 44 Figure 8: Comparing of mSema5B labelling with Nissl staining of E18 OB and OE A , E l 8 O B , coronal section, hybridized with antisense probe to Sema5B, shown at lOx. B , E12 O B , coronal section, stained with Cresyl Violet, lOx. C, E18 O B , 40x image of A . D , E18 O B , 40x image of B . E , E18 O E , coronal section, probed with antisense to Sema5B, 40x. F, E18 O E , coronal section, stained with Cresyl Violet, 40x. C ; cartilage, O E ; olfactory epithelium, O B ; olfactory bulb, L O T ; lateral olfactory tract, G C L ; granule cell layer, M / T ; mitral cell layer with associated tufted cells, G L O M ; glomerular layer. Scale bar = 400u.m in A , B , 200um in C, D , l O O u m i n D , E . 46 Semaphorin 5B expression within the Mouse Forebrain In the E14 forebrain, expression of Sema5B at the V Z is pronounced and labelling is apparent in the cells populating the growing cortical plate (CP; Fig . 9A).The strongest staining is apparent in the most superficial cells that are within or just deep to the marginal zone (MZ) , whereas the intermediate (IZ) and subventricular zones (SVZ) are essentially devoid of labelled cells. In .the E l 8 mouse strong labelling for mSema5B in the forebrain V Z is maintained. The superficial-most layers of the now thicker cortical plate are more densely populated by Sema5B expressing cells where even some neuronal-specific morphology such as apical dendrites is evident (Fig. 9E). In contrast to these superficial layers of cortical plate cells and the subplate cells that continue to express Sema5B, the intervening deep layers of the cortical plate demonstrate a marked reduction in straining that is comparable to the lack of staining of the IZ and S V Z between the V Z and C P . This is even more clearly evident when comparing E l 8 cortex stained with Cresyl Violet with the E l 8 cortical Sema5B expression (Fig. 10A, B) . 47 Figure 9: In situ hybridization of semaphorin 5B in the developing mouse cortex. E14 (A-C, n = 21) and E l 8 (D-F, n = 19) coronal sections of the dorsal mouse forebrain. Medial is to the left, and dorsal is up. A, D: antisense probe hybridization shown at low magnification. B, E: antisense probe hybridization shown at high magnification. C, F: sense probe controls at low magnification. V; ventricle, VZ; ventricular zone, SVZ; subventricular zone, IZ; intermediate zone, SP; subplate, CP; cortical plate, MZ: marginal zone. Boxes; regions of A and D that are shown at high magnification in B and E. Scale bar = 200pm for A, C, D, F and 100pm for B, E. 48 49 Figure 10: Comparison of E18 cortex labelled for mSema5B with Nissl-stained E18 cortex A . Antisense probed E l 8 cortex, 40x. B . Niss l stained E l 8 cortex, 40x. M Z ; marginal zone, C P ; cortical plate, SP; subplate, IZ; intermediate zone. Comparison of the two cortical plates demonstrates the lack o f Sema5B labelling in the deeper regions of the cortical plate. Scale bar = 100pm. 50 51 Chapter 4: Discussion Expression of semaphorin 5B m R N A was determined in four regions o f the developing central nervous system of C57Black mice: the spinal cord and dorsal root ganglia, the forebrain, the eye (lens and retina), and the olfactory system (olfactory epithelium and olfactory bulb). These four regions of focus in this study were initially of interest due to expression profile developed in this lab in the developing chick. The use of non-isotopic in situ hybridization confers some limitations to the study at hand. In some instances, the sense probe hybridized with the same profile as the antisense, only the staining was less intense, as is exemplified in figure 7 (the olfactory bulb and epithelium). The sense staining may be related to the presence of the dixoygenin molecule that was conjugated to the U T P to allow immunological detection of the probe, since when control in situs were performed (no probe added during the hybridization step), there was no labeling of tissue. In addition, the structural integrity of E l 2 tissue was often very poor following the in situ procedure, and in some cases could not be used for descriptive purposes. Finally, this study is investigating the m R N A localization of Sema5B which has a great deal of sequence similarity to the other class 5 member, Sema5A, with which it is 58% identical and 72%o similar in nucleotide sequence (Adams et al 1996). Even within the most divergent region used to synthesize the probe, there is 51.6% identity; however, calculation of melting temperatures reveals that the Sema5B probe should not hybridize to Sema5 A under the experimental parameters used (Appendix A ) . Previous work has been performed illustrating the 52 expression of Sema5A in certain regions of the nervous system; therefore Sema5B expression wi l l be contrasted with that of Sema5A whenever possible throughout the following discussion. Functional Implications of Semaphorin 5B Domains The semaphorin domain is the defining feature of the semaphorin family, and is fundamental to the function of each member. Not only is the semaphorin domain necessary for these molecules to exert their effects, but additionally, the specific nature of each particular semaphorin, whether strongly repellent, mildly repellent or non-repellent, is conferred by its individual semaphorin domain (Koppel et al. 1997), owing to specifically tailored receptor binding sites located within the domain (Antipenko et al. 2003). The closest relatives to the class 5 semaphorins in terms of homology within the semaphorin domain are the class 1 semaphorins (Adams et al. 1996). This may be of great significance, due to the fact that sema-la in the grasshopper has been shown to provide a positive instructive/permissive medium for axon extension and fasciculation, rather than outright inhibition (Bonner and O'Connor 2000). Class 1 semaphorins, however, do not carry any other domain specializations and therefore Sema5B function cannot be greatly inferred from its activity. Many semaphorins contain additional domain structures that are l ikely to modulate or moderate the primary activity of the semaphorin domain. Semaphorin class 5 members contain a unique domain of seven thrombospondin type-1-like repeats. This is an intriguing feature as members of the thrombospondin family of proteins have been shown to provide a permissive substrate to growing neurites; similar to the manner that laminin is permissive (Adams and Tucker 2000). Thrombospondin family members have been shown to act through integrin receptors (deFreitas 53 et al. 1995), as does laminin, and therefore can be classified as adhesion molecules. It is therefore possible that, i f the semaphorin domain of Sema5B is inhibitory via a plexin receptor, the thrombospondin repeats may in certain instances counteract this inhibition through interactions with specific integrins. There is a possibility that Sema5B is cleaved in order to exert its effects, which is the case for other semaphorins (Adams et al. 1997; Artigiani et al. 1999). There is a furin-cleavage recognition sequence that lies between the thrombospondin domain and the transmembrane region (data not shown). If Sema5B is indeed cleaved, this would greatly alter how this molecule might function in the developing nervous system, and this is taken into account in some of the following discussion. Guidance cues are associated with diverse developmental processes Guidance cues are most commonly associated with the directional pathfinding of growing axons in developing nervous systems, and there are a plethora of examples of such action by semaphorins (Goodman and Shatz 1993; Kolodkin et al. 1993; Luo et al. 1993; Luo et al. 1995; Puschel et al. 1996; Kolodkin and Ginty 1997; Isbister etal . 1999; Puschel 1999; Bonner and O'Connor 2000; Goshima et al. 2000; He et al. 2002; de Castro 2003; Dickson 2003), slits (Brose et al. 1999; K i d d et al. 1999; Nic lou et al. 2000; Ringstedt et al. 2000; Zou et al. 2000; de Castro 2003), netrins (Hu and Rutishauser 1996; Steup et al. 2000; Campbell and Holt 2003; Charron et al. 2003; de Castro 2003) and ephrins (Harris and Holt 1995; Monschau et al. 1997; Ohta et al. 1997; Frisen et al. 1998; O'Leary and Wilkinson 1999; Yue et al. 1999; Kullander and Kle in 2002; Hahn and Emmons 2003; Y u n et al. 2003). However, other processes inherent to 54 development are also guidance cue-mediated. Evidence suggests that the post-pathfmding processes of synaptogenesis, axon pruning and apoptosis rely upon so-called guidance cues (Shirvan et al. 1999; Bagri et al. 2003). It is becoming increasingly clear that another crucial process in nervous system development, neuronal migration, is actually based upon very similar, i f not identical processes to those of axon guidance - i.e. migration is also a guidance event requiring the signaling mechanisms of guidance molecules (Hu and Rutishauser 1996; Mar in et al. 2001; Park et al. 2002; Spassky et al. 2002; Tsai and Mi l l e r 2002; Cohen et al. 2003; de Castro 2003; L i u and Rao 2003; Tamamaki et al. 2003). Because the function of murine Sema5B in mice has yet to be investigated by way of in vivo and in vitro experiments, the following pages w i l l discuss the observed expression pattern in terms of potential roles in axon guidance, cell migration, and differentiation. A variety of molecules known to be involved in axon guidance have now been shown to also be involved in providing directional guidance instructions to several cell types within the nervous system. Oligodendrocytes, astrocytes, neurons and their precursors/progenitors are all guided by axon guidance molecules of the four major groups: slit, netrin, semaphorin, ephrins/Eph (Armstrong et al. 1990; Brose et al. 1999; O'Leary and Wilkinson 1999; Bagnard et al. 2001; Park et al. 2002; de Castro 2003). Semaphorin 5B Functions as an Axon Guidance cue in the Spinal Cord and Retina of the Chick Semaphorin 5B in the mouse may be involved in axon guidance, as has been demonstrated with in vitro experiments performed with embryonic chick explants. Chick S E M A 5 B was cloned in 55 the O'Connor laboratory (Wong 1999) and has since been found to be prominently expressed in primary neuroepithelium, spinal cord grey matter and dorsal root ganglia (Wang et al, in progress). Various neuronal explants were assessed in their response to contact with cells expressing full-length recombinant and mutant forms of chicken S E M A 5 B . Retinal ganglion cells, which were found to stain with an antibody developed toward c S E M A 5 B , and sympathetic neurons, which did not stain for c S E M A 5 B , strongly avoid the full-length molecule. Some sensory neurons were found to express S E M A 5 B , and overall the D R G neurites display an intermediate response, where instead of avoidance of expressing cells, many appeared to stall upon contact. Deletion of the semaphorin domain caused neurites of all three cell types to prefer the expressing cells as a growth surface, compared with the laminin-coated substrate. Expression of a construct lacking the thrombospondin repeats caused all neuronal types, including sensory neurites, to strongly avoid the cells. It was proposed that the thrombospondin repeats may modulate the inhibition caused by the semaphorin domain, and the difference in response by different cell types to c S E M A 5 B may depend on neuronal expression, and previous exposure to the molecule. Therefore it may be proposed that guidance of retinal ganglion axons and a subset of sensory axons are mediated by this protein in mice as well as in chick. Potential Role of Semaphorin 5B in Axon Guidance within the Mouse Spinal Cord There are many cues and pathfinding processes that occur in the spinal cord during development (Altman and Bayer 1984). However, owing to the sensory neuron expression and the spinal cord distribution of Sema5B m R N A , the following discussion w i l l focus on potential roles in guidance between the D R G and the sensory axon targets. In the rat D R G neurons are born between E12 and 15 (Altaian and Bayer 1984), corresponding to E10.5 to 13.5 in the developing mouse. Mouse sensory neurons enter the spinal cord at E12 but pause at the dorsal root entry zone until E l 4 at which point they begin to penetrate the grey matter to establish connections (Messersmith et al. 1995; Puschel et al. 1995; Sanes and Yamagata 1999). These neurites can be differentiated based upon their growth factor preferences. Small diameter afferents mediate thermo- and nociception, terminate in the Rexed laminae I and II of the dorsal horn (Rexed 1954) and are nerve growth factor (NGF) responsive (Zhang et al. 1994). The larger diameter afferents that mediate non-noxious cutaneous stimuli and the mono-synaptic stretch reflex, terminating in laminae III/1V and upon motor neurons, respectively (Rexed 1954), are responsive to neurotrophin-3 (NT3; (Zhang et al. 1994; Messersmith et al. 1995)). NT3-responsive sensory axons are the first to penetrate the dorsal grey matter, and NGF-responsive axons enter the last. In an embryonic co-culture of E14-15 ventral spinal cord and D R G , sensory neurites were found to be potently inhibited from growing toward the spinal cord explant (Fitzgerald et al. 1993). Many secreted class 3 semaphorins have patterns of expression within the developing spinal cord that begins around E l l (Puschel et al. 1995). Sema3A is expressed in the ventral half of the spinal cord between E l l and 15, and is generally considered to be responsible for inhibiting growth of N G F - but not NT3-positive axons into the ventral half o f the spinal cord (Messersmith •et al. 1995; Puschel et al. 1995; Wright et al. 1995; Giger et al. 1996; Puschel et al. 1996). In vitro it is a powerful chemorepellent for NGF-responsive sensory axons, and when knocked-out, there is aberrant growth of some sensory fibers toward the ventral root via the dorsal root (Behar et al. 1996; Catalano et al. 1998). Not only is Sema3A expressed in the grey matter, but it is also 57 found in the mesenchyme surrounding the D R G (Puschel et al. 1995), and is thought to be responsible for directing both central and peripheral sensory projections (Wright et al. 1995). SemA, B , C and E were also found to display specific patterns o f expression in the developing spinal cord between E l 1 and E15 (Puschel et al. 1995, 1996); however, their roles have not been assessed to the extent of Sema3A. In spite of all the overlapping expression of the Sema3s in the spinal cord, there is little known o f what mechanism(s) is(are) actually responsible for the determination of laminar-specific terminations of different sensory axon subtypes (Messersmith et al. 1995; Sanes and Yamagata 1999). The present study shows that Sema5B is expressed in an appropriate spatial and temporal context (Messersmith et al. 1995; Sanes and Yamagata 1999) to denote involvement in the establishment of laminar-specific connections in the spinal cord. It is first expressed in the dorsal third of the grey matter, as well as at the central canal and extreme ventral regions o f the motor column around E12 (Fig. 3A) and then spreads throughout the grey matter at E14 (Fig. 3D). B y E l 8 Sema5B expression is crisp and the architecture o f the grey matter is more easily resolved (Fig. 3G) such that the motor columns are identifiable (Fig. 4). In addition, neurons within the D R G from E l 2 to E l 8 also express Sema5B, as do neural crest cells along the dorsal root and at the ventral root entry zone (Fig. 3 A , B , D , E , G , and H ; Altman and Bayer 1984). The expression pattern of Sema5B described in the spinal cord and D R G indicates several possibilities in terms of its function as a protein. 1) A hypothesis that resulted from the chick S E M A 5 B experiments was that expression of Sema5B conferred decreased sensitivity to its effects when encountered from other sources. D R G staining indicates there may be a subset of 58 Sema5B-postive cells - possibly NT3-responsive axons - that are less sensitive to Sema5B. This would permit them to penetrate the spinal cord grey matter in advance while the broad dorsal expression at E12 (Fig. 3A) would inhibit the ingrowth of NGF-responsive axons (assuming they do not express Sema5B). A s Sema5B expression becomes more restricted dorsally, other afferents may be permitted to enter the grey matter due to the contact-requirement of its inhibitory activity. Dense expression in the upper ventral regions may provide a molecular barrier, in addition to that o f Sema3 A , that causes the stereotyped turnabout o f overshooting afferents from low-threshold mechanoreceptors into laminae III and IV of the dorsal horn, and dorsal expression may be adequate to restrict penetration of NGF-responsive afferents to laminae I and II. 2) Sema5B may serve as a homophilic recognition factor involved in target recognition and synaptogenesis between positive D R G axons and positive cells within the grey matter. 3) The strong expression of Sema5B later in development may be in part responsible for the creating an inhibitory domain that maintains the segregation of the surrounding white matter tracts by preventing axons from re-entering the grey matter once they have exited. In contrast, the E l 2 expression in the ventricular zone and the alar plate may reflect a role of Sema5B in the proliferation or differentiation of spinal cord interneurons (Altman and Bayer 1984), a role which may continue throughout the development of the spinal cord; therefore it is possible that Sema5B may serve no guidance purpose for developing sensory projections. Potential Roles for Semaphorin 5B in Axon Guidance within the Developing Mouse Eye In the eye, the lens and the factors it releases are essential for the proper development and differentiation of the neural retina (Graw 1996). In the chick, the lens has been found to potently repel retinal ganglion cells, and is thought to perhaps mediate the appropriate guidance of the R G C axons to the optic disk as they initiate axonogenesis. Because sensory axons are also repelled by the lens, it was assumed that the cue must be collapsin-l/Sema3A. However R G C axons are not responsive to Sema3A (Luo et al. 1993), therefore it was determined that the lens epithelium secretes a novel collapsing factor, perhaps in addition to Sema3A (Ohta et al. 1999). The mouse lens epithelium expresses Sema5B at E14 and E l 8 . Presumably the E l 2 lens would also express Sema5B, and so at E12 when the lens and retina are in close opposition, which is temporally consistent for R G C birth (Rodier 1980). Therefore Sema5B, which as was discussed earlier may be cleaved and released from membrane association, may be involved in the differentiation of R G C s . Alternatively, the lens expression of Sema5B may be involved in maintaining correct directional growth of R G C axons from out of the vitreous humor, or it may be involved in the appropriate centripetal orientation of retinal axons toward the optic disc, as was the postulated role for the unknown secreted inhibitory factor (Ohta et al. 1999). Many cues are involved or are at least present in the developing retina. There are six Eph family members expressed by R G C s (O'Leary and Wilkinson 1999); EphA4, A 5 and B l are expressed ubiquitously throughout the R G C layer, whereas EphA3 is expressed in a high temporal to low nasal gradient and is crucialfor the correct anterior-posterior terminations in the superior colliculus of mice where the ephrins ligands are expressed, and in a similar fashion, the EphB2 and B3 high ventral to low dorsal gradient mediates appropriate dorsal and ventral terminations in the superior colliculus. Ephs and ephrins are therefore involved in the formation of the retinotopic map within the tectum (Harris and Holt 1995; Monschau et al. 1997; Frisen et al. 1998; Kullander and K l e i n 2002), and it is the other cues present, to be discussed next, that must guide these axons out of the eye. 60 Members of the other three major guidance cue families (semaphorins, slits, and netrins) as well as cell adhesion molecules are expressed within the developing retina. Several semaphorins are found to be expressed in the retina ganglion cell layer; the m R N A for secreted Sema3A, 3C and 3E, as well as transmembrane Sema4A and 6A, is detected in the R G C layer between E l 3 and E l 5 (Oster et al. 2003). The inhibitory s l i t l and 2, and their functional receptors, robol and 2, are also expressed in the R G C layer of the mouse retina between E l 3 and E16 (Niclou et al. 2000). On the other hand, the diffusible chemoattractant netrin-1 is expressed by a ring of neuroepithelium at the optic disc, and is thought to promote centripetal R G C axon growth (Deiner et al. 1997). In addition to netrin-1, the permissive cue laminin is expressed at the optic disc and these two proteins are thought to interact in terms of R G C axon guidance (Hopker et al. 1999). L I of the Ig superfamily, known to convert the typical response to Sema3A from repulsion to attraction (Castellani et al. 2002), is also expressed along retinal axons and is thought to maintain their fasciculation in the retina (Brittis et al. 1995; Oster and Sretavan 2003). Each of these cues has been shown to influence R G C neurite growth and is therefore thought to be at least in part responsible for the production of the ultimate R G C projections out of the retina. In addition to the abovementioned cues, in a pattern resembling the slit and robo retinal expression profiles (Niclou et al. 2000), semaphorin 5B m R N A is expressed in the retinal ganglion cell (RGC) layer, and not other retinal layers or the optic disc and nerve, which is first seen at E l 4 (Fig. 5 A , B , D , E). This pattern of expression is consistent with in situ hybridization data from Oster et al. (2003). In contrast to Sema5B, Sema5 A is expressed at the optic disc as well as within the neuroepithelium flanking retinal axons within the optic nerve, and has been 61 shown to elicit an inhibitory response from axons of E l 4 retinal explants. Antibody-mediated disruption of Sema5A function in situ results in defasciculation of retinal axons and aberrant straying from the optic nerve (Oster et al. 2003). A s Sema5 A has been shown to be inhibitory, regardless of its thrombospondin domain, it may be postulated that Sema5B is inhibitory as well . A n d although other repulsive cues are present within the R G C layer, Sema5B may contribute to the inhibition of axons that may erroneously attempt to penetrate the retina. Migration of cells within the retina involves both radial and tangential routes (Reese et al. 1999; Park et al. 2002). The initial disorganized appearance of Sema5B expression in the R G C layer at E14 and the subsequently increased order to the staining pattern at E l 8 may reflect a finalization of the clonal retinal architecture (Goldowotiz and Williams 1996; Cepko 1999) and/or elimination of tangential migration within this layer of the retina (Oster and Sretavan 2003). It is possible that it is the R G C s that express Sema5B and thus may provide a boundary that delimits the thickness of the retina as a whole, controlling any errant migration by other retinal cell types by keeping them separated molecularly from the R G C population and the developing nerve fiber layer. Semaphorin 5B May Serve an Important Role in Cortical Neuron Migration There are two forms o f cell migration that produce the intricate structure o f the nervous system. Radial migration along radial glial fibers is responsible for the bulk of the inside-out migration of neurons that produces the laminae of the cortex and hippocampus whereas tangential migration is important for movement within (Fishell et al. 1993) as well as from proliferative zones (Marin 62 et al. 2001; Tamamaki et al. 2003), and for populating the olfactory bulb (Hu and Rutishauser 1996; de Castro 2003; L i u and Rao 2003), midbrain and brainstem regions (Park et al. 2002). Around E10, the developing cortex consists of three layers; a pseudo stratified epithelium that is the mitotically active ventricular zone (Jacobson 1978; Shoukimas and Hinds 1978; Fishell et al. 1993), the pre-plate which is the first layer of generated neurons (Cajal-Retzius cells which w i l l differentiate in the marginal zone and future subplate neurons (Allendoerfer and Shatz 1994)), and the intermediate zone which forms from the axons of pre-plate neurons (Marin-Padilla 1978). A s development continues, neurons migrate outwards, and cause the pre-plate to be split into three regions (Rakic 1978): the subplate is the innermost; the marginal zone containing Cajal-Retzius cells is the outermost, and; the cortical plate develops in between, being formed from the inside-out with each progressive flux of post-mitotic migrating neurons (Ogawa et al. 1995). The cortical plate is analogous to layers II-IV and the marginal zone becomes layer I (Jacobson 1978), where all other structures are solely embryonic and transitory (Allendoerfer and Shatz 1994). As the mouse develops, Sema5B is expressed within the telencephalic ventricular zone (also demonstrated in (Puschel et al. 1996), as well as in neurons and possibly radial glia within the cortical plate as it begins to develop (Fig. 9A, B) . To a point, as the cortical plate becomes thicker, so do the cortical layers that express Sema5B (Fig. 9G); however, at E l 8 the deeper regions of the cortical plate, which at E14 expressed Sema5B, have ceased to do so. One would then expect that once migration is complete none of the cortical layers should express Sema5B. 63 If it is the cortical neurons that are expressing Sema5B, this dynamic expression pattern would suggest that once migrating neurons have reached the cortical plate and become established in their particular areas there would be a loss of the requirement for them to express Sema5B. Therefore, Sema5B expression in V Z cells may indicate the initiation of migration. Sema5B may thus play a role in the appropriate formation and population of cortical laminae, specifically in terms of forcing neurons to exit the V Z and to migrate past established regions of the cortical plate to its most superficial reaches. Although both slits and netrins have been shown to be involved in neuronal migration (Wu et al. 1999; de Castro 20.03; L i u and Rao 2003), neither is implicated in radial cortical migration and the formation of cortical laminae. There are, however, five Ephs and one ephrin that are expressed in the developing neocortex, and the majority of their expression patterns are static within the expressing embryonic layer ( V Z , S V Z , IZ, or CP) as it develops (Yun et al. 2003). Each layer is thus conferred a different array o f Eph protein expression, and the authors argue that this system of molecular coordinates may confer the precise cytoarchitectonic differentiation of the cortex, much in the manner of homeobox genes pattern the nervous system of a much earlier embryo. Reelin, a secreted protein expressed by Cajal-Retzius cells (Caviness and Sidman 1973), is demonstrably crucial for the inside-out formation of cortical laminae (Ogawa et al. 1995); a Reelin defect results in neurons that cannot penetrate the preplate, and the resulting lamination of the cortex is reversed. Cortical plate neurons express receptors for Reelin (the very low-density lipoprotein receptor and the apolipoprotein E receptor-2 (D'Arcangelo et al. 1999), the signal for 64 which is mediated intracellularly by disabled-1 (Howell et al. 1997). Sema5B expression throughout the upper portions of the cortical plate may ensure that migrating neurons do not detach from the radial glia scaffold prematurely; once expressed on the cell membrane, it may cause neurons to continue migrating until a "stop" signal is reached, which may perhaps be mediated by Reelin signalling. It would therefore be of interest to determine whether there is an interaction between Reelin and Sema5B expressing cortical neurons, or whether Reelin exerts it activity independently of whether a cell expresses Sema5B. Semaphorin 5B may play a Role in the Guidance of Mouse Olfactory Bulb Axons The olfactory bulb is populated in development by interneurons (periglomerular and granule cells) that migrate via the rostral migratory stream from the lateral ganglionic eminence of the telencephalon (L iu and Rao 2003). The subventricular zones giving rise to these cells do not express Sema5B (Fig. 9 A , C ; (Puschel et al. 1996)), nor do the majority of olfactory bulb neurons (Fig. 7A, C and Fig . 8A, C). The Sema5B antisense probe strongly labels the glomerular layer of the O B at E14 and E l 8, as does the sense probe. A s such it is unlikely that Sema5B is involved in this particular migratory behaviour, or any other kind of axon guidance within or to the O B . Slit is thought to repel neurons from the subventricular zone (Wu et al. 1999) and from the septum (Hu and Rutishauser 1996), while the O B contains a chemoattractive activity that is essential for this migration to occur (Liu and Rao 2003). Other semaphorins are involved in the guidance of olfactory neuron axons from the O E to the O B , as wel l as the projection from the O B along the lateral olfactory tract (LOT, shown in Fig. 7, 8) (de Castro et al. 1999; Schwarting et al. 2000; de Castro 2003). There appears to be Sema5B expressed at or within the L O T (Fig. 65 8A, C), which may reflect a role for Sema5B in the guidance of olfactory bulb axons that parallels the role played by Sema5A in the retina (Oster et al. 2003). Potential Role for Semaphorin 5B in the Mouse Olfactory Epithelium Within the olfactory epithelium (OE), olfactory receptor neurons (ORN) are known to begin life at the basal lamina as undifferentiated "basal" cells. These cells differentiate and then extend to span the entire depth of the O E . Labeling for Sema5B appears to correspond to ORNs and based upon its distribution may therefore be involved in their migration, or in the migration of other cell types, depending on whether it is functioning as a cell adhesion molecule or a chemotactic repellent. One prediction of this hypothesis is that Sema5B labeling should persist into the adult regenerating O E . Confirmation of this prediction awaits further experiments. 66 Chapter 5: Summary Guidance cues were first designated as such owing to their ability to influence the directional outgrowth of neuritic processes during nervous system development. There are four major families of proteins that fall into this category: slits, netrins, ephrins and semaphorins (Dickson 2003). It is becoming apparent, however, that guidance cues are not only involved in the establishment of pathways and connections that are so crucial for a functioning nervous system, but may additionally provide directive guidance information for the cell migration that creates the basic cytoarchitecture that w i l l underlie the establishment of connectivity (de Castro 2003). Evidence also points towards guidance cues influencing other stages of nervous system development, such as proliferation and differentiation, synaptogenesis, pruning and apoptosis. Members of the semaphorin family have been implicated in many of these atypical guidance cue functions. This thesis describes the spatial and temporal m R N A expression pattern of semaphorin 5B in C57Black mice. This expression pattern is suggestive of several different potential functions of Sema5B during nervous system development. 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In order to determine whether the Sema5B probe could possibly be hybridizing to Sema5 A transcripts based on hybridization at 55°C in a solution containing 50% formamide ( D I G Easy Hyb; Roche Biochemical) I calculated the melting temperature of the probe and their target m R N A s . The equation for calculation of melting temperature (T m ) is: T m = 49.82 + 0.41 (% G + C) - (600/L) where L is length of hybrid in base pairs (321bp). Tm for Sema5B probe hybridizing with Sema5B m R N A = 69.53°C. T o p t (for hybridization) = T m - 20°C = 49.53°C This allows for 18%> mismatches between the probe and the target. Therefore, as the probe is 100% identical to the target region of Sema5B, the stringency of hybridization was increased to 55°C (determined empirically to produce the best ratio o f signal to noise), and was followed by washes between 50 and 55°C. To calculate the T m between the Sema5B probe and Sema5A, the length of the hybrid changes to 175bp (which corresponds to the amount of identity between the probe and the homologous target region of 5A). T m for Sema5B probe hybridizing with Sema5A m R N A = 65.04°C T o p t (5A) - T m - 20°C = 45.04°C 82 Therefore, with hybridization and wash steps taking place 5-10°C above the temperature that would be optimal for hybridization between the Sema5B probe and the target Sema5A region, it is unlikely for the data presented to have resulted from errant hybridization between the probe and Sema5A. 

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