Neural function depends on precise wiring of axon during development. Previous studies have demonstrated that the erythropoietin producing hepatocellular carcinoma (Eph) family of tyrosine receptor kinases is crucial for the proper development of a number of neural circuits in the mammalian central nervous system (CNS). Mice lacking Eph receptors have been shown to exhibit deficits in pathways which include the thalamocortical, callosal, retinal and corticospinal tract. Due to the large number of Eph family members, the relative contribution of each receptor to axon pathfinding and neural function remains elusive. In this thesis, I have addressed the function of EphA4, EphB2 and EphB3 in the regulating the formation of interhemispheric projections within the forebrain and motor axon connections within the spinal cord using EphA4, EphB2 and EphB3and combinatorial null mice. To perform a detailed examination of the process of axon guidance regulated by these receptors within the forebrain, high resolution magnetic resonance imaging (MRI), immunofluorescence and in vivo stereotactic fluorescent labeling were performed. This work resulted in the development and validation of MRI-based analytic tools performed using EphB2 mutants which we have previously shown to exhibit specific morphologic defects in the anterior commissure (AC). Analysis of EphA4 null mice using high resolution MRI revealed for the first time that in addition to errors of midline crossing, loss of EphA4 activity results in a positional reorganization of the rostral AC. Results demonstrate that while EphB2 and A4 each regulate distinct aspects of guidance within ACpp, these receptors also operate cooperatively to control the guidance of axons in the pars anterior of the AC, a pathway not been previously implicated in Eph-mediated guidance. With respect to the spinal cord, mice deficient in EphB2 and EphA4 display prominent axon guidance errors in the medial subsets of the lateral motor column (LMCm); neurons which normally innervate ventral limb musculature. Finally, I have addressed the functional effect which Eph mutants exhibit with respect to motor behavior by examining a detailed set of motor coordination parameters

Axon guidance is the developmental process where developing neurons navigate their processes based on attractive and repulsive cues. C. elegans has been an instrumental model in the study of neurobiology with one of the key benefits being a relatively simple nervous system which comprises of only 302 neurons. The Eph Receptors are a canonical class of axon guidance molecules and in C. elegans there is only Eph receptor VAB-1. To understand axon guidance it is useful to study the mechanosensory neurons, and in particular a pair of neurons called the PLM (Posterior Lateral Microtubule). In this thesis I undertook a series of projects involving new techniques, and identified gene products that may interact with VAB-1 in the PLMs. I demonstrate that the use of the PLM Length and PLM/Body length ratio at the L1 stage offers an improved way of detecting axon guidance phenotypes. I show proof of concept that use of a light induced cell ablation technique can help study the developing nervous system. Further, I show that with the use of a tissue specific RNAi technique the role of lethal genes in axon guidance can be analyzed. Finally I conducted a screen that identified new effectors of the VAB-1 signal transduction pathway. / Thesis (Master, Biology) -- Queen's University, 2012-10-29 11:31:54.764

During neural development, each neuron sends an axon out from its cell body. Extending axons are guided by interactions between environmental factors and axonal receptors for these factors. It has been suggested that certain proteins of the immunoglobulin-like superfamily are among the molecules involved in axon guidance. In particular, TAG-1, Ll and NrCAM have previously been implicated in the guidance of dorsal spinal commissural axons at the ventral midline region known as the floor plate. To establish whether these molecules have such roles in mice, the dorsal spinal axons of TAG-1, L1 or NrCAM mutant mouse embryos were traced. There were no significant differences between the results from mutant embryos and their wild type counterparts. This indicated that these three proteins are individually not essential for the normal development of mouse dorsal spinal projections. However, results from TAG-MLI double mutant embryos suggested that TAG- I and LI might affect the ability of commissural axons to extend out of the floor plate. Analysis of ephrin B3 mutant embryos indicated that ephrin B3 might also be important for floor plate exit. As the TAG-1 null mutation includes a lacZ construct, this reporter gene was used to further investigate the roles of TAG-1. Its expression was used to determine distribution of TAG-1 gene activity in the developing mouse nervous system. As the pattern of reporter expression was found to be comparable with that of TAG-1 protein, the TAG-1 null allele was used as a marker for TAG-1-expressing cells in mutant embryos. Most of the structures that normally express TAG-1 seemed to be unaffected by an absence of the protein. However, the hypoglossal nerve was significantly less likely to extend towards the tongue in TAG-1 null homozygous embryos than in heterozygotes. This suggested that TAG-1 might be important for the guidance of hypoglossal axons.

Neural function depends on precise wiring of axon during development. Previous studies have demonstrated that the erythropoietin producing hepatocellular carcinoma (Eph) family of tyrosine receptor kinases is crucial for the proper development of a number of neural circuits in the mammalian central nervous system (CNS). Mice lacking Eph receptors have been shown to exhibit deficits in pathways which include the thalamocortical, callosal, retinal and corticospinal tract. Due to the large number of Eph family members, the relative contribution of each receptor to axon pathfinding and neural function remains elusive. In this thesis, I have addressed the function of EphA4, EphB2 and EphB3 in the regulating the formation of interhemispheric projections within the forebrain and motor axon connections within the spinal cord using EphA4, EphB2 and EphB3and combinatorial null mice. To perform a detailed examination of the process of axon guidance regulated by these receptors within the forebrain, high resolution magnetic resonance imaging (MRI), immunofluorescence and in vivo stereotactic fluorescent labeling were performed. This work resulted in the development and validation of MRI-based analytic tools performed using EphB2 mutants which we have previously shown to exhibit specific morphologic defects in the anterior commissure (AC). Analysis of EphA4 null mice using high resolution MRI revealed for the first time that in addition to errors of midline crossing, loss of EphA4 activity results in a positional reorganization of the rostral AC. Results demonstrate that while EphB2 and A4 each regulate distinct aspects of guidance within ACpp, these receptors also operate cooperatively to control the guidance of axons in the pars anterior of the AC, a pathway not been previously implicated in Eph-mediated guidance. With respect to the spinal cord, mice deficient in EphB2 and EphA4 display prominent axon guidance errors in the medial subsets of the lateral motor column (LMCm); neurons which normally innervate ventral limb musculature. Finally, I have addressed the functional effect which Eph mutants exhibit with respect to motor behavior by examining a detailed set of motor coordination parameters

Directed regeneration of axons in the CNS has potential for the treatment of CNS disorders and injuries. In contrast to mammals, following optic nerve lesion zebrafish regenerate axons that navigate to their correct targets and form new synapses leading to functional recovery. Correct pathfinding is thought to rely on a range of molecular cues in the CNS which the growing axon expresses receptors for. However, the specific guidance cues are not well elucidated. It is likely that a proportion of them will be the same as during development, while some may be specific to regeneration. Alternatively, axons may simply retrace former trajectories guided by the molecular environment or mechanical constraints of degenerating tracts, as demonstrated in the mammalian PNS. To elucidate this, we investigated regeneration in the astray/robo2 knockout mutant which exhibits misprojection of optic axons during development leading to the establishment of ectopic tracts. We show that degenerating tracts do not provide a strong guidance cue for regenerating axons in the CNS as ectopic tracts in the astray mutant are not repopulated following lesion despite presenting a similar environment to entopic degenerating tracts. We also find that as astray mutant (knockout) and robo2 morphant (transient knockdown) projection and termination errors persist in the adult, it is clear that there is not an efficient correction mechanism for large-scale pathfinding errors of optic axons during development. In addition, we find a reduced importance of the axon guidance receptor Robo2 and its repellent ligand Slit2 for pathfinding during regeneration as specific developmental pathfinding errors of optic axons in astray mutants are corrected during adult optic nerve regeneration and global overexpression of Slit2 elicits pathfinding defects during development but not regeneration. To address regeneration-associated gene regulation in axotomised retinal ganglion cells, we carried out a microarray analysis. We found that many genes detected as a gradient in the adult retina during regeneration are not differentially expressed in the embryonic eye, despite having distinct expression patterns in other embryonic tissues. Of the genes which exhibit strong differential expression in the retina of both regenerating adults and developing embryos, foxI1 is one of the most interesting candidates as other fox genes have been implicated in axon guidance and due to its highly restricted retinal expression pattern. Surprisingly, further investigation has revealed that foxI1 knockout mutant embryos have retinotectal projections which appear normal in terms of axon pathfinding and mapping. Another family of genes indicated by the array, which are cytosolic phosphoproteins known to be involved in the signal transduction cascade of multiple inhibitory guidance cues during axon growth, are the crmps. Knocking down crmp2 with morpholinos during development resulted in a sparser innervation of the tectum with individual axons which trend towards having less complex arbors with shorter branches and reduced overall axon length. As a whole this work adds to our current knowledge of optic axon guidance during development and regeneration and the relative importance and effect of selected potential guidance cues, which may help toward informing future mammalian CNS regeneration research.

UNC-6/Netrin guides circumferential migrations along the dorso-ventral axis in C.elegans. Its receptors, UNC-40/DCC and UNC-5, mediate both attraction and repulsion of migrating cells or growth cones from sources of UNC-6. seu-2(ev523)and seu-3(ev555) (suppressor of ectopic unc-5) were identified as suppressors of ectopic UNC-5 in the touch receptor neurons(Colavita and Culotii, 1998). Like other components of UNC-6 signaling, seu-2 and seu-3 have roles not just in the migration of axon growth cones, but also in the repulsive migration of other cell types, specifically the distal tip cells (DTCs). Similar to observations in the touch receptor neurons, both seu-2 and seu-3 are able to suppress ectopic expression of the UNC-5 receptor in the DTCs. Genetic analysis of seu-2; seu-3 double mutants reveals that these genes function within the same signaling pathway for repulsive unc-6 guidance. seu-2 also appears to act in attractive unc-6 guidance. Mutations in seu-2 result in ventral to dorsal axon guidance defects in the HSN and ray 1 neurons. Double mutant analyses of seu-2 with either unc-40 or unc-6 null mutations exhibited HSN and ray 1 axon guidance defects at similar penetrance to either single mutant. These results suggest that seu-2 functions in the attractive unc-6-unc-40 dependent signaling pathway for HSN and ray 1 axon guidance. seu-2 was found to encode a G protein coupled receptor. Whole genome sequencing was used to identiy that seu-3 encodes the novel protein K09C6.9. K09C6.9 is predicted secreted protein that is expressed throughout development. Taken together, the phenotypes, method of isolation and genetic interactions of seu-2 and seu-3 make them interesting candidate mediators of UNC-6 signaling. I utilized genes, such as seu-2 and seu-3, to further elucidate other signaling components governing cell migration and axon guidance.

Healthy pre-natal development of the mammalian visual system requires that retinal ganglion cell (RGC) axons navigate a precise path to their targets in the thalamus and superior colliculus by making a precise series of turns determined by the complex interactions between growth cone and extracellular environment. One important choice point for RGC axons is the crossing of the midline at the optic chiasm, where ipsilateral/ contralateral sorting takes place. In this thesis a novel image analysis method using steerable filters for quantifying the gross orientation and turning of axons from a static image (such as from DiI filled axons) is presented. This method was applied to understanding Slit dependent axon guidance at the mouse optic chiasm. It was possible to quantify the differences at the chiasm between the wildtype and various classes of mutants involving heterzygous or homozygous knockout of the Slit1 and the Slit2 genes. Assessment was in terms of the spatial distributions in axon density and axon orientation as derived from DiI labeled RGCs originating from one eye. The animals were assessed at embryonic day 13.5. To my knowledge this is the first quantification of its kind in the field of axon guidance. It was found that there were strong statistical differences from wildtype in both the Slit1-/-;Slit2-/- and Slit1+/+;Slit2-/- knockouts in terms of both axon density and axon orientation across large extents of the chiasm. In both these knockouts it was found that the changes in axon density were localised to the anterior region of the chiasm, but the changes in axon orientation were spread across almost the entire extent of the chiasm. No other combination of the Slit1 and Slit2 knockouts for which embryos could be generated showed significant differences from wildtype in terms of spatial changes in axon density or axon orientation. No embryos were generated for the Slit1+/-;Slit2-/- combination. No changes in the spatial distribution of axon density or axon orientation were found between the Slit1-/-;Slit2-/- and Slit1+/+;Slit2-/- knockouts, suggesting that in terms of these two quantities, the two phenotypes are indistinguishable. This evidence suggests that the role of Slit2 is more important than the role of Slit1 at the optic chiasm in terms iii of axon guidance. In addition, the gradients of mRNA expression of Slit1 and Slit2 were quantified using in situ hybridisation, and these data were used to compare the mRNA gradients with the orientation and turning of axons in both the wildtype and Slit1/Slit2 knockout chiasms. Although this provided a powerful visualisation tool, no simple mathematical relationship was found between the mRNA gradient of Slit1 or Slit2 and the orientation or turning of axons at the optic chiasm. These approaches now provide an important suite of methods for spatial analysis of axon tracts and molecular gradients in axon guidance.

UNC-6/Netrin guides circumferential migrations along the dorso-ventral axis in C.elegans. Its receptors, UNC-40/DCC and UNC-5, mediate both attraction and repulsion of migrating cells or growth cones from sources of UNC-6. seu-2(ev523)and seu-3(ev555) (suppressor of ectopic unc-5) were identified as suppressors of ectopic UNC-5 in the touch receptor neurons(Colavita and Culotii, 1998). Like other components of UNC-6 signaling, seu-2 and seu-3 have roles not just in the migration of axon growth cones, but also in the repulsive migration of other cell types, specifically the distal tip cells (DTCs). Similar to observations in the touch receptor neurons, both seu-2 and seu-3 are able to suppress ectopic expression of the UNC-5 receptor in the DTCs. Genetic analysis of seu-2; seu-3 double mutants reveals that these genes function within the same signaling pathway for repulsive unc-6 guidance. seu-2 also appears to act in attractive unc-6 guidance. Mutations in seu-2 result in ventral to dorsal axon guidance defects in the HSN and ray 1 neurons. Double mutant analyses of seu-2 with either unc-40 or unc-6 null mutations exhibited HSN and ray 1 axon guidance defects at similar penetrance to either single mutant. These results suggest that seu-2 functions in the attractive unc-6-unc-40 dependent signaling pathway for HSN and ray 1 axon guidance. seu-2 was found to encode a G protein coupled receptor. Whole genome sequencing was used to identiy that seu-3 encodes the novel protein K09C6.9. K09C6.9 is predicted secreted protein that is expressed throughout development. Taken together, the phenotypes, method of isolation and genetic interactions of seu-2 and seu-3 make them interesting candidate mediators of UNC-6 signaling. I utilized genes, such as seu-2 and seu-3, to further elucidate other signaling components governing cell migration and axon guidance.

During development the nuclei of the thalamus form reciprocal connections with specific regions within the cortex. These connections give rise to the thalamocortical tract. The processes by which axons of the thalamocortical tract are guided to their target regions are poorly understood. It has been shown that diffusible or membrane bound factors can have a chemoattractive or chemorepulsive effect on the tip or growth cone of the axon. Thalamocortical axons may also be guided along ‘pioneer’ axon populations that form a scaffold along which axons may grow. The transcription factor Pax6 has been shown to have a role in a variety of developmental processes such as neuronal patterning, proliferation, migration and axon guidance. It is known that Pax6 is involved in the development of the thalamocortical tract but its exact role is unknown. To explore the role that Pax6 plays in the development of the thalamocortical tract I have used two different mouse models, the small eye (Pax6Sey/Sey) mouse which lacks functional Pax6, and a conditional Pax6 knock-out (Pax6cKO) mouse made using a Gsh2 Cre line that specifically reduces Pax6 expression in the ventral telencephalon and prethalamus. Using the Pax6Sey/Sey mouse I show that thalamocortical axons do not enter the ventral telencephalon in the absence of Pax6 and that a small number of axons incorrectly enter the hypothalamus. In addition axons found within the ventral telencephalon of the mutant do not originate from the thalamus but instead originate from cells within the ventral telencephalon itself. I have found that the expression of guidance molecule Robo2 is reduced in the Pax6Sey/Sey mouse, which may explain why thalamocortical axons enter the hypothalamus. When Pax6 expression is reduced at the prethalamus and ventral telencephalon using the Pax6cKO mouse I show that the majority of thalamocortical axons reach the cortex normally but some axons become disorganized within the thalamus. Pioneer axons which emanate from the prethalamus normally guide thalamocortical axons through the diencephalon but in the Pax6cKO I report that these axons are reduced which may explain the disorganization of thalamocortical axons within the thalamus. Taken together the data from these two models demonstrate that for the thalamocortical tract to form normally Pax6 expression is required in both the cells of the thalamus and in cells that lie along the route of the tract. In addition I provide evidence that Pax6 may influence axon guidance by controlling the expression of guidance molecules and the development of pioneer axon tracts.

Development of the visual system involves the precise orchestration of neural connections between the retina of the eye, the thalamus (dorsal lateral geniculate nucleus; dLGN) and the superior colliculus (SC). During early development, receptor molecules on the growth cones of retinal ganglion cell (RGC) axons sense molecular guidance cues in the extra cellular matrix (ECM) that define their route and branching behaviour within the visual system. Heparan sulphate proteoglycans (HSPGs) are ECM molecules composed of a core protein and a variable number of disaccharide residues that have been implicated in mediating axon guidance. HSPGs are modified by a number of enzymes that contribute to their structural diversity. Based on this structural diversity; the “heparan sulphate code” hypothesis of Bulow and Hobert (2004) postulated that different HSPG modifications confer different axon navigation responses as the growth cones traverse the local environment. To investigate the roles played by specific modifications of HSPG molecules in the guidance of axons, we examined two lines of mutant mice harbouring mutations in the genes encoding HSPG modifying enzymes, Heparan sulphate-6-O-sulphotransferase-1 (Hs6st1) and Heparan sulphate-2-O-sulphotransferase (Hs2st). These two mutant lines were generated through the use of gene trapping. Previous observations of RGC axon development in the two mutant lines revealed distinct axon guidance errors at the optic chiasm. Loss of Hs6st1 sulphation resulted in RGC axons navigating ectopically into the contralateral eye. Loss of Hs2st sulphation resulted in RGC axons navigating outside the normal boundary of the optic chiasm. Early observations suggested that both Hs2st sulphation and Hs6st1 sulphation have distinct, non-overlapping actions and thus, influence different axon guidance signalling pathways at the optic chiasm. Based on our findings and previous work describing the expression patterns and functions of the chemo-repellent axon guidance molecules, Slit1 and Slit2 at the optic chiasm and their Robo2 in the retina, we formulated the hypothesis of an HSPG sulphation code where Hs2st sulphation is specifically required for Slit1-Robo2 signalling and Hs6st1 sulphation is specifically required for Slit2-Robo2 signalling at the optic chiasm. To further our understanding of the roles Hs2st sulphation and Hs6st1 sulphation have on axon guidance, we looked at a number of key choice points that navigating axons encounter and are known to be influenced by Slit signalling. Further observations of RGC axons at the optic chiasm of Hs2st-/- mutants and Hs6st1-/- mutants showed distinct axon guidance phenotypes, both resulting in statistically significant increases in the width of the optic chiasm at the midline. While Hs6st1 sulphation had no effect on RGC axon navigation within the eye (possibly due to 6-O-sulphation compensation by Hs6st3); the loss of Hs6st1 sulphation at the dLGN resulted in a significant increase in the defasciculation of the optic tract. Observations of other axonal tracts influenced by Slit signalling revealed the importance of Hs2st and Hs6st1 sulphation in aiding callosal axons to successfully traverse the midline in corpus callosum development. Observations of the thalamocortical (TCA)/corticothalamic (CTA) tracts revealed that neither Hs2st sulphation nor Hs6st1 sulphation was required for the development of the mouse TCA tract (the latter may be explained by 6-O-sulphation compensation by Hs6st2). To test whether Hs2st and Hs6st1 enzymes have redundant functions in optic chiasm development, we attempted to create Hs2st-/-/Hs6st1-/- double mutants. A PCR genotyping strategy was developed for the identification of Hs6st1 animals and showed that Hs6st1-/- mutants had high postnatal lethality with only 3% of the offspring surviving to weaning while Hs2st-/-/Hs6st1-/- double mutants all died very early during embryonic development. Observations of Hs2st-/-/Hs6st1+/- mutants and Hs2st+/-/Hs6st1-/- mutants that lacked three of the four Hst alleles showed no differences when compared to single Hst knockouts. Finally, we showed that altered Slit expression at the optic chiasm and Robo expression in the retina could not explain the mutant phenotypes observed in Hs2st-/- mutants and Hs6st1-/- mutants, and therefore we hypothesized that Hs2st sulphation and Hs6st1 sulphation regulate distinct aspects of Slit-Robo signalling at the surface of the navigating axon growth cone.