Spermatogonial Stem Cell Maintenance

项目摘要/摘要 大多数成体组织由常驻的成体干细胞维持，这些干细胞维持着 组织。当旧细胞死亡或受损时，成年干细胞会产生新的细胞。在许多组织中都有 新出现的数据表明，既存在快速循环干细胞，也存在静止干细胞。快速循环细胞是 积极参与组织修复的慢循环或G0停滞细胞是储备细胞。最近出版的 这一提议中的工作和初步研究支持了精原干细胞(SSCs)在 睾丸在其周期状态中是不同的，正常细胞周期的中断可能会干扰 既有自我更新，又有差异化。我们已经确定了一个表达Eome的精原细胞亚群， T盒转录因子。使用血统追踪，我们已经证明了它们对稳定状态的贡献 白花丹消融生殖细胞后的精子发生和再生。在PLZF突变小鼠中，这表明 年龄相关的SSCs的耗竭，Eome细胞周期更快，这表明年龄相关的 SSCs的枯竭是由增殖性衰竭引起的。这项提议的中心假设是 精原干细胞(SSCs)在其周期状态方面也是异质性的，而且有 无论是快自行车还是慢自行车SSCs。我们认为，细胞周期的适当调节对于 维持自我更新和分化以及细胞周期丧失之间的动态平衡 由于关键的自我更新基因的错误调节，调节可导致SSCs的年龄依赖性丢失。在……里面 具体目标1我们将量化细胞周期异步化的频率，SSC标记的一致性 以及PLZF基因突变对这两个参数的影响。《特定目标2》 我们将利用Ki67(Mki67-RFP)等位基因从更大的 GFRA1种群。单细胞RNA测序(ScRNAseq)和转座酶的单核检测 周期细胞和非周期细胞的可及染色质(SnATACseq)将提供独立的 评估SSCs的非周期G0群体，Eome和其他SSCs细胞的标志物是否 在种群中丰富，以及是否存在以前未确定的G0细胞种群 SSC泳池。最后，在特定的目标3中，我们将评估Eome表达的调节因子BATF的突变， 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 驱动高增殖表型的基因。

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.