UC Santa Barbara

Neural tube closure (NTC) is a fundamental process in the development of all chordates. During NTC, the developing central nervous system is transformed from a flat sheet of neural precursor cells into a hollow neural tube, which is the basic form that persists into the mature central nervous system. When the neural tube fails to properly close, neural tube defects (NTDs) result. NTDs are the most prevalent category of human birth defects, and they occur in about 1 out of every 1000 births. Previously, we discovered a NTC mutant (named bugeye) in the basal chordate Ciona savignyi that results from a defect in the gene encoding a T-Type Calcium Channel (Cav3). We found that Cav3s are also required for proper NTC in the amphibian Xenopus suggesting a conserved function among chordates. Loss of Cav3 leads to defects in anterior NTC characterized by the brain protruding from the larval head. Here, I present a study expanding these findings to a new Cav3 mutant in the related species C. robusta. RNAseq analysis of bugeye mutants from both species reveals misregulation of several transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane protein are aberrantly upregulated in the mutant. They are expressed in the closing neural tube and knockdown of either in wildtype larvae phenocopies the bugeye mutant. We speculate that these molecules play a transitory role in tissue separation and adhesion during NTC, and the failure to downregulate them properly leads to an open neural tube. One hypothesis surrounding Cav3’s role in NTC, is that it acts as a mechanical developmental checkpoint, detecting the completion of NTC and signaling through calcium to move on to the next developmental process. To further explore this, I used various pharmacological inhibitors as well as manual disruption of the progression of NTC to recapitulate the open brain phenotype. The RNAseq investigation revealed a suite of transcripts that are abnormally upregulated in the bugeye mutant, but their expression during normal development declines sharply following neurulation. Other transcripts displaying similar expression profiles may play a role in NTC. Using publicly available RNA-sequencing data I identified several additional candidate genes. Further work is necessary to determine what role these genes play in NTC, but this demonstrates a valid approach for identifying genes with roles in specific developmental processes in Ciona.