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

• 批准号: 10318955
• 负责人: Ondine B Cleaver
• 金额: $ 40.13万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-10 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318955
• 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; Embryo; Embryonic Development; Endocrine; Epithelial; 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; 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; directed differentiation; embryonic stem cell; experimental study; improved; insight; islet; mouse model; non-muscle myosin; novel; pancreas development; pancreas imaging; progenitor; regenerative therapy; stem cells; three dimensional cell culture; trafficking; type I diabetic

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.

项目概要 胰腺内分泌细胞，包括产生胰岛素的β细胞，在生长过程中逐步获得它们的命运。 胚胎发育。事实证明，理解并重述这些步骤对于定向 ES细胞分化为β细胞。许多学术和生物制药团体报告称，情况有所改善 将培养方法从 2D 培养转变为 3D 培养时 β 细胞生成的效率。这一观察表明 β细胞生成的细胞生态位的结构至关重要，然而，这个想法目前仍然存在 未经探索。我们之前报道过，当胰腺首次出现时，内胚层上皮经历 瞬时分层，然后是微腔形成和融合，生成互连的 3D 网络 上皮管称为胰“丛”[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神经丛中。目前尚不清楚神经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。自从我们最初提出建议以来，我们发布了 研究结果表明，Afadin 对于胰腺形态发生和内分泌命运至关重要（Azizoglu 等人，2017）。 在这里，我们建议阐明 Afadin 控制上皮细胞的细胞和分子机制。 管腔形成和神经丛形态发生。 在之前的工作中，我们生成了一种缺失了连接和细胞骨架调节因子 Afadin 的突变小鼠 （AfapancKO）未能解决其短暂神经丛。 Afadin 和 RhoA 共同耗尽（AfaRhoApancKO 或 AfaRhoDKO）表现出多个管腔缺陷。令人惊讶的是，它还能增加内分泌细胞数量， 包括β细胞。然而，RhoApancKO 没有表现出胰腺缺陷。 Afadin 和 RhoA 通路如何相互作用 仍然未知。 AfapancKO 和 AfaRhoDKO 中的一项引人注目的观察结果是核心神经丛仍然存在。我们 提出祖细胞池和最终内分泌质量由核心丛的持久性决定。 这是如何发生的是该提案的核心问题。我们假设 Afadin 和 RhoA 驱动上皮细胞 通过调节细胞过程（例如囊泡）实现管腔形态发生（形成/延伸/解析） 运输（目标 1），以及细胞分裂和/或细胞迁移（目标 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（目标 3）。这些过程共同协调 建立一个有利于内分泌细胞产生的生态位。 这些研究的完成将扩大我们对胰腺发育的了解，并将导致增强 产生内分泌细胞（包括β细胞）的策略，可能有助于开发新型治疗方法 我是糖尿病患者。