Anatomic and functional characterization of the intestinal crypt-villus niche

项目说明 肠上皮的完整性、周转和功能需要来自底层间充质的信号。 NIDDK和NIAID的肠干细胞联盟(ISCC)的新焦点是在 提供关键支持的间充质细胞，它们在组织中的组织，以及它们的 分子特征，包括基本的分泌因子。该协会一直是该联盟的成员。 此申请是响应RFA-DK-18-507，一个有限的竞争，以 续签ISCC研究中心。尽管该领域的最新进展涉及到各种细胞群体 (CD34、Foxl1、PDGFRA、Gli1)作为关键的生态位元素，它们的准确身份、重叠和贡献 仍然不确定。通过综合考虑高分辨率整体安装的共焦显微镜和 通过流式细胞术分离的明确定义的细胞群体的总体和单细胞(Sc)RNA-seq分析， 我们已经确定了三种不同的间充质细胞类型作为基本利基的主要候选细胞。 功能。在过去的两年里，我们分享了关于这些端粒细胞和两个截然不同的(解剖学和 分子)PDGFRAlo细胞群体在几个ISCC中刺激了一个重要的研究流 实验室。在这一经验和社区的基础上，在富有成效地完成所有目标之后 从目前的获奖期来看，我们提出了三条调查路线。特定目标1将生成 详细的小鼠基质细胞群体的分子、解剖和功能图谱，有三个目的： (A)确定WNT、BMP、EGF和其他高空间关键间充质信号的来源 决议，(B)为选定的重要间充质细胞建立健壮和可重复的培养条件 细胞类型，以及(C)通知相应功能人体细胞的识别和验证 人口。这些研究将整个小肠作为一个单位，目标2将推进目的 (C)进一步考虑每个肠道区域(十二指肠、空肠、回肠以及升降 冒号)分开。这两个目标都将整合高分辨率的解剖和分子分析，以产生 准确的3D地图，叠加了对间充质细胞功能的评估 与ISCC其他小组的协作。目标3解决了外地的迫切需要和具体的区域金融行动 调用以生成新的Cre驱动程序和荧光报告小鼠。基于广泛的分子图谱 针对1和2，我们将确定新的细胞类型特异性基因座，并使用CRISPR/Cas9编辑插入报告 构建在多达3个这样的基因座上。每一项目标都涉及与ISCC实验室的广泛合作 全面协作管理计划的保护伞。此外，我们还将贡献和采用 标准化方案，并与ISCC调查人员共享数据、方案、试剂和小鼠品系。

Project Description Intestinal epithelial integrity, turnover, and function require signals from the underlying mesenchyme. The new focus of the Intestinal Stem Cell Consortium (ISCC) of the NIDDK and NIAID is to investigate in depth the mesenchymal cells that provide crucial support, their organization within the tissue, and their molecular signatures, including essential secreted factors. The PI has been a member of the Consortium for the last 4 years and this application is made in response to RFA-DK-18-507, a Limited Competition to renew the ISCC Research Centers. Although recent progress in the field implicates various cell populations (CD34+, Foxl1+, PDGFRA+, Gli1+) as key niche elements, their precise identities, overlap, and contributions remain uncertain. By integrated consideration of high-resolution whole-mount confocal microscopy and ensemble and single-cell (sc) RNA-seq analysis of well-defined cell populations isolated by flow cytometry, we have identified three distinct mesenchymal cell types as leading candidates for essential niche functions. Over the last 2 years, our sharing of data on these telocytes and two distinct (anatomic and molecular) PDGFRAlo cell populations has stimulated one important stream of investigation in several ISCC laboratories. Building on this experience and community, and following productive completion of all Aims from the current award period, we propose three lines of investigation. Specific Aim 1 will generate detailed molecular, anatomic, and functional maps of mouse stromal cell populations, with three purposes: (a) to identify the sources of Wnt, BMP, EGF, and other key mesenchymal signals at high spatial resolution, (b) to establish robust and reproducible culture conditions for selected important mesenchymal cell types, and (c) to inform identification and validation of the corresponding functional human cell populations. These studies consider the whole small intestine as a unit, and Aim 2 will advance purpose (c) further by considering each gut region (duodenum, jejunum, ileum, and ascending and descending colon) separately. Both Aims will integrate high-resolution anatomic and molecular analyses to generate accurate 3D maps, superimposed with assessments of mesenchymal cell functions conducted in collaboration with other ISCC groups. Aim 3 addresses an urgent need in the field and the specific RFA call to generate new Cre-driver and fluorescent reporter mice. Based on extensive molecular profiling in Aims 1 and 2, we will identify novel cell type-specific loci and use CRISPR/Cas9 editing to insert reporter constructs in up to 3 such loci. Each Aim involves extensive collaboration with ISCC laboratories under the umbrella of a comprehensive Collaborative Management Plan. In addition, we will contribute and adopt standardized protocols and share data, protocols, reagents, and mouse strains with ISCC investigators.