Spermatogonial Stem Cell Maintenance

项目概要/摘要 大多数成体组织由驻留的成体干细胞维持，成体干细胞维持组织的功能和完整性。 组织.当旧细胞死亡或受损时，新细胞由成体干细胞产生。在许多组织中， 新出现的数据表明存在快速循环和静止干细胞。快速循环细胞是 当慢循环或G 0停滞的细胞是储备细胞时，它们积极参与组织修复。最近发表 这项工作和这项提议的初步研究支持了精原干细胞（SSCs）在胚胎发育中的假设。 睾丸在其周期状态中是异质，且正常细胞周期的破坏可干扰 自我更新和分化。我们已经确定了一个表达EOMES的精原细胞亚群， T盒转录因子。使用谱系追踪，我们已经表明，它们有助于稳态 精子发生和白消安去除生殖细胞后再生。在Plzf突变小鼠中， SSC的年龄依赖性耗竭，EOMES+细胞周期更快，表明年龄依赖性 SSC的耗竭是由增殖性耗竭引起的。这一提议的核心假设是， 精原干细胞（SSCs）在其循环状态方面也是异质的， 快速循环和慢速循环SSC。我们认为，细胞周期的适当调节是至关重要的 维持自我更新和分化之间的稳态平衡， 由于关键的自我更新基因的错误调节，这种调节可导致SSC的年龄依赖性损失。在 具体目标1我们将量化细胞周期紊乱的频率，SSC标志物的一致性， 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Ki 67的等位基因（Mki 67-RFP）从较大的细胞库中流式分选循环和非循环细胞。 GFRA 1+人群。转座酶的单细胞RNA测序（scRNAseq）和单核分析 循环和非循环细胞的可染色质（snATACseq）将提供和独立的染色质标记。 评估SSC的非循环G 0群体，Eomes和SSC细胞的其他标志物是否 在群体内富集，以及在群体内是否存在先前未鉴定的G 0细胞群体。 SSC池。最后，在具体目标3中，我们将评估如何突变的Batf，一个调节Eomes表达， 增强Plzf lu/lu突变体中生殖细胞损失表型，并评估细胞周期错误调控的作用 基因在驱动过度增殖表型。

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.