UC Santa Barbara

My dissertation focuses on identifying the role of T-type voltage- gated Ca2+ channels (VGCC) in neural development. This research uses invertebrate and vertebrate models - specifically, the ascidian Ciona and the frog Xenopus, to span chordate development. Our lab previously identified a Ciona savignyi (C. savignyi) mutant, bugeye, that phenocopies exencephaly in humans by displaying an open brain. In C. savignyi bugeye, we see a dramatic decrease of Cav3 expression using qPCR. The Cav3 gene encodes for the single T-type VGCC in the Ciona genome. T-type VGCCs are part of a larger family of VGCCs, and are distinguished as being “low- voltage” because they are activated by small depolarizations in membrane potential to increase Ca2+ permeability. T-type VGCCs had not been implicated in embryonic development prior to these findings. Moreover, our lab found that EphrinA-d, a cell repulsion protein, was overexpressed in bugeye embryos, and thus was likely regulated by Ca2+. We found that overexpression of EphrinA-d phenocopied the open brain phenotype in wild-type embryos.

More recently the Smith lab discovered a new exencephaly mutant in a related species, Ciona robusta (C. robusta). To identify the causative gene, I performed linkage analysis and a complementation test which pointed toward Cav3, the causative gene in C. savignyi bugeye. RT-PCR analysis of Cav3 indicated no change in expression levels compared to wild- type siblings. However, sequence analysis of the mutant C. savignyi Cav3 gene revealed multiple amino acid changes, many in areas of functional importance. The Smith lab followed up on these observations and performed RNAseq on both C. savignyi bugeye and C. robusta bugeye embryos to identify novel genes involved in neural tube closure. I extended research on Cav3 by examining a vertebrate model, the African clawed frog Xenopus laevis, which provided more tools to study morphogenesis. In Xenopus, I found that morpholino oligonucleotide knockdown of Cav3.3 caused a wide range of developmental defects, including delays in gastrulation and neurulation, craniofacial defects, a dorsal flexure phenotype, and an apparent mis-migration of melanocytes.

To identify a cellular pathway, I investigated Calpain2, a Ca2+ -dependent protease involved in the Wnt/Ca2+ pathway during gastrulation and neurulation. Knockdown of Calpain2 shows similar phenotypes as knockdown of Cav3.3, and I observed abolishment of tagged Calpain2L fluorescence, mislocalization of Calpain2L in nearby cells, and increased surface area for cells affected by the knockdown. Furthermore, we found loss of apical actin localization along the hinge points of the folding neural tube. This apical constriction is essential for normal neural tube closure. Our results indicate that Cav3.3 is necessary for neural induction and/or differentiation and mechanical aspects of gastrulation and neurulation. Future research will aim to identify the mechanisms through which Cav3.3 affects these events.