P-element Induced Wimpy Testis (PIWI) Expression in the Central Nervous System of Ciona intestinalis is Moderated by Transcription Factors Otx and Bsh

Abstract

Ciona intestinalis is an ascidian species that has been a popular subject of research due to its proximity to vertebrates in evolution. The motile, swimming larvae of Ciona turn into sessile adults via metamorphosis, requiring the regeneration and restructure of cells after reaching maturity. Recent research suggested that ependymal cells in the Ciona central nervous system (CNS) may serve as neural stem cells by demonstrating that part of the adult neurons differentiate from these cells. Our lab’s single-cell RNA transcriptomics data has shown P-element–induced wimpy testis (PIWI) expression in the CNS, including some overlapping expression with the ependymal marker opsin3. Based on previous reports of PIWI function in maintaining genome integrity in the germline and neural progenitor cells, I investigated the regulation of PIWI expression in the Ciona central nervous system. Serial deletion of the endogenous piwi 5 kb intronic region led to a 300 bp region that drove expression. I have analyzed binding motifs in the 300 bp region of piwi enhancer and found multiple Otx binding sites. Otx overexpression and target gene repression, as well as binding site mutations, suggested that Otx directly binds to the piwi enhancer to activate transcription. I have also discovered that brain-specific transcription factor Bsh acts upstream of piwi to repress expression. Surprisingly, evidence shows that Otx activates bsh as well as piwi, suggesting a regulatory mechanism that modulates piwi expression. By investigating how piwi is regulated in the CNS, this study opens up the question of whether the properties of progenitor-like ependymal cells can be explained through known piwi function.