Cellular and molecular mechanisms of vertebrate testis homeostasis

多样性实现的项目摘要是可持续发展应用程序 项目摘要 原始生殖细胞在脊椎动物发育的早期就在体细胞性腺中播种，并一起发育，产生一个生殖细胞。 功能性睾丸充满精原干细胞（SSCs）。这些SSC群体维持自身， 在动物的一生中产生惊人数量的分化精子。我们缺少一个 理解SSC中维持干细胞活跃群体的细胞和分子机制 细胞脊椎动物中精原干细胞的研究仅限于群体或单细胞快照研究， 以获取各个SSC对这一过程的贡献。 在这里，我建议应用最近开发的CRISPR谱系追踪，CRISPR诱变和单细胞 RNA测序方法揭示维持脊椎动物睾丸的机制。一是 研究单个生殖系干细胞在睾丸稳态中的动态。我们将使用CRISPR谱系 在斑马鱼中进行追踪，以确定个体SSC对自我更新和分化的贡献，以及 对个体动物进行连续采样，以跟踪长期动态。第二，我们将新 SSC体内平衡的调节剂。使用SSC基因表达的单细胞图谱，我们将使用我们的快速 使用单细胞表型分析来诱变候选调节基因和表型的CRISPR方法， 高分辨率成像。第三，我们将研究产生睾丸的进化机制 脊椎动物亚门的表型多样性。我们将生成单细胞，空间分辨的地图集， 在硬骨鱼类系统发育树中， 基因表达分析以确定SSC和小生境细胞中的细胞和分子差异。 这些项目将定义维持干细胞群体在细胞内的分子和细胞机制。 睾丸，与人类不育的影响。他们还将揭示干细胞稳态的一般原理 在组织和器官中。

PROJECT SUMMARY FOR DIVERSITY SUPPLEMENT IS SAME AS PARENT APPLICATION PROJECT SUMMARY Primordial germ cells seed the somatic gonad early in vertebrate development, and develop together to yield a functional testis replete with spermatogonial stem cells (SSCs). This population of SSCs maintains itself while generating astonishing numbers of differentiated sperm across the lifetime of the animal. We lack an understanding of the cellular and molecular mechanisms in SSCs that maintain an active population of stem cells. Studies of SSCs in vertebrates have been limited to population or single-cell snapshot studies, which fail to capture the contributions of individual SSCs to this process. Here, I propose to apply recently developed CRISPR lineage tracing, CRISPR mutagenesis, and single-cell RNA sequencing methods to uncover the mechanisms that maintain the vertebrate testis. First, we will investigate the dynamics of individual germline stem cells in testis homeostasis. We will use CRISPR lineage tracing in zebrafish to determine the contributions of individual SSCs to self-renewal and differentiation, and serial sampling from individual animals to track long-term dynamics. Second, we will characterize new regulators of SSC homeostasis. Using single-cell atlases of SSC gene expression, we will use our rapid CRISPR methods to mutagenize candidate regulator genes and phenotype using single-cell phenotyping and high-resolution imaging. Third, we will examine the evolutionary mechanisms that have generated testis phenotypic diversity across the vertebrate subphylum. We will generate single-cell, spatially resolved atlases of the juvenile, adult and aged testis from species across a phylogenetic tree of teleosts, and use comparative gene expression analysis to determine cellular and molecular differences in SSCs and niche cells. These projects will define the molecular and cellular mechanisms that maintain stem cell populations within the testis, with implications for human infertility. They will also uncover general principles of stem cell homeostasis in tissues and organs.