Genetic regulation of active and reserve intestinal stem cell states

摘要：肠上皮的完全生理更新大约每周发生一次，并且是 由位于上皮隐窝基底的活跃增殖的 ISC (aISC) 驱动。罕见的细胞亚群 地穴尚未完全定义，但统称为“保留”ISC（rISC）， “静止”或缓慢分裂，当某些健康状况或无线电或 化疗暴露会损害并耗尽天然 aISC 池。 rISC 到 aISC 的转换 传统上在小鼠中研究这一过程，其中使用辐射 (IR) 来消耗 aISC 并诱导 rISC。 在这里，rISC 的定义是它们对 IR 诱导死亡的抵抗力，然后是它们的“可塑性”能力 产生活跃分裂的 ISC 后代，补充 aISC 池并驱动后续的上皮细胞 再生。赋予 rISC 放射抗性和可塑性的机制尚不清楚。在之前的 我们的工作证明了转录因子 Sox9 是生成和发挥功能所必需的 小鼠体内的 rISC。使用 Sox9CreERT2 驱动程序进行谱系追踪表明，IR 后，所有再生上皮细胞 源自表达 Sox9 的细胞，Sox9 的上皮特异性基因消融深刻 IR 后阻碍上皮再生和细胞存活。这些发现表明 Sox9 依赖性 控制 RISC 功能的机制。干细胞和放射领域越来越多的证据表明 减慢分裂细胞的细胞周期速率可以增强红外线照射后的放射抗性，并且可以 调节细胞命运承诺与自我更新决策。我们发现 Sox9 水平升高 与隐窝中缓慢分裂的细胞有关，并且快速分裂中的 Sox9 过度表达 aISC 可以减缓或阻止其增殖。我们假设 Sox9 表达水平调节 细胞周期进展以确定 rISC (Sox9HI) 和 aISC (Sox9LO) 功能并使其多样化 肠隐窝。如果我们的实验结果支持的话，这项研究将揭示 控制 RISC 辐射抗性和可塑性的基本途径，并可以提供统一的方法 描述 rISC 特性是否以及如何存在于广泛的细胞类型中的机制 地穴。目标 1：评估增加 Sox9 水平对 ISC 细胞周期进展的影响。目标 2： 确定 Sox9 介导的 G1 延伸是否具有放射抗性。目标 3：确定 Sox9- 介导的 G1 延伸赋予“分泌前体”rISC 表型。

ABSTRACT: Complete physiologic renewal of intestinal epithelium occurs about every week and is driven by actively proliferating ISCs (aISCs) located in the epithelial-crypt base. A rare subset cells in the crypt, still not completely defined but that are collectively known as `reserve' ISCs (rISCs), are `quiescent' or slowly dividing, and can convert into aISCs when certain health conditions or radio- or chemotherapeutic exposures damage and deplete the native aISC pool. The rISC to aISC conversion process is traditionally studied in mice where irradiation (IR) is used to deplete aISCs and induce rISCs. Here, rISCs are defined by their resistance to IR-induced death, and then by their `plastic' ability to generate actively dividing ISC progeny, which replenish the aISC pool and drive subsequent epithelial regeneration. The mechanisms conferring rISC radio-resistance and plasticity are unknown. In prior work we demonstrated that the transcription factor Sox9 is required for the generation and function of rISCs in mice. Lineage tracing with a Sox9CreERT2 driver showed that after IR, all regenerating epithelium is derived from cells that expressing Sox9, and epithelium-specific genetic ablation of Sox9 profoundly impeded epithelial regeneration and cell survival post-IR. These findings indicate that Sox9-dependent mechanisms govern rISC function. Growing evidence in the stem cell and radiation fields suggest that slowing the cell-cycle rate in dividing cells can enhance radioresistance after IR-exposure, and can modulate cell fate commitment versus self-renewal decisions. We have found that elevated Sox9 levels are associated with slowly dividing cells in the crypt, and that Sox9-overexpression in rapidly dividing aISCs can slow or halt their proliferation. We hypothesize that Sox9-expression levels modulate cell-cycle progression to determine and diversify rISC (Sox9HI) and aISC (Sox9LO) functions in the intestinal crypt. If supported by the results of our experiments, this study will uncover the underlying pathways governing rISC radioresistance and plasticity, and could provide a unifying mechanism describing whether and how rISC properties exist among a broad range of cell types in the crypt. Aim 1: Assess the effects of increasing Sox9 levels on cell-cycle progression in ISCs. Aim 2: Determine if Sox9-mediated G1-elongation confers radio-resistance. Aim 3: Determine if Sox9- mediated G1-elongation confers a `secretory precursor' rISC phenotype.