The Role Of Tp53I11 In Regulating Apoptosis Of Rohon-Beard Neurons During Zebrafish Development

During development of the nervous system, far more cells are generated during embryogenesis than are needed in the adult organism. These surplus cells undergo a natural process of elimination called Programmed Cell Death (PCD). Multiple signaling pathways are responsible for regulating PCD, including the evolutionarily conserved tumor suppressor gene, TP53. Activation of the TP53 pathway begins a cascade of signals that can stop the cell cycle from turning on pro-apoptotic and repressing pro-mitotic genes. In zebrafish, Rohon-Beard primary sensory neurons undergo PCD during the early larval stages. These developmental cells participate in the embryonic escape response, and help to guide the growth of motor axons and nerves that innervate the trunk and tail of the fish. The RB cells are short-lived, and typically undergo apoptosis by 5 days post fertilization, after the spinal cord and branching tail nerves have begun developing normally. Recent work has shown that RB cells express the TP53- induced gene, TP53i11. Of the TP53-induced genes, relatively little research has been published about TP53i11. Since RB cells apoptose naturally only a few days after developing and TP53- induced genes on the whole play a critical role in signaling PCD, understanding the role of TP53-Induced Gene 11 in the apoptosis of RB cells may help our understanding of this evolutionarily conserved signaling pathway. Using Crispr/Cas9, we will knock out TP53i11 and assess the timing of PCD in RB cells. If TP53i11 is necessary for PCD in RB cells, we predict that these cells will not undergo apoptosis in the normal timeframe. To assess this potential phenotype, we will use fluorescent antibody immunostaining and confocal microscopy to count the number of RB cells at 5 days post fertilization with and without expression of TP53i11, when RB cells have normally been eliminated from the spinal cord. We expect a higher number of RB cells in the absence of TP53i11 if this gene is indeed necessary for developmentally regulated PCD to occur in these cells. In parallel, we will use in situ whole-mount mRNA hybridization to determine when and where TP53i11 is expressed during normal zebrafish development. These studies will advance our understanding of PCD during development, as well as providing a clearer picture of TP53 and its associated signaling pathways