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.

项目摘要 胰腺内分泌细胞，包括产生胰岛素的β细胞，在 胚胎发育。事实证明，理解和概括这些步骤对于指导 胚胎干细胞向β细胞的分化。一些学术和生物制药团体报告说，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。这一观察结果表明 产生β细胞的细胞生态位的结构是至关重要的，然而，这个想法目前仍然存在 未被开发的。我们之前曾报道，当胰腺第一次出现时，内胚层上皮经历 瞬时分层，随后微腔形成和融合，以生成相互连接的3D网络 上皮管被称为胰腺‘神经丛’[1]。有趣的是，最近的研究表明，内分泌 祖细胞在这个短暂的核心神经丛中诞生。目前还不清楚丛状结构对命运的影响 胰腺祖细胞，包括内分泌血统的那些。自从我们最初的提议以来，我们发布了 研究发现，Afadin对胰腺形态发生和内分泌命运至关重要(Azizoglu等人，2017年)。 在此，我们建议阐明Afadin控制上皮细胞的细胞和分子机制 管腔形成和神经丛的形态发生。 在之前的工作中，我们产生了一个缺失连接和细胞骨架调节因子Afadin的突变小鼠 (AfapancKO)，未能解决其暂时性神经丛。Afadin和RhoA的共同耗竭(AfaRhoApancKO或 AfaRhoDKO)表现出多个管腔缺陷。令人惊讶的是，它还会增加内分泌细胞的数量， 包括β细胞。然而，RhoApancKO显示没有胰腺缺陷。Afadin和RhoA通路如何相互作用 仍然不为人知。在AfapancKO和AfaRhoDKO中都有一个引人注目的观察结果，即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神经丛的耐力决定。 如何做到这一点是这项提议的核心问题。我们假设Afadin和RhoA驱动上皮细胞 管腔形态发生(形成/延伸/分解)通过调节细胞过程，如小泡 贩运(目标1)和细胞分裂和/或细胞迁移(目标2)。此外，我们假设阿法丁和罗亚 通过调节细胞骨架组织来控制这些过程(目标3)。总的来说，这些过程相互协调 建立一个有利于内分泌细胞生成的利基环境。 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 产生内分泌细胞的策略，包括β细胞，这可能有助于类风湿关节炎的新治疗 我是糖尿病患者。