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

温度通过控制胚胎性腺发育为睾丸还是卵巢来调节许多爬行动物的雄性或雌性发育，包括红海龟Trachemys scripta elegans。然而，自50年前发现这一现象以来，温度影响性腺发育的机制一直是一个谜。此前，卡佩尔实验室确定了两个基因，在雄性和雌性生产温度下，它们在海龟性腺中表现出强烈的表达差异。在这个项目中，实验室将确定这些基因的表达如何受温度控制，以及这些基因的活性如何控制睾丸与卵巢的发育。这项工作的结果将建立温度对雄性或雌性发育影响的机制。该实验室积极参与利用该项目作为一个平台，让年轻学生和公众参与科学研究。该项目也是一个强大的平台，倡导以证据为基础的科学制定环境政策，以减轻气候变化的影响，预计将强烈偏见的雄性/雌性比例的物种，温度影响性腺发育。卡佩尔实验室以前表明，转录的表观遗传调节KDM 6 B和RNA剪接因子，RBM 20，在海龟性腺在雄性生产温度升高。KDM 6 B通过去除DMRT 1（睾丸发育的保守调节因子）上的抑制性组蛋白标记来激活睾丸途径。在这个项目中，该实验室计划确定将温度与KDM 6 B和RBM 20转录和剪接变化联系起来的分子途径。该实验室假设，KDM 6 B的一种已知阻遏物pSTAT 3在高雌性产生温度下通过激活TRP通道、Ca 2+内流和CAMK的高活性而差异磷酸化。为了检验这一假设，将在性腺中表达的TRP通道敲低后、干扰Ca 2+信号传导后或pSTAT 3抑制剂后测量雄性和雌性途径的标志物。实验将（1）确定RBM 20是否也是pSTAT 3的靶标或KDM 6 B的下游，和（2）使用敲低方法测试RBM 20的作用。剪接变异体将在两个温度下使用实验室的新T。scripta基因组组装。结果将建立温度依赖性性腺测定的分子基础，并可能揭示一个保守的作用剪接在性腺发育。该实验室通过创建将尖端研究纳入当地课堂的课程计划，以及鼓励女孩追求STEM职业的计划，让学生参与科学研究。该实验室还通过播客采访、为杜克正在进行的“科学家的艺术”展览和杜克的“科学咖啡馆”系列做出贡献，将这一科学引起公众的关注。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.