Elucidating the regulation and function of FoxL1+ cell-derived Wnt5a during intestinal development

项目摘要 本研究的目的是探讨FoxL1+端粒细胞在肠道发育过程中的作用。 其生产的WNT5a。WNT5A是一种非正则的WNT配体，对许多器官的发育是必不可少的， 包括肠道。Wnt5a在Wnt5a-/-小鼠的早期肠道发育中的作用已经被研究， 在中肠伸长过程中，由于顶端基础极性(ABP)受损，表现为肠管缩短。然而， 关于Wnt5a的表达是如何调节的，或者它在出生后发育过程中的功能，人们知之甚少。最近， 我们的实验室显示了一群罕见的间充质细胞，即FoxL1+上皮下端粒细胞(FoxL1+端粒细胞)，以 是干细胞室中典型的Wnt信号的重要来源。此外，这些FoxL1+ 成年小鼠的端细胞沿隐窝壁和隐窝远端区域表达Wnt5a。因此，我 建立FoxL1-Cre；Wnt5af/f小鼠和FoxL1-CreERT2；Wnt5af/f小鼠，以阐明表达调控和 Wnt5a在FoxL1+端粒细胞中的作用这是一种创新的方法，因为之前所有的WNT5a研究都缺乏 对表达该基因的不同细胞群体的了解，限制了功能研究的深度 都可以被执行。我的模型是第一个在肠道中以特定细胞类型的方式删除Wnt5a的模型，用于 在生物学背景下对Wnt5a功能的活体评估。目标1将确定FoxL1是否调节Wnt5a 在肠道发育过程中建立ABP的表达。使用FoxL1-Cre；Wnt5af/f小鼠，我将评估ABP 在中肠伸长过程中的完整性，并进行蛋白质印迹和下拉试验以阐明 涉及的机制。使用FoxL1-/-小鼠，我将检验FoxL1与Wnt5a启动子和Wnt5a结合的假设。 通过qRT-PCR、ChIP-SEQ和荧光素酶分析直接调节Wnt5a的表达。Aim 2将使用 条件性Wnt5a基因敲除小鼠模型FoxL1-CreERT2；Wnt5af/f，以探讨Wnt5a在新生儿中的作用 地穴形成，这是一个以前从未被描述过的过程。我会用组织学的方法看看 这一过程需要来自FoxL1+端粒细胞的Wnt5A。我还会进行免疫组织化学分析， Western blotting和qRT-PCR来确定Wnt5a是否拮抗典型的Wnt/b-catenin信号 在地穴形成过程中。最后，我将利用3D器官培养系统对胎儿隐窝进行体外分析 发展。总之，这些目的将阐明FoxL1和Wnt5a信号在调控中的作用 哺乳动物肠道发育，这可能为肠道发育提供新的治疗靶点 疾病和炎症性疾病。

Project Summary The objective of this proposal is to investigate the role of FoxL1+ telocytes during intestinal development through its production of Wnt5a. Wnt5a is a non-canonical Wnt ligand that is essential for development of many organs, including the intestine. The role of Wnt5a in early intestinal development has been examined in Wnt5a-/- mice, which exhibit shortened intestines due to impaired apical basal polarity (ABP) during midgut elongation. However, little is known about how Wnt5a expression is regulated, or its functions during postnatal development. Recently, our lab showed a rare population of mesenchymal cells, the Foxl1+ subepithelial telocytes (FoxL1+ telocytes), to be the essential source of canonical Wnt signals in the stem cell compartment. Furthermore, these FoxL1+ telocytes were found to express Wnt5a along the crypt wall and crypt-distal regions in adult mice. Therefore, I generated FoxL1-Cre; Wnt5af/f mice and FoxL1-CreERT2; Wnt5af/f mice to elucidate expression regulation and function of Wnt5a in FoxL1+ telocytes. This is an innovative approach because all previous Wnt5a studies lacked knowledge of the distinct cell population expressing this gene, which limited the depth of functional studies that could be performed. My model is the first to delete Wnt5a in a cell type-specific manner in the intestine, for an in vivo assessment of Wnt5a function in its biological context. Aim 1 will determine whether FoxL1 regulates Wnt5a expression to establish ABP during intestinal development. Using FoxL1-Cre; Wnt5af/f mice, I will assess ABP integrity during midgut elongation, and perform western blotting and pulldown assays to elucidate the mechanisms involved. Using FoxL1-/- mice, I will test the hypothesis that FoxL1 binds at the Wnt5a promoter and is a direct regulator of Wnt5a expression by qRT-PCR, ChIP-seq, and luciferase assays. Aim 2 will use a conditional Wnt5a knockout mouse model, FoxL1-CreERT2; Wnt5af/f, to investigate the role of Wnt5a in neonatal crypt formation, a process that has never been characterized previously. I will use histological methods to see if Wnt5a from FoxL1+ telocytes are required for this process. I will also perform immunohistochemical analyses, western blotting, and qRT-PCR to determine whether Wnt5a antagonizes canonical Wnt/b-catenin signaling during crypt formation. Finally, I will utilize a 3D organoid culture system for an ex vivo analysis of fetal crypt development. Together, these Aims will elucidate the roles of FoxL1 and Wnt5a signaling in regulating mammalian intestinal development, which may point to novel therapeutic targets for intestinal developmental diseases and inflammatory diseases.