Spermatogonial Stem Cell Maintenance

项目概要/摘要 大多数成体组织由常驻成体干细胞维持，这些细胞维持着成体组织的功能和完整性。 组织。当旧细胞死亡或受损时，成体干细胞就会产生新细胞。许多组织中都有 新出现的数据表明存在快速循环干细胞和静止干细胞。快速循环细胞是 积极参与组织修复，而慢周期细胞或 G0 停滞细胞是储备细胞。最近发表 这项工作和该提案中的初步研究支持以下假设：精原干细胞（SSC） 睾丸的循环状态是异质的，正常细胞周期的破坏会干扰 既有自我更新，又有分化。我们已经鉴定出表达 EOMES 的精原细胞亚群， T盒转录因子。使用谱系追踪，我们已经证明它们有助于稳态 白消安消融生殖细胞后的精子发生和再生。在 Plzf 突变小鼠中，显示 由于 SSC 的年龄依赖性消耗，EOMES 细胞的循环速度更快，这表明年龄依赖性 SSC 的耗竭是由增殖衰竭引起的。该提案的中心假设是 精原干细胞 (SSC) 的循环状态也存在异质性，并且存在 快速循环和慢速循环的SSC。我们认为细胞周期的适当调节对于 维持自我更新和分化与细胞周期丧失之间的稳态平衡 由于关键自我更新基因的错误调节，调节可能导致年龄依赖性的 SSC 损失。在 具体目标 1 我们将量化细胞周期异步的频率、SSC 标记的一致性 表达和循环状态，以及 Plzf 突变对这两个参数的影响。具体目标 2 我们将利用 Ki67 等位基因 (Mki67-RFP) 从更大的细胞库中对循环细胞和非循环细胞进行流式分类 GFRA1 人群。单细胞 RNA 测序 (scRNAseq) 和转座酶单核测定 循环细胞和非循环细胞的可及染色质 (snATACseq) 将提供独立的 评估 SSC 的非循环 G0 群体，Eomes 和 SSC 细胞的其他标记是否 群体内富集，以及群体内是否存在先前未识别的 G0 细胞群体 SSC 池。最后，在特定目标 3 中，我们将评估 Eomes 表达调节因子 Batf 的突变如何， 增强 Plzf lu/lu 突变体的生殖细胞丢失表型，并评估细胞周期错误调节的作用 驱动过度增殖表型的基因。

PROJECT SUMMARY/ABSTRACT Most adult tissues are maintained by resident adult stem cells that maintain the function and integrity of the tissue. As old cells die or are damaged, new cells are produced from adult stem cells. In many tissues there is emerging data suggesting the presence of both rapid-cycling and quiescent stem cells. Rapid cycling cells are actively engaged in tissue repair while slow-cycling, or G0-arrested cells, are reserve cells. Recently published work, and preliminary studies in this proposal, support the hypothesis that spermatogonial stem cells (SSCs) in the testis are heterogeneous in their cycling status and that disruption of the normal cell cycle can interfere with both self-renewal and differentiation. We have identified a subpopulation of spermatogonia that express EOMES, a T box transcription factor. Using lineage tracing we have shown that they contribute to steady-state spermatogenesis and to regeneration following germ cell ablation by busulfan. In Plzf mutant mice, which show an age-dependent depletion of SSCs, EOMES+ cells cycle more rapidly, suggesting that age-dependent depletion of SSCs is caused by proliferative exhaustion. The central hypothesis of this proposal is that spermatogonial stem cells (SSCs) are also heterogeneous with respect to their cycling status and that there are both rapid cycling and slow cycling SSCs. We propose that proper regulation of the cell cycle is critical for maintaining the homeostatic balance between self-renewal and differentiation and that loss of cell cycle regulation can lead to age-dependent loss of SSCs due to the mis-regulation of critical self-renewal genes. In Specific Aim 1 we will quantify the frequency of cell cycle asynchrony, the concordance of SSC marker expression and cycling status, and the effect of mutation of Plzf on both of these parameters. In Specific Aim 2 we will utilize an allele of Ki67 (Mki67-RFP) to flow sort cycling and non-cycling cells from the larger pool of GFRA1+ population. Single cell RNA sequencing (scRNAseq), and single nuclei Assay for Transposase Accessible Chromatin (snATACseq) of the cycling and non-cycling cells will provide and independent assessment of the non-cycling G0 population of SSCs, whether Eomes and other markers of SSCs cells are enriched within the population, and whether there is a previously unidentified population of G0 cells within the SSC pool. Lastly, In Specific Aim 3 we will assess how mutation of Batf, a regulator of Eomes expression, enhances the germ cell loss phenotype in Plzf lu/lu mutants, and assess the role of the mis-regulation of cell cycle genes in the driving the hyperproliferative phenotype.