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

在许多爬行动物中，温度通过控制胚胎性腺是以睾丸还是卵巢的形式发育来调节雄性或雌性的发育，包括驯化的滑龟Trachemys scripta elegans。然而，自从50年前发现这种现象以来，温度影响性腺发育的机制一直是一个谜。此前，卡佩尔实验室发现了两个基因，它们在雄性和雌性产生温度的海龟性腺中显示出强烈的表达差异。在这个项目中，实验室将确定这些基因的表达如何受到温度的控制，以及这些基因的活动如何控制睾丸和卵巢的发育。这项工作的结果将建立温度对男性或女性发育影响的潜在机制。该实验室积极参与将该项目作为一个平台，让青年学生和公众参与科学研究。该项目也是一个强有力的平台，在制定环境政策以减轻气候变化的影响时倡导循证科学，预计会在温度影响性腺发育的物种中强烈地偏向男性/女性比例。卡佩尔实验室之前表明，表观遗传调节因子KDM6B和RNA剪接因子RBM20的转录在雄性产生温度的海龟性腺中升高。KDM6B通过去除DMRT1的抑制性组蛋白标记来激活睾丸途径，DMRT1是一种保守的睾丸发育调节因子。在这个项目中，实验室计划确定将温度与KDM6B和RBM20转录和剪接的变化联系起来的分子途径。该实验室假设，已知的KDM6B抑制物pSTAT3在高雌性产生温度下，通过激活色氨酸通道、钙离子内流和高活性的CAMK而被差异磷酸化。为了验证这一假设，在性腺中表达的色氨酸通道被敲除后，在干扰钙信号之后，或者在pSTAT3抑制剂之后，将测量男性和女性通路的标志物。实验将(1)确定RBM20是否也是pSTAT3的目标，或KDM6B的下游，以及(2)使用击倒方法测试RBM20的作用。将使用实验室新的Scripta基因组组件在两种温度下分析Splicoform变体。结果将为依赖温度的性腺测定奠定分子基础，并可能揭示剪接在性腺发育中的保守作用。该实验室通过创建将尖端研究纳入当地课堂的教案，以及鼓励女孩从事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、对杜克大学正在进行的科学家艺术展览的贡献以及杜克大学的科学咖啡馆系列节目，使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.