A systematic analysis of iron and iglr genes’ function in axon guidance within the ventral nerve cord of Caenorhabditis elegans

For the nervous system to develop properly, axons must connect neurons into networks by navigating to their target destinations. A large proportion of genes containing extracellular Leucine-Rich Repeats (eLRRs) function in neurodevelopment, including in axon guidance. The objective of this thesis is to identify novel eLRR genes in the Caenorhabditis elegans’ lron and iglr gene families that function in axon guidance. Animals with mutations in these genes were observed with pan-neuronal and pioneer markers to identify mutations that induced axon guidance defects. Six mutants had significant axon guidance defects. In addition, iglr-2 mutants were found to have fasciculation defects in the left ventral nerve cord. lron-11 mutants had the most penetrant axon guidance defects. Therefore, lron-11 animals were further characterized with several inter and motor neuron markers and further axon guidance defects were identified. This research suggests that lron-11 and possibly other lron/iglr genes function as receptors in axon guidance

Drosophila semaphorin2b is Required for the Axon Guidance of a Subset of Embryonic Neurons

Background: The process of axon guidance is important in establishing functional neural circuits. The differential expression of cell-autonomous axon guidance factors is crucial for allowing axons of different neurons to take unique trajectories in response to spatially and temporally restricted cell non-autonomous axon guidance factors. A key motivation in the field is to provide adequate explanations for axon behavior with respect to the differential expression of these factors. Results: We report the characterization of a predicted secreted semaphorin family member, semaphorin2b (Sema-2b) in Drosophila embryonic axon guidance. Misexpression of Sema-2b in neurons causes highly penetrant axon guidance phenotypes in specific longitudinal and motoneuron pathways; however, expression of Sema-2b in muscles traversed by these motoneurons has no effect on axon guidance. In Sema-2b loss-of-function embryos, specific motoneuron and interneuron axon pathways display guidance defects. Specific visualization of the neurons that normally express Sema-2b reveals that this neuronal cohort is strongly affected by Sema-2b loss-of-function alleles. Conclusions: While secreted semaphorins have been implicated as cell non-autonomous chemorepellants in a variety of contexts, here we report previously undescribed Sema-2b loss-of-function and misexpression phenotypes that are consistent with a cell-autonomous role for Sema-2b. Developmental Dynamics 242:861–873, 2013. © 2013 Wiley Periodicals, Inc. Key Findings Misexpression of the secreted semaphorin Sema-2b in neurons results in specific axon guidance phenotypes. Both Sema-2b loss-of-function and misexpression phenotypes are congruent with a cell-autonomous role for Sema-2b. Novel axon guidance phenotypes caused by Sema-2b loss-of-function mutations are characterized

Regulation of CNS and motor axon guidance in Drosophila by the receptor tyrosine phosphatase DPTP52F

Receptor-linked protein tyrosine phosphatases (RPTPs) regulate axon guidance and synaptogenesis in Drosophila embryos and larvae. We describe DPTP52F, the sixth RPTP to be discovered in Drosophila. Our genomic analysis indicates that there are likely to be no additional RPTPs encoded in the fly genome. Five of the six Drosophila RPTPs have C. elegans counterparts, and three of the six are also orthologous to human RPTP subfamilies. DPTP52F, however, has no clear orthologs in other organisms. The DPTP52F extracellular domain contains five fibronectin type III repeats and it has a single phosphatase domain. DPTP52F is selectively expressed in the CNS of late embryos, as are DPTP10D, DLAR, DPTP69D and DPTP99A. To define developmental roles of DPTP52F, we used RNA interference (RNAi)-induced phenotypes as a guide to identify Ptp52F alleles among a collection of EMS-induced lethal mutations. Ptp52F single mutant embryos have axon guidance phenotypes that affect CNS longitudinal tracts. This phenotype is suppressed in Dlar Ptp52F double mutants, indicating that DPTP52F and DLAR interact competitively in regulating CNS axon guidance decisions. Ptp52F single mutations also cause motor axon phenotypes that selectively affect the SNa nerve. DPTP52F, DPTP10D and DPTP69D have partially redundant roles in regulation of guidance decisions made by axons within the ISN and ISNb motor nerves

Doctor of Philosophy

dissertationBrain wiring is critical for normal brain function, and brain connection defects have been described in many neurodevelopmental disorders. Accurate central nervous system connectivity relies on precise axon navigation. The cellular and molecular mechanisms governing axon guidance during development are important but not well understood. In this dissertation, I utilize zebrafish, a powerful vertebrate model in developmental and genetic studies, to determine the molecular mechanisms regulating axon guidance in vivo. We specifically determine the role of the two molecules FoxP2 and serotonin (5-HT) in axon guidance. We demonstrate that FoxP2, a forkhead-domain transcription factor, is not required for axon guidance, but it reduces the expression levels of a synaptic protein Cntnap2. We also establish that 5-HT and its receptor Htr2a are important for axon guidance. The regulation of 5-HT on axon guidance is mediated by the tyrosine kinase Ephrinb2a. Moreover, we discover that axon guidance defects induced by environmental hypoxia can be rescued by serotonin reuptake inhibitor fluoxetine. I have also developed and implemented a PCR-based high resolution melting analysis (HRMA) for large-scale genotype screening and characterization of genes involved in axon development. Taken together, this work identifies a novel mechanism involved in central nervous system connectivity, and indicates raphe 5-HT neurons act as a sensor to alterations in the developmental environment

Insights into the Control of Growth and Axon Guidance by the Drosophila Insulin Receptor

The Drosophila insulin receptor (DInR) regulates a diverse array of biological processes, including growth, axon guidance, and sugar homeostasis. Growth regulation by DInR is mediated by the adaptor protein Chico, the Drosophila homolog of vertebrate Insulin-Receptor-Substrate (IRS) proteins. In contrast, DInR regulation of photoreceptor axon guidance in the developing visual system is mediated by the SH2/SH3 domain adaptor protein Dreadlocks (Dock). In vitro studies by others suggested that different parts of DInR interact with different adaptor proteins: five NPXY motifs, one situated in the juxtamembrane region and four in the signaling C-terminal tail (C-tail), were important for interaction with Chico. Yeast two-hybrid assays suggested that a different region in the DInR C-tail interacts with Dock. To test whether these sites are required for growth or axon guidance in the animal, in vivo add-back type experiments were conducted. A panel of DInR proteins, in which the putative Chico and Dock interaction sites had been mutated individually or in combination, were tested for their ability to rescue viability, growth, and axon guidance defects of dinr mutant flies. Sites important for viability were identified. In addition, mutation of all five NPXY motifs drastically decreased growth in both male and female adult flies, but did not affect photoreceptor axon guidance, showing that different binding sites on DInR control growth and axon guidance. Unexpectedly, mutation of both putative Dock binding sites, either individually or in combination, did not lead to defects in photoreceptor axon guidance. Finally, none of the seven putative ligands for DInR, the Drosophila insulin-like peptides (dilps), was required for directing photoreceptor axon guidance, although we found that dilp1, -2, -3, -4, and/or -5 are required for controlling whole animal allometry. Importantly, we showed that the developmental delay exhibited by dinr mutants is not a factor underlying their photoreceptor axon guidance defects. Together, these studies confirmed the role of Chico-interacting regions of DInR in regulation of growth in vivo. They demonstrated that DInR is necessary to control axon guidance in vivo and showed that this role is not simply a function of developmental timing. However, they leave open the mechanisms activating DInR in regulating axon targeting

Characterizing the Role of Key Planar Cell Polarity Pathway Components in Axon Guidance

An essential process to the development of the neural network of the nervous system is axon guidance. The noncanonical Wnt/Planar Cell Polarity pathway has been identified as an integral component in controlling the projection of axons during axon guidance. Prickle, ROR1 and ROR2 are PCP related proteins that do not have clearly defined roles in the process. This study aims to use zebrafish CoPA neurons as a model to study the roles of Prickle, ROR1, and ROR2 in axon guidance. Using in situ hybridization, morpholino knockdown, and CRISPR/Cas9 loss of function experiments were able to identify ror1, ror2 and prickle as potential required components in CoPA neuron axon guidance. Elucidating the role of these protein in axon guidance not only will increase our knowledge of the PCP pathway but it will also increase our understanding of the development of the nervous system

Where does axon guidance lead us?

During neural circuit formation, axons need to navigate to their target cells in a complex, constantly changing environment. Although we most likely have identified most axon guidance cues and their receptors, we still cannot explain the molecular background of pathfinding for any subpopulation of axons. We lack mechanistic insight into the regulation of interactions between guidance receptors and their ligands. Recent developments in the field of axon guidance suggest that the regulation of surface expression of guidance receptors comprises transcriptional, translational, and post-translational mechanisms, such as trafficking of vesicles with specific cargos, protein-protein interactions, and specific proteolysis of guidance receptors. Not only axon guidance molecules but also the regulatory mechanisms that control their spatial and temporal expression are involved in synaptogenesis and synaptic plasticity. Therefore, it is not surprising that genes associated with axon guidance are frequently found in genetic and genomic studies of neurodevelopmental disorders