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: 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
• 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

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，在胰腺癌发生过程中以逐步的方式获得它们的命运。 胚胎发育理解和概括这些步骤已被证明是至关重要的定向 ES细胞分化为β细胞。一些学术和生物制药团体报告说， 在将培养方法从2D培养转移到3D培养时的β细胞生成效率。这一观察结果表明 β细胞生成的细胞小生境的结构是至关重要的，然而，这个想法目前仍然存在， 未开发的我们以前报道过，当胰腺第一次出现时，内胚层上皮经历 瞬时分层，然后是微腔形成和融合，以生成互连的3D网络。 称为胰腺“丛”的上皮管[1]。有趣的是，最近的研究表明， 祖细胞在这个短暂的核心神经丛中诞生。目前还不清楚神经丛结构如何影响 胰腺祖细胞，包括那些内分泌谱系。自从我们最初提出建议以来， 发现Afadin对胰腺形态发生和内分泌命运是必需的（Azizoglu等，2017年）。 在这里，我们建议阐明阿法丁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。 在以前的工作中，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）未能解决其短暂丛。Afadin和RhoA的共消耗（AfaRhoApancKO或 AfaRhoDKO）表现出多个管腔缺陷。令人惊讶的是，它还增加了内分泌细胞的数量， 包括β细胞然而，RhoApancKO没有显示胰腺缺陷。Afadin和RhoA通路如何相互作用 仍然未知。在AfapancKO和AfaRhoDKO中的一个引人注目的观察结果是核心神经丛持续存在。我们 提出了祖先池和最终的内分泌质量是由核心丛的持久性决定的。 如何做到这一点是本提案的核心问题。我们假设Afadin和RhoA驱动上皮细胞 管腔形态发生（形成/延伸/消退）通过调节细胞过程，如囊泡 运输（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（目的3）。这些过程共同协调 以建立一个有利于内分泌细胞生成的生态位。 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 产生内分泌细胞的策略，包括β细胞，这可能有助于新型治疗方法。 我是糖尿病患者。