The physical and molecular mechanisms of intestinal villus morphogenesis and repair

摘要 绒毛是指状突起，排列在小肠的管腔内。绒毛在营养吸收中起着关键作用 通过将肠道吸收表面积增加几个数量级。失去了这种吸收能力 体表通过绒毛萎缩会导致主要的消化并发症和营养吸收不良。异常 绒毛中存在于许多胃肠道疾病中，如炎症性肠病和乳糜泻， 还有放射、化疗和感染的副作用。退化的绒毛有时可以完全改造，但在 其他情况下，再生受损，导致持续性绒毛萎缩和患者痛苦。 在发育过程中，从最初平坦的肠道表面长出绒毛。绒毛形成的机制 形成和修复仍然没有得到很好的描述，对这些过程的理解对于开发是至关重要的 新疗法。这个提议的长期目标是建立对分子和力的理解，这些分子和力 在发育和再生过程中塑造绒毛，以改进生长和再生的策略 人类病人的肠道。我们实验室的最新数据表明，间充质在 雕刻着别墅的建筑。具体地说，我们的初步数据表明，有一群专门的自闭症患者 将凝聚在形成的绒毛下方的亚上皮间充质细胞组织为来源 形成覆盖的上皮并将其折叠成绒毛所需的物理力量。我们调查了 使用单细胞RNAseq导致这些细胞凝结的分子机制，并发现他们还 表达一个独特的转录程序。这个程序与调控钙离子的基因有不同寻常的重叠。 介导了平滑肌细胞的收缩，但不表达平滑肌肌动蛋白。我们提供证据 抑制这一程序中的关键蛋白会导致间充质凝聚和绒毛的丧失 外翻。在这些初步数据的指导下，我们建议检验与互补性相关的两个假设 绒毛形成的物理和分子方面。我们将定量测量和计算相结合 对绒毛凝结物的形成类似于相分离的假设进行建模检验 物理学家和材料科学家广泛研究的现象，以及凝聚体施加物理力 在上面的上皮细胞上启动其折叠。然后，我们测试了内皮素从血管内皮细胞释放出来的假设 上皮细胞触发上皮下间充质细胞内钙信号的增加，这些信号是同步的 通过缝隙连接来驱动细胞的收缩，导致相分离和凝聚。 这个项目对我们从根本上理解肠道的发育和 肠道组织的组织工程学，因为我们不知道哺乳动物的肠道绒毛是如何构建的。因此，这些 这些发现将是有影响力的，因为它们为这一过程提供了一种新的机械蓝图，其中包括 既有信号也有力量。这些发现也将为未来的再生研究奠定基础：我们发现 在成人中，亚上皮间充质保留了许多这些分子特征的表达。此外， 这些特征在受伤后上调。

ABSTRACT Villi are finger-like projection that line the lumen of the small intestine. Villi play a critical role in nutrient uptake by increasing the intestinal absorptive surface area by several orders of magnitude. Loss of this absorptive surface through villus atrophy causes major digestive complications and nutrient malabsorption. Abnormalities in villi are found in many gastrointestinal maladies, such as inflammatory bowel and celiac diseases, and are also side effects of radiation, chemotherapy, and infection. Degenerated villi can sometimes fully reform, yet in other situations regeneration is impaired, resulting in persistent villus atrophy and patient suffering. Villi emerge during development from an initially flat intestinal surface. The mechanisms underlying villus formation and repair remain poorly described, and an understanding of these processes is essential to develop new therapies. The long term goal of this proposal is to build an understanding of the molecules and forces that sculpt the villus during development and regeneration so as to improve strategies for growing and regenerating the intestine for human patients. Recent data from our labs suggest that the mesenchyme plays a central role in sculpting the architecture of the villus. Specifically, our preliminary data implicate a specialized population of self- organizing sub-epithelial mesenchymal cells that condense immediately below the forming villus as the source of the physical forces necessary to pattern and fold the overlying epithelium into villi. We investigated the molecular mechanisms leading to condensation of these cells using single cell RNAseq and found they also express a unique transcriptional program. This program has an unusual overlap with genes regulating Ca2+ mediated contractility in smooth muscle cells but without expressing smooth muscle actin. We provide evidence that inhibition of key proteins in this program results in a loss of mesenchymal condensation and villus evagination. Guided by these preliminary data, we propose to test two hypotheses related to the complementary physical and molecular aspects of villus formation. We combine quantitative measurements and computational modeling to test the hypothesis that the formation of villus condensates occurs analogously to phase separation phenomena studied extensively by physicists and material scientists, and that condensates exert physical forces on the overlying epithelium initiating its folding. We then test the hypothesis that Endothelin released from the epithelium triggers increased calcium signaling in the subepithelial mesenchyme, which are synchronized through gap junctions to drive cell contractility leading to phase separation and condensation. This project has major implications for our fundamental understanding of the developmental of the gut and for tissue engineering of intestinal tissue, as we do not know how mammalian intestinal villi are built. Thus, these findings will be impactful because they provide a new mechanistic blueprint for this process that incorporates both signals and forces. These findings will also lay the groundwork for future studies of regeneration: we find that in adults, the sub-epithelial mesenchyme retains expression of many of these molecular features. Further, these features are upregulated following injury.