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。虽然它发生在生命的每个王国，但最熟悉和最美丽的例子之一是蜗牛壳。蜗牛壳的多样化被认为与这一动物群体的丰富和成功有关（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升高，我们也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。壳在发育过程中开始形成，此时壳腺细胞表达一系列转录因子蛋白，这些转录因子蛋白反过来调节壳基质蛋白；基质蛋白形成控制碳酸钙结晶的支架。这个项目使用一种被充分研究过的海蜗牛——Crepidula来检验贝壳基质的形成。利用工具来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的结构和生长。这些研究将是首次在软体动物中进行此类研究。该项目的另一个创新之处在于，基质蛋白将在活体动物中进行研究。这项研究将作为探究性实验课程的一部分带入课堂，本科生、研究生和高中生可以研究软体动物壳的发育并测试他们自己的假设。尽管人们对软体动物壳的生物材料特性、微观结构和进化了解很多，但在发育过程中它们是如何形成的主要问题仍然存在。本项目的总体目标是首次对海洋蜗牛Crepidula atrasolea外壳中嵌入的细胞外基质蛋白的表达和功能进行全面研究。目的1将利用scRNAseq、原位杂交和蛋白质组学揭示壳腺表达调控基因和套组织表达基质基因的时空表达模式。Aim 2将使用谱系追踪来展示壳腺细胞如何成熟为成年套组织，并使用CRISPR/Cas9基因组编辑制作基质蛋白- gfp融合物来研究体内壳的形成。Aim 3将鉴定基质蛋白的上游调节因子，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软体动物生物矿化基因调控网络（GRN）。Aim 4将这项研究整合到一个面向本科生、研究生和高中生的新的细胞和发育生物学CURE实验课中。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识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.