An Analysis of the Tweety Gene Family Members 1 and 3 and Their Role During Neural Development in Xenopus laevis

Neural development is a complex process regulated by numerous cell signaling pathways. The Notch signaling pathway plays an essential role in guiding development and regulating stem cell potency. One gene linked to the Notch pathway is tweety 1 (ttyh1), a member of the highly conserved tweety gene family. In vertebrates, the tweety gene family has 3 paralogous members: tweety 1 (ttyh1), tweety 2 (ttyh2), and tweety 3 (ttyh3). These genes are thought to encode calcium-dependent and independent, volume-sensitive chloride channels. Previous research has shown ttyh1 and ttyh3 to be highly expressed in the nervous system during development and adulthood, with ttyh3 showing broader expression in maturity. These genes have been further shown to have clinical relevance, with ttyh1 being associated with numerous diseases and cancers, and ttyh3 found to drive the proliferation and metastasis of tumor cells. This thesis will investigate the role of tweety genes 1 and 3 during neural development in Xenopus laevis by knocking them out using a CRISPR/Cas9 system and overexpressing them through the injection of synthetic tweety mRNA. Analysis of mutation efficiency following knockout injection showed significant induction of INDEL mutations at the target site, indicating the ability of this system to induce gene knockout in Xenopus laevis. Following tweety 1 overexpression, chromogenic in situ hybridization revealed changes in the expression of tubb2b and sox2 consistent with a function in maintaining neural progenitor cell potency. Tweety 3 overexpression induced less obvious and, in comparison, somewhat contrasting changes in sox2 and tubb2b expression. Overexpression of both genes induced distinct, prominent changes in embryo morphology. Together, these results indicate tweety 1 and tweety 3 as proteins that play a significant role in development.