Molecular Analysis of Tweety Family Genes in Development and Tissue Homeostasis

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Identified in animals, plants, and even simple eukaryotes, the tweety gene family is thought to encode gated chloride channels important in the maintenance of cell volume. In vertebrates, the tweety gene family is highly conserved and consists of three members, ttyh1, ttyh2, and ttyh3, all of which have strikingly different expression patterns in the developing embryos. ttyh1 and ttyh3, display almost mutually exclusive expression patterns in the developing nervous system, with ttyh1 expressed in the proliferative zone and ttyh3 present almost exclusively in post-mitotic regions. ttyh2 is expressed in the eye and cranial nerves. Additionally, both ttyh1 and ttyh3 are differentially expressed in RNA-Seq screens for genes that regulate the ability of embryos to mount a profound compensatory or regenerative response following genetic and physical perturbations. Despite the strong conservation in the vertebrate lineage, and being implicated in a wide range of human diseases, particularly aggressive cancers, this family of genes has received relatively minimal attention. Given the dynamic expression of ttyh1 and ttyh3 genes in embryogenesis, adult pathologies, and regenerative responses, the overarching goal of this proposal is to elucidate their role in the developing embryo and in developmental plasticity and homeostasis. Specific Aim #1 will focus on determining the roles of ttyh1 and ttyh3 in normal embryogenesis using gain-of-function and loss of function experiments. Experiments will employ Xenopus laevis, a classic model system for dissecting the role of developmental genes and an emerging model for human disease, to perform standard, well-vetted approaches of ectopic expression using mRNA injection and CRISPR to generate loss-of-function. Embryos will be assayed at key developmental stages using whole mount in situ hybridization with a range of marker genes for the major developmental pathways and cell types and single cell RNA-Seq to obtain global gene expression at a single cell level with the goal of identifying the precise cell types and networks affected by the gain- and loss-of-function experiments. Specific Aim #2 will focus on determining the role of ttyh1 and ttyh3 in early embryonic neural plasticity, specifically their role in the recovery that occurs following physical (rotation of the anterior-posterior axis) or genetic (overexpression of the Notch signaling pathway) perturbation. Genetic and physical perturbation will be performed in a ttyh1 or ttyh3 mutant background generated via a CRISPR approach. The effect of ttyh1 and ttyh3 knockouts on the recovery response will be assayed using the complementary approaches of whole mount in situ hybridization and single cell RNA-Seq. Taken together, the proposed experiments will engage an eager cadre of undergraduate students—both in course-based research and mentored lab research—in an effort to elucidate the role of the tweety gene family and address fundamental, yet poorly studied, problems in developmental biology that have broader implications for regenerative medicine.