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

确定 Sox9 和 XVIII 胶原蛋白在维持肠道干细胞身份方面的关系

基本信息

批准号：
8980462
负责人：
Bailey Zwarycz
金额：
$ 3.1万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8980462
关键词：
Affect Binding Cell Maintenance Cell Proliferation Cell Survival Cell Therapy Cell physiology Cells Collagen Type XVIII Data Disease Environment Epithelial Epithelial Cells Epithelium Extracellular Matrix Extracellular Matrix Proteins Extracellular Protein Fibroblasts Future Gene Expression Profile Gene Expression Profiling Genes Goals Growth Heterogeneity Homeostasis Immunohistochemistry In Vitro Individual Influentials Injury Intestines Ligands Location Maintenance Maps Mass Spectrum Analysis Morphology Mus Myofibroblast Natural regeneration Organoids Paneth Cells Pathway interactions Pattern Population Porifera Production Proteins RNA Sequences Regulation Signal Transduction Small Interfering RNA Stem cells Testing Tissue Engineering Tissues Villus Western Blotting base cell behavior cell type collaborative environment differential expression extracellular intestinal crypt intestinal epithelium matrigel novel public health relevance stem cell biology stem cell niche theories transcription factor transcriptome sequencing

项目摘要

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.