Mesenchymal regulation of fetal intestinal development and adult regeneration

项目摘要 炎症性肠病代表上皮修复不足和结直肠癌（CRC）， 这是美国癌症死亡的第二大原因，反映了肠道干细胞的破坏 （ISCs）稳态。ISCs部分依赖于潜在的间充质信号梯度， 包括ISC在内的隐窝细胞的增殖与分化能力。此外，ISC可以再生 在消融后通过隐窝细胞的去分化而有效。尚不清楚哪些信号或间质 细胞促进这种再生过程。我的初步研究发现了三种不同的间充质细胞 两种类型，其中两种协调BMP信号梯度。高PDGFRA表达细胞， 对应于最近描述的端细胞，位于最接近上皮的位置，在绒毛处最丰富 碱基，并表达高水平的BMP配体。低PDGFRA表达细胞含有两个 亚群：仅在隐窝下方发现并表达高水平BMP的CD 81+部分 拮抗剂Gremlin 1和CD 81-部分（CP细胞），其位于隐窝上方和周围，但缺乏 Grem 1.在分离的隐窝上皮与这些严格纯化的细胞类型中的每一种的共培养物中， CD 81 + PDGFRA 10细胞在功能上替代了所有补充生长因子的培养基，而CP细胞则是如此。 最低限度，而telocyte则没有。此外，体内Grem 1+细胞的消融导致ISC的损失， 因此我称这些细胞为滋养细胞。这些不同的间充质细胞群如何调节 胚胎ISC在发育过程中的可塑性和ISC在丧失过程中的再生尚不清楚。我假设 端细胞、CP和滋养细胞功能在发育过程中进化，导致稳定的隐窝形成， 绒毛生态位和一些相同的人口促进隐窝再生后ISC消融。在 目的1、研究端细胞、CP细胞和 小鼠胚胎ISC发育过程中的滋养细胞。我将确定这些细胞何时出现， 开发，研究其完整的转录谱，并使用已建立的隐窝共培养 方法来定义他们的支持作用。在目标2中，我将测试这些间充质细胞如何响应 减少ISC丢失并促进隐窝再生。使用ISC再生的体内测定，结合 通过大量和单细胞RNAseq以及体内基因敲除，我将测试哪些细胞促进ISC 再生，特别是如果这通过超生理BMP抑制发生。总的来说，这些 这些研究将为胎儿ISCs的发育和调节提供基本的见解， 发育和再生，对肠道生物学和疾病具有广泛的影响。

Project Summary Inflammatory bowel disorders represent insufficient epithelial repair and colorectal cancer (CRC), the second leading cause of cancer deaths in the United States, reflects disruption of intestinal stem cell (ISCs) homeostasis. ISCs depend in part on underlying mesenchymal signaling gradients that balance proliferation versus differentiation capacity of crypt cells, including ISCs. Moreover, ISCs regenerate efficiently after ablation, by de-differentiation of crypt cells. It is unknown which signals or mesenchymal cells promote this regenerative process. My preliminary studies uncover three distinct mesenchymal cell types, two of which orchestrate BMP signaling gradients. High PDGFRA- expressing cells, which correspond to recently described telocytes, lie closest to the epithelium, are most abundant at the villus base, and express high levels of BMP ligands. Low PDGFRA-expressing cells contain two subpopulations: a CD81+ fraction found exclusively below crypts and expressing high levels of the BMP antagonist Gremlin1, and a CD81- fraction (CP cells) which resides above and around crypts but lacks Grem1. In co-cultures of isolated crypt epithelium with each of these strictly purified cell types, CD81+PDGFRAlo cells functionally replace all growth factor-supplemented media, while CP cells do so minimally and telocytes do not. Furthermore, ablation of Grem1+ cells in vivo results in loss of ISCs, hence I call these cells trophocytes. How each of these distinct mesenchymal cell populations regulates fetal ISC plasticity during development and ISC regeneration during loss, is unknown. I hypothesize that telocyte, CP, and trophocyte functions evolve during development, leading to genesis of a stable crypt- villus niche and that some of the same populations promote crypt regeneration after ISC ablation. In Aim 1, I will investigate the molecular profiles and functions of telocytes, CP cells, and trophocytes during mouse fetal ISC development. I will determine when these cells arise during development, investigate their full transcriptional profiles, and use an established crypt co-culture method to define their supportive roles. In Aim 2, I will test how these mesenchymal cells respond to ISC loss and promote crypt regeneration. Using in vivo assays of ISC regeneration, combined with bulk and single-cell RNAseq and in vivo genetic knockouts, I will test which cells promote ISC regeneration and specifically if this occurs through supraphysiologic BMP inhibition. Collectively, these studies will provide fundamental insights into the development and regulation of ISCs in fetal development and regeneration, with broad implications for intestinal biology and disease.