Cellular and molecular mechanisms of vertebrate testis homeostasis

项目总结 原始生殖细胞在脊椎动物发育的早期为体细胞性腺播种，并共同发育以产生 功能性睾丸充满精原干细胞(SSCs)。这样的SSC群体在保持自身的同时 在动物的一生中产生了数量惊人的分化精子。我们缺少一个 SSCs维持干细胞活性群体的细胞和分子机制的理解 细胞。脊椎动物SSCs的研究仅限于群体或单细胞快照研究，均以失败告终 捕捉各个SSC对这一进程的贡献。 在这里，我建议应用最近开发的CRISPR谱系追踪、CRISPR突变和单细胞 RNA测序方法，以揭示维持脊椎动物睾丸的机制。首先，我们将 研究个体生殖系干细胞在睾丸动态平衡中的动态。我们将使用CRISPR谱系 在斑马鱼中进行追踪，以确定单个SSCs对自我更新和分化的贡献，以及 对单个动物进行连续采样，以跟踪长期动态。第二，我们将描述新的 SSC动态平衡的调节。使用SSC基因表达的单细胞图谱，我们将使用我们的快速 CRISPR方法用单细胞表型突变候选调控基因和表型 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。我们将生成单个细胞的空间分辨率地图集 来自硬骨鱼系统发育树上物种的幼年、成年和老年睾丸，并使用比较 基因表达分析以确定SSCs和NICE细胞的细胞和分子差异。 这些项目将确定维持干细胞数量的分子和细胞机制。 睾丸，与人类不孕不育有关。他们还将揭示干细胞动态平衡的一般原理 在组织和器官中。

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.