Origin and functional significance of the spermatogonial stem cell transcriptome barcode

项目摘要 哺乳动物的雄性生殖力取决于睾丸中基础SSC库的形成。缺陷 SSC的形成或维持被认为是仅支持细胞（SCO）综合征的主要原因， 导致非阻塞性无精子症（NOA）和男性不育。啮齿类动物的SSC起源于棘原细胞 在生命的第一周，然而，负责其规格的机制一直逃避男性 几十年来，生殖领域。在这个项目中，我们将询问两个替代假设：1-基础 SSC是预先确定的，并且从表达独特转录组的前体亚群中出现 条形码（“预定”），或2-SSC是从原球茎诱导的，使得独特的 转录组来自等价前体（“选择”）。通过测试这些替代假设，我们将 解决了我们对精子发生基础如何形成的知识中的一个关键空白。 初步研究结果表明，P6 ID 4-EGFP+精原细胞表达基因 异质性，定义离散的亚群，包括那些高度富集和耗尽的森林 基础SSC。我们定义了一个独特的SSC转录组条形码，该条形码是基于 独特的基因表达特征揭示了一个群体的近纯单细胞RNA-seq分析 SSC。这种SSC条形码似乎也由分离的雄性生殖细胞亚群表达。 从胎儿和新生儿的男性生殖细胞。最后，我们发现，丰富的人类未分化群体， 精原细胞表现出与小鼠精原细胞相似的异质性， 神秘的人类SSC这些结果表明，SSC条形码通过小鼠雄性细胞是保守的。 生殖系发育可用于广泛区分哺乳动物基础SSC。 我们提出了三个具体的目标来测试交替的预先决定和选择假设， 检查SSC条形码的时间起源及其对SSC库形成的功能意义。 目的1将区分个体小鼠生殖细胞中表达SSC条形码的细胞的出现， 胎儿晚期至出生后早期睾丸发育及其与SSC相关表型的出现 功能移植。目的2将确定是否需要表达这种SSC条形码的胎儿生殖细胞 用于在小鼠中使用谱系消除和追踪方法形成基础SSC库。目标3将 确定小鼠SSC条形码在高等灵长类动物（狒狒和人类）中是否保守，并询问是否 它在灵长类动物中的暂时出现与在小鼠中看到的相似。 在整个提案中，我们将使用单细胞分辨率检查基因表达，并利用SSC 移植和谱系追踪/消融以提供SSC命运的关键功能读数。拟议 这项研究将使我们能够解决男性生殖中的一个关键问题--精原干细胞库是如何建立的？

PROJECT SUMMARY Male fertility in mammals depends on formation of a foundational SSC pool in the testis. Defects in formation or maintenance of SSCs are considered primary causes of Sertoli cell-only (SCO) syndrome, which results in non-obstructive azoospermia (NOA) and male infertility. Rodent SSCs arise from prospermatogonia in the first week of life, yet the mechanisms responsible for their specification have eluded the male reproduction field for decades. In this project, we will interrogate two alternate hypotheses: 1- that foundational SSCs are predetermined and emerge from a precursor subpopulation expressing a unique transcriptome barcode (“predetermination”), or 2- that SSCs are induced from prospermatogonia such that unique transcriptomes emerge from equipotent precursors (“selection”). By testing these alternate hypotheses, we will address a critical gap in our knowledge of how the foundation of spermatogenesis is formed. Results of preliminary studies demonstrated that P6 ID4-EGFP+ spermatogonia exhibit gene expression heterogeneity which defined discrete subpopulations, including those highly-enriched and depleted for foundational SSCs. We defined a distinct SSC transcriptome barcode which was identified based on the unique gene expression signature revealed by single-cell RNA-seq analyses of a population of nearly pure SSCs. This SSC barcode also appeared to also be expressed by a subpopulation of male germ cells isolated from fetal and neonatal male germ cells. Lastly, we found that enriched populations of human undifferentiated spermatogonia exhibited similar heterogeneity to mouse spermatogonia which could enable future discernment of the elusive human SSC. These results suggest that the SSC barcode is conserved through mouse male germline development could potentially be used to broadly distinguish mammalian foundational SSCs. We propose three Specific Aims to test the alternate predetermination and selection hypotheses by examining the temporal origin the SSC barcode and its functional significance to the formation of the SSC pool. Aim 1 will distinguish emergence of cells expressing the SSC barcode among individual mouse germ cells from late fetal through early postnatal testis development and correlate appearance of this phenotype with SSC function by transplant. Aim 2 will determine whether fetal germ cells expressing this SSC barcode are required for formation of the foundational SSC pool using lineage ablation and tracing methods in mice. Aim 3 will establish whether the mouse SSC barcode is conserved in higher primates (baboons and humans) and ask if its temporal emergence in the primate parallels that seen in the mouse. Throughout this proposal, we will examine gene expression with single-cell resolution and leverage SSC transplantation and lineage tracing/ablation to provide pivotal functional readouts of SSC fate. The proposed studies will allow us to address a key question in male reproduction– how is the pool of SSCs established?