CAREER: Developmental origins of the shell gland and genetic networks for biomineralization in the mollusk Crepidula atrasolea

生物矿化是生物体产生矿物质的过程。 虽然它发生在每一个生命王国，最熟悉和美丽的例子之一是蜗牛壳。 蜗牛壳的多样化被认为与这类动物的丰富和成功有关（就物种数量而言，这是仅次于节肢动物的第二大门）。 随着全球海洋温度的上升，迫切需要了解贝壳是如何形成和生长的，以保护牡蛎、蛤蜊和贻贝等水产养殖物种。 贝壳开始形成于发育过程中，此时贝壳腺细胞表达一套转录因子蛋白，这些转录因子蛋白反过来调节贝壳基质蛋白;基质蛋白形成控制碳酸钙结晶的支架。 本项目使用一种研究充分的海洋蜗牛Crepidula来研究壳基质的形成。 利用工具修改该物种的基因组，该项目研究了哪些特定的转录因子控制基质蛋白的产生，以及它们如何影响壳结构和生长。这些研究将是第一次在软体动物中进行。 该项目的另一个创新方面是将在活体动物中研究基质蛋白。 这项研究将被带入课堂，作为基于探究的实验室课程的一部分，本科生，研究生和高中生可以学习软体动物壳的发育并测试自己的假设。 尽管人们对软体动物外壳的生物材料特性、微观结构和进化有了很多了解，但关于它们在发育过程中是如何形成的，仍然存在一些主要问题。 该项目的总体目标是首次全面研究嵌入海洋蜗牛Crepidula atrasolea外壳中的细胞外基质蛋白的表达和功能。 目的1利用scRNAseq、原位杂交和蛋白质组学技术，揭示贝壳腺表达的调控基因和外套膜组织表达的基质基因的时空表达模式。 目标2将使用谱系追踪来显示壳腺细胞如何成熟为成体外套膜组织，并使用CRISPR/Cas9基因组编辑来制备基质蛋白-GFP融合物，以研究体内壳的形成。 目标3将鉴定基质蛋白的上游调控因子，并敲除它们以测试它们的功能。 这项工作将导致第一个真正的基因调控网络（GRN）的生物矿化的软体动物。 目标4将这项研究整合到一个新的细胞和发育生物学CURE实验室课程中，面向本科生、研究生和高中生。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。

Biomineralization is the process by which living organisms produce minerals. Although it occurs in every kingdom of life, one of the most familiar and beautiful examples is the snail shell. The diversification of the snail shell is thought to be related to the abundance and success of this group of animals (the second largest phylum in terms of species number, after arthropods). With ocean temperatures increasing worldwide, there is also an urgent need to understand how shells form and grow to protect aquaculture species like oysters, clams, and mussels. Shells begin forming during development, when shell gland cells express a suite of transcription factor proteins that in turn regulate shell matrix proteins; matrix proteins form a scaffold that controls calcium carbonate crystalization. This project examines shell matrix formation using a well-studied marine snail, Crepidula. Taking advantage of tools to modify the genome of this species, this project examines which specific transcription factors control matrix protein production, and how they influence shell structure and growth. These studies will be the first of their kind among molluscs. Another innovative aspect of this project is that matrix proteins will be studied in the living animal. The research will be brought into the classroom as part of an inquiry-based laboratory course where undergraduates, graduate students, and high school students can study mollusc shell development and test their own hypotheses. Although much is known about the biomaterial properties, micro-structure, and evolution of molluscan shells, major questions remain about how they are made during the process of development. The overarching goal of this project is to make the first comprehensive study of the expression and function of extracellular matrix proteins embedded in the shell of the marine snail Crepidula atrasolea. Aim 1 will uncover the spatiotemporal expression patterns of shell gland-expressed regulatory genes and mantle tissue-expressed matrix genes, using scRNAseq, in situ hybridization, and proteomics. Aim 2 will use lineage tracing to show how the shell gland cells mature into the adult mantle tissue, and use CRISPR/Cas9 genome editing to make matrix protein-GFP fusions to study shell formation in vivo. Aim 3 will identify the upstream regulators of matrix proteins, and knock them down to test their function. This work will lead to the first bona fide gene regulatory network (GRN) for biomineralization in a mollusc. Aim 4 integrates this research into a new cell and developmental biology CURE lab class for undergraduates, graduate students, and high school students.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.