Determining the role of cellular forces in endoderm differentiation and development

项目摘要/摘要 胚胎发育期间组织和器官的形成涉及到仔细协调的组装。 将众多单个细胞组合成一个连贯的、更高层次的结构。虽然分子和遗传因素 这些因素调节的细胞生物学过程，如细胞 对迁移、运动和粘合的了解要少得多，对机械的作用更是知之甚少。 在这一过程中的力量。内胚层是三个主要胚层之一，最终导致 胃肠道和呼吸道上皮以及其他器官，如甲状腺和胸腺。在他们的 在发育过程中，内皮细胞经历了细胞表型的根本性变化。在原肠发育过程中，它们是 高度迁徙，但彼此之间的协调程度最低，我们将这种行为称为“单细胞迁移”。最终 然而，它们在执行重要屏障的连贯组织内分化为成熟的上皮细胞。 身体内部的功能。这一关键的过渡期，从动态的单细胞迁移到上皮片 由于其光学透明性和适应性，在斑马鱼胚胎中特别容易形成 长期显微镜检查。在初步实验中，我们发现这一过渡期伴随着 由细胞内物理力驱动的质膜气泡的变化，即膜到膜 皮质附着(MCA)和皮质张力力。在这份提案中，我们将量化地描述这些 物理力量和决定它们对内胚层发育的意义有两个特定的目标。在第一个 目的：我们将采取体外和体内互补的方法来定量确定是否和对什么 内皮细胞从单细胞向内皮细胞转化的过程中，MCA和收缩力的变化 向板状地层迁移。在第二个目标中，我们将确定与起泡相关的体内意义。 通过实验控制分化过程中的气泡频率，特异性地促进内胚层发育 斑马鱼胚胎内的内胚层细胞。这项工作将促进我们对细胞的作用的理解 内胚层形态发生中的机械力，重要的是，建立了一个定量的实验 可以延伸到其他组织和未来研究的流水线。

PROJECT SUMMARY/ABSTRACT The formation of tissues and organs during embryonic development involves the carefully coordinated assembly of numerous individual cells into a coherent, higher-order structure. Although the molecular and genetic factors of organogenesis are well-studied, the cell biological processes that these factors regulate, such as cell migration, motility, and adhesion, are much less understood, and even less is known about role of mechanical forces in this process. The endoderm is one of the three primary germ layers that ultimately gives rise to the gastrointestinal and respiratory epithelia as well as other organs such as the thyroid and thymus. During their development, endodermal cells undergo a radical change in cellular phenotype. During gastrulation, they are highly migratory but minimally coordinated with each other, a behavior we term “single-cell migration.” Eventually however, they differentiate into mature epithelial cells within a coherent tissue that performs important barrier functions within the body. This critical transition period, from dynamic, single-cell migration to epithelial sheet formation, is especially accessible in the zebrafish embryo owing to its optical transparency and amenability to long-term microscopy. In preliminary experiments, we found that this transition period is accompanied by changes in plasma membrane blebbing that are driven by intracellular physical forces, namely membrane-to- cortex attachment (MCA) and cortical tension forces. In this proposal, we will quantitatively characterize these physical forces and determine their significance to endoderm development with two specific aims. In the first aim, we will take complementary in vitro and in vivo approaches to quantitively determine whether and to what extent MCA and contractile forces are changing during the transition of endodermal cells from single-cell migration to sheet formation. In the second aim, we will determine the in vivo significance of blebbing-associated forces specifically to endoderm development by experimentally manipulating blebbing frequency in differentiating endodermal cells within zebrafish embryos. This work will advance our understanding of the role of cellular mechanical forces in endodermal morphogenesis, and, importantly, establish a quantitative experimental pipeline that can be extended to other tissues and future studies.