(Georgia Institute of Technology, 2021-05)
Shearer, Tanner Reid
Bipolar tail neurons (BTNs) in Ciona robusta offer a unique chance to assess regulatory networks that give rise to cell behaviors during development. Like most cells within the embryo of Ciona robusta, BTNs develop following highly stereotyped yet dynamic programs. Neurogenin, a transcription factor, was found to be necessary and sufficient for BTN specification, and SIPA1l was observed to be downstream of Neurogenin activation. The present study sought to elucidate the role of SIPA1l in BTN development, and hypothesized that it was required for collective cell migration of BTNs. The gene encoding SIPA1l, Sipa1l, was reconstructed prior to analyzing its domains and its relatedness to the human orthologs Sipa1l1, Sipa1l2, and Sipa1l3. It was found that this gene in Ciona closely resembles its human counterparts in both the domains present in the protein it encodes, as well as, its amino acid sequence alignment. Sipa1l was then targeted using CRISPR/Cas9 in an attempted knock out condition wherein mixed results were obtained that are limited in interpretation. The fluorescent protein used to assess BTN development only labeled anterior BTNs and thus collective cell migration could not be assessed. Moreover, it was observed that Sipa1l did not affect anterior BTN migration or morphology but did appear to affect axonal outgrowth in select embryos. In all, the results of this investigation provide insights into the role of Sipa1l in BTN development and serve as a preliminary study that will prove useful to future researchers seeking to understand BTN development.
(Georgia Institute of Technology, 2021-05)
Gurgis, Alexandra Marie
The tunicate Ciona is a marine chordate whose biphasic life cycle, relatively simple transcriptome, and ability to be genetically manipulated via electroporation makes it an ideal model for studying phenomena of neurodevelopment and regeneration. During the metamorphosis of larvae to adults, nearly the entire central nervous system is eliminated and rebuilt, with the notable exception of the “Neck”, a compartment of quiescent neural progenitor cells. The mechanism by which these cells are spared from the wave of programmed cell death that occurs around them is not currently understood, but could reveal important principles of cell cycle regulation. In this work, I re-examine single-cell RNA sequencing data to further characterize differential gene expression in the Neck, with a particular emphasis on the nitric oxide signaling pathway as a potential suppressor of apoptosis. I also provide experimental constructs for future investigations of genes I believe to be relevant to understanding Neck cell survival, including designs for in situ hybridization probes and GFP reporters to verify gene expression in vivo, sgRNA primers for CRISPR/Cas9 knockouts, and peakshift primers to verify the efficacy of sgRNAs.