Identification of a Genetic Pathway Linking Temperature with Epigenetic Control of Gonad Determination in T. scripta

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Temperature regulates male or female development in many reptiles, including the redeared slider turtle Trachemys scripta elegans, by controlling whether the embryonic gonad develops as a testis or an ovary. However, the mechanism through which temperature influences gonad development has been a puzzle since the discovery of this phenomenon 50 years ago. Previously, the Capel lab identified two genes that show strong expression differences in the turtle gonad at male- versus female-producing temperatures. In this project, the lab will determine how the expression of these genes is controlled by temperature, and how the activity of these genes controls testis versus ovary development. Results from this work will establish the mechanism underlying the effect of temperature on male or female development. The lab is actively involved in using this project as a platform to engage young students and the public in scientific research. This project is also a strong platform to advocate for evidence-based science in setting environmental policies to mitigate the effects of climate change, expected to strongly bias male/female ratios in species where temperature influences gonad development.The Capel lab previously showed that transcription of the epigenetic regulator KDM6B and the RNA-splicing factor, RBM20, are elevated in the turtle gonad at male-producing temperature. KDM6B activates the testis pathway by removing repressive histone marks at DMRT1, a conserved regulator of testis development. In this project, the lab plans to identify the molecular pathway linking temperature with changes in KDM6B and RBM20 transcription and splicing. The lab hypothesizes that a known repressor of KDM6B, pSTAT3, is differentially phosphorylated at high female-producing temperatures through activation of TRP channels, Ca2+ influx, and high activity of CAMK. To test this hypothesis, markers of the male and female pathways will be measured after knockdowns of TRP channels expressed in the gonad, after interference with Ca2+ signaling, or after of pSTAT3 inhibitors. Experiments will (1) determine whether RBM20 is also a target of pSTAT3, or downstream of KDM6B, and (2) test the role of RBM20 using a knock-down approach. Splicoform variants will be analyzed at the two temperatures using the lab's new T. scripta genome assembly. Results will establish the molecular basis for temperature-dependent gonad determination, and may uncover a conserved role for splicing in gonad development. The lab engages students in science research through creation of lesson plans that incorporate cutting edge research into local classrooms, and programs that encourage girls to pursue STEM careers. The lab also brings this science to public attention through podcast interviews, contribution to Duke's ongoing Art of the Scientist exhibition, and Duke's science cafe' series.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.