Determining the relationship between Sox9 and Collagen XVIII in maintaining intestinal stem cell identity

 描述（由申请人提供）：肠干细胞（ISC）位于隐窝底部，在含有支持性潘氏细胞和隐窝周围肌成纤维细胞的小生境内。细胞外基质（ECM），一个未充分研究的组成部分的生态位，也密切相关的ISC和可能会影响ISC的行为，因为它是在其他组织。虽然关键机制，本质上控制ISC的维护开始被确定，很少有信息存在的外部因素，如ECM，影响ISC。转录因子Sox 9差异标记小肠隐窝内的ISC，是正确ISC所需的 功能，并已被证明是必要的适当的上皮细胞增殖。Sox 9还与ECM的调节相关，其中胶原蛋白XVIII A型（Col 18 a1）在缺乏Sox 9的肠上皮（Sox 9-KO）中下调。有趣的是，Col 18 a1具有结合Wnt的能力，Wnt是Sox 9和ISC增殖的已知调节因子。Sox 9、Col 18 a1和Wnt信号之间的关联表明，Sox 9可能是ISC生态位内动态互惠的驱动因素，允许ISC与周围ECM相互作用。因此，该提议的中心假设是Sox 9通过在小肠隐窝中产生Wnt梯度来驱动Col 18 a1的产生以维持ISC身份。为了检验这一假设，我们将首先确定的细胞起源的Col 18 a1的ISC生态位内的野生型肠隐窝上皮细胞和支持性围胞肌成纤维细胞的单细胞RNA测序。收集的数据将提供有关Col 18 a1的细胞起源以及任何其他差异表达基因的信息，供未来研究使用。然后，我们将通过无细胞野生型和Sox 9-KO ECM的质谱法确定Sox 9对小生境ECM内的Col 18 a1蛋白的影响。Western印迹和免疫组织化学将鉴定Col 18 a1的丰度和位置以及Sox 9-KO ECM中的任何异常表达模式。最后，我们将通过在培养的ISCs中慢病毒转导Col 18 a1 siRNA沉默Col 18 a1表达来确定Col 18 a1对ISC功能的影响。确定Sox 9和Col 18 a1之间的关系对于了解内稳态，损伤后再生和疾病期间的ISC/ECM动力学以及开发新的肠组织工程策略至关重要。

 DESCRIPTION (provided by applicant): Intestinal stem cells (ISCs) reside at the base of the crypt, within a niche that contains supportive Paneth cells and pericryptal myofibroblasts. The extracellular matrix (ECM), an understudied component of the niche, is also intimately associated with ISCs and may be influential on ISC behavior, as it is in other tissues. Although key mechanisms that intrinsically control ISC maintenance are starting to be identified, little information exists on how extrinsic factors, such as the ECM, impact ISCs. The transcription factor Sox9 differentially marks ISCs within the small intestinal crypt, is required for proper ISC function, and has been shown to be essential for proper epithelial proliferation. Sox9 has also been associated with regulation of ECM, with Collagen XVIII Type A (Col18a1) down-regulated in intestinal epithelium lacking Sox9 (Sox9-KO). Interestingly, Col18a1 has the ability to bind Wnt, a known regulator of Sox9 and ISC proliferation. Association between Sox9, Col18a1 and Wnt signaling suggests that Sox9 may be a driver of dynamic reciprocity within the ISC niche, allowing ISCs to interact with the surrounding ECM. Therefore, the central hypothesis for this proposal is that Sox9 drives the production of Col18a1 to maintain ISC identity by creating a Wnt gradient in the small intestinal crypt. To test this hypothesis, we will first determine the cellular origin of Col18a1 within the ISC niche by single-cell RNA sequencing of wild type intestinal crypt epithelium and supportive pericryptal myofibroblasts. Data collected will provide information about the cellular origin of Col18a1 as well as any other differentially expressed genes for future studies. We will then determine the effect of Sox9 on Col18a1 proteins within the niche ECM by mass spectrometry of acellular wild type and Sox9-KO ECM. Western blots and immunohistochemistry will identify abundance and location of Col18a1 and any aberrant expression patterns in Sox9-KO ECM. Finally, we will determine the influence of Col18a1 on ISC function by silencing Col18a1 expression by lentiviral transduction of Col18a1 siRNA in cultured ISCs. Identifying the relationship between Sox9 and Col18a1 is critical for understating ISC/ECM dynamics during homeostasis, regeneration following injury, and disease, as well as developing novel intestinal tissue engineering strategies.