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.
Intestinal stem cells are responsible for constant renewal of the epithelial lining of the intestine and are surrounded by a supportive environment of cells and extracellular matrix. The cellular origin of individual extracellular matrix proteins and their interaction with intestinal stem cells is unknown, but is important to completely understand intestinal stem cell function. Determining this relationship is critical for understanding how intestinal stem cells regenerate following injury and disease, as well as developing stem cell based therapies for intestinal tissue engineering.
| Kechele, Daniel O; Blue, R Eric; Zwarycz, Bailey et al. (2017) Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation. J Clin Invest 127:593-607 |
