Genetic regulation of active and reserve intestinal stem cell states

摘要：肠上皮的完全生理性更新大约每周发生一次。 由位于上皮-隐窝基底部的活跃增殖的ISCs(AISCs)驱动。一种稀有的子集细胞 加密仍未完全定义，但统称为“保留”ISCs(RISCs)， 静止性的或缓慢分裂的，当某些健康状况或无线电-或 化疗暴露会损害和耗尽本地的AISC池。RISC到AISC的转换 这一过程传统上是在小鼠身上研究的，辐射(IR)被用来耗尽aISCs和诱导rISCs。 在这里，rISCs是通过它们对IR诱导的死亡的抵抗力来定义的，然后由它们的‘可塑性’能力来定义 产生积极分裂的ISC后代，补充AISC池并驱动后续的上皮细胞 再生。赋予RISC辐射抗性和可塑性的机制尚不清楚。在之前 我们的工作证明了转录因子Sox9是产生和发挥功能所必需的 小鼠的rISCs。Sox9CreERT2驱动程序的谱系追踪显示，IR后，所有再生的上皮细胞 是从表达Sox9的细胞中获得的，并深刻地对Sox9进行了上皮特异性的基因消融 阻碍IR后的上皮再生和细胞存活。这些发现表明，Sox9依赖于 机制支配着RISC的功能。越来越多的干细胞和辐射领域的证据表明 减缓细胞分裂的细胞周期速率可以增强IR暴露后的辐射抵抗力，并可以 调节细胞命运的承诺与自我更新的决定。我们发现，SOX9水平升高 与隐窝中缓慢分裂的细胞有关，而Sox9-在快速分裂中过度表达 AISCs可以减缓或阻止它们的增殖。我们假设Sox9的表达水平调节 细胞周期进程决定和多样化RISC(Sox9HI)和AISC(Sox9LO)的功能 肠道隐窝。如果得到我们实验结果的支持，这项研究将揭示 控制RISC辐射抗性和可塑性的潜在途径，并可能提供一个统一的 描述RISC属性是否以及如何存在于 地窖。目的1：评价Sox9水平升高对ISCs细胞周期进程的影响。目标2： 确定Sox9介导的G1延长是否具有辐射抗性。目标3：确定Sox9是否- 介导的G1延长赋予了一种“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.