Role of Afadin in 3D epithelial plexus morphogenesis and beta cell mass

Afadin 在 3D 上皮丛形态发生和 β 细胞质量中的作用

基本信息

批准号：
9983885
负责人：
Ondine B Cleaver
金额：
$ 2.07万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-10 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9983885
关键词：
3-Dimensional Actins Actomyosin Affect Apical Architecture Beta Cell Binding Sites Cell Count Cell division Cell physiology Cell-Cell Adhesion Cells Cytoskeletal Modeling Cytoskeleton Defect Development Duct (organ) structure Ductal Embryo Embryonic Development Endocrine Epithelial Epithelium Exhibits Exocytosis F-Actin Future Generations Genetic Epistasis Genetic Models Image In Vitro Insulin Islet Cell Islets of Langerhans Knowledge Mediating Methods Molecular Morphogenesis Mosaicism Mus Mutant Strains Mice Myosin Type II Pancreas Pancreatic Bud Pancreatic duct Pathway interactions Pharmacology Process Publishing Replacement Therapy Reporter Reporting Resolution Role Shapes Stem cells Stratification Structure of beta Cell of islet Testing Three-Dimensional Imaging Time Tomatoes Tube Vesicle Work afadin apical membrane cell growth regulation cell motility cell type diabetes mellitus therapy diabetic patient embryonic stem cell experimental study improved insight islet mouse model non-muscle myosin novel pancreas development pancreas imaging progenitor regenerative therapy three dimensional cell culture trafficking type I diabetic

项目摘要

Project Summary Pancreatic endocrine cells, including insulin-producing beta cells, acquire their fate in a step-wise manner during embryonic development. Understanding and recapitulating these steps has proven essential for directed differentiation of ES cells into beta cells. A number of academic and biopharma groups have reported improved efficiency of beta cell generation upon shifting culture methods from 2D to 3D cultures. This observation suggests that architecture of the cellular niche for beta cell generation is critical, however, this idea currently remains unexplored. We previously reported that when the pancreas first emerges, the endodermal epithelium undergoes transient stratification, followed by microlumen formation and fusion to generate a 3D network of interconnected epithelial tubes called the pancreatic `plexus' [1]. Interestingly, recent studies demonstrate that endocrine progenitors are born within this transient core plexus. It is unclear how the plexus architecture impacts the fate of pancreatic progenitors, including those of endocrine lineage. Since our initial proposal, we published the findings that Afadin is essential to pancreas morphogenesis and endocrine fate (Azizoglu et al., 2017). Here, we propose to elucidate the cellular and molecular mechanisms by which Afadin controls epithelial lumen formation and plexus morphogenesis. In previous work, we generated a mutant mouse with deletion of the junctional and cytoskeletal regulator Afadin (AfapancKO) that fails to resolve its transient plexus. Co-depletion of Afadin and RhoA (AfaRhoApancKO or AfaRhoDKO) exhibits multiple lumen defects. Surprisingly, it also produces an increase in endocrine cell numbers, including beta cells. RhoApancKO however, show no pancreatic defects. How Afadin and RhoA pathways interact remains unknown. One striking observation in both AfapancKO and AfaRhoDKO is that the core plexus persists. We propose that the progenitor pool and final endocrine mass is determined by the perdurance of the core plexus. How this occurs is the central question of this proposal. We hypothesize that Afadin and RhoA drive epithelial lumen morphogenesis (formation/extension/resolution) via regulation of cellular processes, such as vesicle trafficking (Aim 1), and cell division and/or cell migration (Aim 2). Further, we hypothesize that Afadin and RhoA control these processes by regulating cytoskeletal organization (Aim 3). Together, these processes coordinate to build a niche propitious for generation of endocrine cells. Completion of these studies will expand our knowledge of pancreatic development, and will lead to enhanced strategies for generating endocrine cells, including beta cells, which may contribute to novel treatments for type I diabetic patients.

项目摘要胰腺内分泌细胞，包括产生胰岛素的β细胞，以逐步的方式获得其命运胚胎发展。理解和概括这些步骤已被证明对指导至关重要 ES细胞分化为β细胞。许多学术和生物制药组报告有所改善 β细胞产生的效率从2D培养物转移到3D培养物中。这个观察表明但是，beta细胞生成的细胞生态市场的结构至关重要，但是，目前仍然存在这个想法未探索。我们先前报道说，当胰腺首次出现时，内皮胚皮上皮会发生瞬态分层，然后是微木材形成和融合，以生成一个互连的3D网络上皮管称为胰腺“ plexus” [1]。有趣的是，最近的研究表明内分泌祖细胞诞生于这个短暂的核心丛中。目前尚不清楚丛结构如何影响命运胰腺祖细胞，包括内分泌谱系的祖细胞。自我们最初的建议以来，我们发表了结果表明，阿法蛋白对于胰腺形态发生和内分泌命运至关重要（Azizoglu等，2017）。在这里，我们提议阐明Afadin控制上皮的细胞和分子机制管腔形成和丛形式发生。在先前的工作中，我们生成了一个突变小鼠，并删除了连接和细胞骨架调节剂afadin （Afapancko）无法解决其瞬态丛。 Afadin和Rhoa（Afarhoapancko或 Afarhodko）表现出多个管腔缺陷。令人惊讶的是，它也会产生内分泌细胞数量的增加，包括β细胞。但是，Rhoapancko没有显示胰腺缺陷。 Afadin和Rhoa途径如何相互作用仍然未知。在Afapancko和Afarhodko中，一个惊人的观察结果是核心丛持续存在。我们建议祖细胞池和最终内分泌质量取决于核心神经丛的渗透率。这是该提案的主要问题。我们假设Afadin和Rhoa Drive上皮腔形态发生（形成/延伸/分辨率）通过调节细胞过程，例如囊泡贩运（AIM 1）和细胞分裂和/或细胞迁移（AIM 2）。此外，我们假设Afadin和Rhoa 通过调节细胞骨架组织来控制这些过程（AIM 3）。这些过程在一起协调为生成内分泌细胞建立一个利基市场。这些研究的完成将扩大我们对胰腺发展的了解，并将导致增强生成内分泌细胞（包括β细胞）的策略，这可能有助于新型治疗类型我的糖尿病患者。