Assembly of neuronal networks with distinct patterns of connectivity during nervous system development involves the growth, extension and branching of axons and dendrites. Over the years genetic and biochemical studies in model organisms have contributed significantly in identifying mechanisms regulating axon growth and extension. However the molecular mechanisms underlying axon branching remain unclear. The egg-laying neuronal circuitry in C. elegans has proven to be a robust system for identifying and characterizing novel genes involved in neuronal morphology. This circuitry which mediates egg-laying behavior in nematodes is composed of two families of motorneurons, HSNs and VCs, which are among the most branched neurons in C. elegans. A genetic screen for axon branch defects in the egg-laying neurons identified png-1 to disrupt neuronal morphology including axon branching. png-1 encodes a Peptide: N-glycanase (PNGase), a conserved cytosolic enzyme that removes N-linked sugar moieties from glycoproteins. In this thesis I present my work characterizing and examining png-1 and its role in mediating axon branching. Mutations in png-1 resulted in excessive ectopic axon branching in the VC4 and VC5 egg-laying neurons as well as branching in the normally unbranched AVL and DVB neurons. Behavioral analysis in these mutants revealed defects in egg-laying behavior and mild in-utero egg retention phenotypes. Cellular characterization shows ubiquitous expression of png-1 in many tissues including vulva cells, muscles, gonads, and neurons. My analysis also shows that png-1 acts both cell-autonomously and cell non-autonomously from neurons and epithelial cells to restrict axon branching around the vulva. Using a candidate gene approach I identified a deletion allele of the DNA repair gene, rad-23, to display axon branching defects and interact with png-1 within a common pathway to regulate axon branching. Additionally, through a genetic modifier screen for enhancers and suppressors of VC4-5 branching defects in png-1, I identified a new allele of sax-2 as an enhancer mutation. sax-2 encodes a scaffolding protein that regulates the activity and localization of sax-1, an NDR kinase. Examination of neuronal phenotypes in sax-1 and sax-2 mutants revealed similar png-1 like defects in VC4-5. Genetic analysis of the double mutants png-1;sax-1 and png-1;sax-2 revealed strong synergistic phenotypes suggesting that png-1 and sax-1/sax-2 function in parallel pathways to regulate axon branching. In summary, this thesis reveals novel components and pathways in the regulation of neuronal branching.

circRNAs are covalently closed RNA molecules recently re-discovered thanks to the advances in RNA-seq technology. They are produced by the canonical spliceosome in a non-canonical splicing process, named back-splicing. Heterogeneous in internal composition and highly stable, circRNAs regained the attention of neuronal biologists because of their enrichment in brain and neuronal compartments. Moreover, several pioneering studies revealed a fine orchestration of circRNA expression in crucial stages of neuronal development, such as synaptogenesis. The growing evidence of circRNA enrichment in synapses raises the intriguing question as to the yet unknown molecular mechanisms leading to this unique neuronal sub-compartmentalization. In addition, in which of the compartments composing the synapse – dendrites and axon – circRNAs preferentially localize, is still largely elusive. Here, I have focused specifically on the pre-synaptic compartment – the axon – during specific stages of neuronal development. The proper development of the axon is crucial to guarantee the correct synapse formation. Impairments in this process can lead to severe neurodevelopment diseases. I explored circRNA expression in the axonal compartment, shedding light on circRNA distribution in and trafficking to the neuronal distal compartment. To reach these goals, I used Xenopus laevis retinal ganglion cell (RGC) developing axons as a model. The results presented in this thesis highlight an abundant expression of circRNAs in the axonal compartment, where they are enriched compared to the somatic one. The enrichment in axons led me to deeper explore their preferential axonal sub localization, by investigating how they reach the neuronal distal compartment. circDDX17 was selected as reference model of axonal circRNAs. Its tracking within the axon revealed an heterogenous distribution and shape. circDDX17 trafficking along the axon displayed an anterograde preferential directionality and slow speed giving hints to uncover the molecular mechanisms of circRNA translocation to the axonal compartment. Bioinformatic analysis revealed that the RNA-RBP complex formation, the most common and described mechanism of axonal transport, could underlie circRNA intracellular translocation. Taken together, my data uncover the axonal circRNA population and characterize their localization in the neuronal distal compartment.

Neurons establish complex networks within the nervous system allowing for rapid cell-cell communication via their long, thin axonal processes. These wire-thin projections are susceptible to a number of insults or injuries, and axonal damage can lead to disruption in signal propagation and an overall dysfunction of the neural network. Recent research focused on investigating the underlying mechanisms of injury-induced axon degeneration led to the discovery of a number of endogenous, pro-degenerative molecules such as dSarm/Sarm1, Highwire/Phr1, and Axundead. These signaling molecules are thought to execute axon degeneration in response to injury locally within the distal severed axon, but the exact mechanism of action is unclear. To further identify novel participants of the axon death signaling cascade, we performed an unbiased forward genetic mutagenesis screen using the sensory neurons within the adult wing of Drosophila melanogaster. We identified a novel role for the C2H2 zinc finger transcription factor, Pebbled (Peb)/Ras-responsive element binding protein 1 (RREB1) in partially suppressing injury-induced axon degeneration. Loss of function peb mutant glutamatergic neurons present two distinct axon degeneration defects: either complete protection from axotomy, or they exhibit a novel phenotype in which axons fragment into long, continuous pieces instead of undergoing complete degeneration. Additionally, we show an enhancement of the peb protective phenotype when dSarm levels are decreased, but not with reduced levels of axundead. These data provide the first evidence of a transcription factor involved in regulating injury-induced axon degeneration signaling in vivo.

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.

Thesis advisor: Laura Anne Lowery / Thesis advisor: Eric Folker / In order to form the exact synaptic connection required for proper neurological function, the growing tip of the neuron hosts an orchestra of hundreds of different proteins interacting with extracellular cues to steer neuron growth in the right direction. The goal of our current research is to study several of the components of this pathway, known as the TACC family. Here, we present a detailed structure/function analysis of the TACC family in regards to binding and activity with other proteins in the growth cone. We investigate the function of TACC3 in mediating neuron outgrowth and guidance in vivo. We have found structural elements of the TACC family that enable their activity. Studying these conserved structures and functions of the TACC family will enable greater understanding of the entire process of cytoskeletal regulation and neurodevelopment. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.

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.

Universiẗat, Diss., 1998--Konstanz.

Universiẗat, Diss., 2003--Münster (Westfalen).