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A Forward Genetic Study on Neural Tube Development


My dissertation work has focused on mapping and characterizing a spontaneous neural tube closure mutant, bugeye, in the marine, invertebrate chordate organism, Ciona savignyi. The work involved developing a new and rapid method of mutation mapping by next-generation DNA sequencing. This new method of mapping was tested on a previously identified Ciona intestinalis mutant whose genomic data was used to calibrate mapping parameters and the mapping program for Ciona genomes. After successful testing of the mapping program, it was applied to the new bugeye mutant in Ciona. Mapping revealed a unique and narrow signature of linkage in the Ciona bugeye mutant genomes over a predicted T-type calcium channel gene. Requirement for the T-type calcium channel gene in neural tube closure was tested by CRISPR mediated knockout in wild type embryos and definitively established causality of the gene for the bugeye mutant phenotype. The bugeye mutant was investigated for previously known characteristics of neural tube closure mutants such as neural specification, cellular polarity and apical constriction defects. My results did not support any of these possibilities and rather support the hypothesis that bugeye is a failure to maintain a sealed anterior neuropore. To further explore and expand this previously uncharacterized role for T-type calcium channel genes, I tested for conservation of function in the vertebrate model Xenopus. Knockdown of the orthologous and earliest expressed T-type calcium channel gene, CAV3.2, in Xenopus laevis embryos via splice-disrupting morpholinos resulted in an open brain phenotype with characteristics similar to that of the bugeye mutants – a failure to seal the anterior neuropore. The function of T-type calcium channels in neural tube closure was tested in both Xenopus and Ciona: cellular calcium fluctuations, using the genetically encoded calcium indicator, GCaMP3, were investigated in the context of neurulation and T-type calcium channel disruption. I also investigated a downstream target of T-type calcium channels and a previously known neural tube closure gene family, the EphrinA family of ligand-receptor signaling molecules. My results found that the bugeye mutant phenotype, open brain, could be rescued by down regulation of EphrinA signaling in the neural tube. The cumulative results allowed us to propose a model of T-type calcium channel function in neural tube closure via EphrinA signaling and suggest future directions to further explore this newly established role for T-type calcium channels in sealing the anterior neuropore.

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