Collaborative Research: Signaling Mechanisms in the Crustacean Molting Gland

蟹、龙虾和虾是海洋环境中具有重要生态和经济价值的甲壳类动物。坚硬、钙化的外骨骼提供保护和支持，但限制生长。因此，这些动物必须定期脱落外骨骼，这一过程称为蜕皮。在蜕皮时，动物在新的外骨骼变硬之前拉伸它，为组织生长提供更多的空间。整个过程由一对蜕皮腺体产生的蜕皮激素控制。蜕皮腺的活动受神经系统介导的环境信号控制。关于控制蜕皮腺的信号基因，特别是（1）使动物蜕皮和（2）在动物蜕皮后抑制蜕皮腺所需的基因，还有很多东西有待了解。承诺是一个“不归路”的决定，对所有甲壳类动物的生存和生长至关重要。抑制阻止蜕皮诱导，直到外骨骼的合成和钙化完成。该合作项目涉及来自四所大学的研究人员团队，他们将使用最先进的DNA和肽测序技术来识别对蜕皮腺的激活，承诺和抑制及其环境信号调节至关重要的基因和蛋白质。所产生的数据将提供给研究人员，以便他们能够更好地了解如何管理渔业，开发有效的水产养殖实践，并减轻污染物和气候变化的影响。三名博士后研究员，四名研究生和6-8名本科生将接受先进分子技术和生物信息学的培训。一个教育推广计划的教师招聘从初中和高中在东北部科罗拉多将制定。在甲壳类动物，成对的蜕皮腺（Y-器官或YOs）产生蜕皮类固醇必要的系统蜕皮周期控制。蜕皮抑制激素（MIH）产生于眼柄神经节，通过G蛋白偶联受体（GPCR）和环核苷酸第二信使抑制YO合成蜕皮激素。该项目将使用转录组学和蛋白质组学工具来确定在蜕皮周期中驱动YO表型变化的信号传导机制;这些信号传导机制被指定为在蜕皮间期的基础，在蜕皮前早期激活，在蜕皮前中期和晚期承诺，并在蜕皮后抑制。具体目标是：（1）确定MIH信号传导如何抑制雷帕霉素（mTOR）活性的机制靶点;（2）确定转化生长因子β/激活素信号传导在YO定型中的作用;（3）确定蜕皮类固醇对YO进入和退出抑制状态的作用;和（4）表征MIH受体。前三个目标将量化实验信号操纵对YO状态转换的表型效应。RNA序列和液相色谱串联质谱数据的网络分析将确定实验处理对基因相互作用和信号通路下游靶点的影响。目的#4使用转录组学工具和荧光素酶配体/受体结合试验与重组神经肽来表征受体功能的候选GPCR。全面了解十足类蜕皮和生长的激素调节对于管理渔业、发展有效的水产养殖做法以及减轻污染物和环境因素对具有经济和生态重要性的海洋生物群体的潜在影响至关重要。三名博士后、四名研究生和6-8名本科生将接受先进分子技术和生物信息学的培训，一名6-12年级的教师将参与该研究课题的教育推广。该奖项反映了NSF的法定使命，并被认为值得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来支持。

Crabs, lobsters, and shrimp are ecologically and economically important crustaceans in marine environments. The hard, calcified exoskeleton provides protection and support, but restricts growth. As a result, these animals must periodically shed the exoskeleton, a process called molting. Upon molting, animals stretch the new exoskeleton before it hardens, providing more space for tissue growth. The entire process is controlled by molting hormone produced by a pair of molting glands. The activity of the molting gland is controlled by environmental signals mediated by the nervous system. Much remains to be known about the signaling genes that control the molting gland, in particular the genes required for (1) committing the animal to molt and (2) repressing the molting gland after the animal molts. Commitment is a “point of no return” decision that is critical for survival and growth in all crustaceans. Repression prevents molt induction until synthesis and calcification of the exoskeleton is completed. This collaborative project involves a team of investigators from four universities who will use state-of-art DNA and peptide sequencing technologies to identify genes and proteins essential for the activation, commitment, and repression of the molting gland and its regulation by environmental signals. The data generated will be made available to researchers so that they can better understand how to manage fisheries, develop effective aquaculture practices, and mitigate the effects of pollutants and climate change. Three postdoctoral fellows, four graduate students, and 6-8 undergraduates will receive training in advanced molecular techniques and bioinformatics. An educational outreach program for teachers recruited from junior high and high schools in northeastern Colorado will be developed.In crustaceans, paired molting glands (Y-organs or YOs) produce ecdysteroids necessary for systemic molt cycle control. Molt-inhibiting hormone (MIH), produced in the eyestalk ganglia, inhibits the synthesis of ecdysteroids by the YO via a putative G protein-coupled receptor (GPCR) and cyclic nucleotide second messengers. The project will use transcriptomic and proteomic tools to determine the signaling mechanisms that drive YO phenotypic changes over the molt cycle; these are designated basal in intermolt, activated in early premolt, committed in mid and late premolt, and repressed in postmolt. The specific aims are to: (1) determine how MIH signaling inhibits Mechanistic Target of Rapamycin (mTOR) activity; (2) determine the role of Transforming Growth Factor beta/Activin signaling in YO commitment; (3) determine the role of ecdysteroids on YO entry into, and exit from, the repressed state; and (4) characterize the MIH receptor. The first three aims will quantify the phenotypic effects of experimental signaling manipulations on YO state transitions. Network analysis of RNA-sequence and liquid chromatography-tandem mass spectrometry data will determine the effects of experimental treatments on gene interactions and downstream targets of signaling pathways. Aim #4 uses transcriptomic tools and a luciferase ligand/receptor binding assay with recombinant neuropeptides to characterize candidate GPCRs for receptor function. A thorough understanding of the hormonal regulation of decapod molting and growth is essential to manage fisheries, develop effective aquaculture practices, and mitigate potential effects of pollutants and environmental factors on an economically and ecologically important group of marine organisms. Three postdocs, four graduate students, and 6-8 undergraduates will receive training in advanced molecular techniques and bioinformatics, and a grade 6-12 teacher will be involved in educational outreach on the research topic.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.