Investigating Conserved Function of Male and Female Developmental Genes in a Model Bivalve

双壳类，如贻贝、牡蛎和文蛤，属于第二个最多样化的动物门。它们在沿海生态系统中发挥着关键作用，有助于维持水质，并被广泛用于水产养殖。尽管它们很重要，但在双壳类中，许多发育过程并没有被很好地理解。有几个基因已经被确定与雄性和雌性的指定和发育有关；然而，它们的确切作用尚不清楚。该项目的目标是描述这些基因在双壳类动物发育过程中的特征，并确定它们在区分雄性和雌性生殖器官过程中的作用。我们的方法包括记录不同组织中In基因的表达水平，并通过实验降低其表达水平，以观察发育过程中的变化。该项目将为男性和女性指定和区分生殖器官的过程提供重要的洞察力。我们的项目还将帮助那些在课外义务阻碍他们攻读STEM学位的学生获得更多机会。研究表明，在线实验室课程，特别是那些包括DIY工具包和手册的课程，可以与面对面实验室一样有效，并减少那些难以将时间花在面对面实验室课程上的学生的障碍。该项目旨在创建一个完全在线的海洋生物学和生理学DIY实验室，作为增加STEM实验室的可及性和扩大参与的典范。尽管已经提出了一些关于软体动物雄性和雌性特定性腺发育的机制的假设模型，但很少有关于这一过程中基因功能的实验测试。利用系统转录组学方法，我们已经在双壳类中鉴定了几个推测的性腺发育基因，其中包括软体动物特有的DMRT基因：DMRT1L。我们提出的项目的主要目的是通过两个特定的目标来表征性腺发育基因在蓝色贻贝成体双壳动物模型中的作用：(1)利用RT-qPCR和原位杂交技术确定雄性和雌性性腺发育基因在配子发生中的时间和组织特异性表达模式。(2)利用RNA干扰技术(SoxH、DMRT1L、FOXL2、β-catenin)评价关键性腺发育基因(SoxH、DMRT1L、FOXL2、DMRT1L)敲除后雄性和雌性性腺发育的变化。这些基因已经在双壳类中进行了研究；然而，这项工作依赖于雄性和雌性之间基因表达的相关性以及从远亲物种获得的知识。这项拟议的工作将验证这些假定的雄性和雌性发育基因的作用，并使我们能够开发一种基于转录的双壳类性腺发育途径。这项拟议的工作将对理解远缘后生动物物种的雄性和雌性发育路径之间的关系以及它们是如何进化的具有变革性的。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Bivalves, such as mussels, oysters, and clams, belong to the second most diverse animal phylum. They play a critical role in coastal ecosystems, help maintain water quality, and are widely used in aquaculture. Despite their importance, many developmental processes are not well understood in bivalves. Several genes have been identified to be correlated with male and female specification and development; however, their exact role is not yet known. The goal of this project is to characterize these genes in bivalve development and identify their roles in the process of differentiating male and female reproductive organs. Our methods involve documenting levels of in gene expression in different tissues and experimentally lowering their expression level to observe changes in development. This project will provide critical insight into the process of male and female specification and differentiation of reproductive organs. Our project will also help to increase opportunities for students that have obligations outside of the classroom that hinder their ability to pursue a STEM degrees. Research shows that online laboratory courses, especially those that include “Do it yourself” (DIY) kits and manuals, can be as effective as in person labs and decrease barriers for students that have difficulty devoting time to in person laboratory courses. This project aims to create a completely online DIY laboratory in marine biology and physiology that will act as a model for increasing accessibility to STEM labs and broaden participation. Although some hypothetical models have been proposed for mechanisms underlying male and female specific gonadal development in mollusks, there have been very few experimental tests of gene function in this process. Using phylotranscriptomics, we have identified several putative gonadal development genes in bivalves, including a mollusk specific DMRT gene: DMRT1L. The primary objective of our proposed project is to characterize the role of putative gonadal development genes in a model, gonochoristic, adult bivalve, the blue mussel M. edulis through two specific aims: (1) Determine the temporal and tissue-specific expression patterns of male and female gonadal development genes using RT-qPCR and in situ hybridization in relation to gametogenesis. (2) Evaluate changes in gonadal development in males and females following knockdown of key gonadal development genes using RNAi (SoxH, DMRT1L, FoxL2, β-catenin). These genes have been studied in bivalves; however, this work has relied on correlations in gene expression between males and females and knowledge gained from distantly related species. The proposed work will verify the roles of these putative male and female developmental genes and will enable us to develop a transcript-based gonadal development pathway for bivalves. This proposed work will be transformative to understanding the relationships between male and female development pathways across distantly related metazoan species and how they evolved.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.