Single-cell RNA Sequencing Analysis and Experimental Designs to Investigate the Role of Nitric Oxide Signaling in Progenitor Cell Survival in Ciona
Gurgis, Alexandra Marie
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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.