Cellular and molecular mechanisms of vertebrate testis homeostasis

脊椎动物睾丸稳态的细胞和分子机制

• 批准号: 10276262
• 负责人: James Alan Gagnon
• 金额: $ 38.13万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10276262
• 关键词: Adolescent; Adult; Animals; Atlases; CRISPR/Cas technology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Gene Expression; Gene Expression Profiling; Gonadal structure; Homeostasis; Human; Individual; Infertility; Longevity; Male Infertility; Methods; Molecular; Mutagenesis; Organ; Phenotype; Phylogenetic Analysis; Population; Process; Regulator Genes; Sampling; Seeds; Structure of primordial sex cell; Testis; Tissues; Trees; Vertebrates; Zebrafish; aged; comparative; germline stem cells; high resolution imaging; male; self-renewal; single-cell RNA sequencing; sperm cell; stem cell genes; stem cell homeostasis; stem cell niche; stem cell population; stem cells; teleost

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.

项目概要 原始生殖细胞在脊椎动物发育早期播种体细胞性腺，并共同发育产生 功能性睾丸充满精原干细胞（SSC）。这个 SSC 群体在维持自身的同时 在动物的一生中产生数量惊人的分化精子。我们缺少一个 了解 SSC 维持活跃干细胞群的细胞和分子机制 细胞。脊椎动物 SSC 的研究仅限于群体或单细胞快照研究，这些研究失败了 捕捉各个南南合作委员会对此进程的贡献。 在这里，我建议应用最近开发的CRISPR谱系追踪、CRISPR诱变和单细胞技术 RNA 测序方法揭示维持脊椎动物睾丸的机制。首先，我们将 研究睾丸稳态中个体生殖干细胞的动态。我们将使用 CRISPR 谱系 追踪斑马鱼以确定个体 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。其次，我们将描述新的 SSC 稳态的调节因子。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和突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 整个脊椎动物亚门的表型多样性。我们将生成单细胞、空间分辨的图谱 硬骨鱼系统发育树上各物种的幼年、成年和老年睾丸，并使用比较 基因表达分析以确定 SSC 和生态位细胞的细胞和分子差异。 这些项目将定义维持干细胞群的分子和细胞机制。 睾丸，对人类不育有影响。他们还将揭示干细胞稳态的一般原理 在组织和器官中。