Determining the role of cellular forces in endoderm differentiation and development

项目概要/摘要 在胚胎发育过程中，组织和器官的形成涉及到精心协调的组装， 将众多的单个细胞整合成一个连贯的、更高层次的结构。尽管分子和遗传因素 器官发生的机制已经得到了充分的研究，这些因素调节的细胞生物学过程，如细胞 迁移，运动和粘附，了解得少得多，甚至更少的是关于机械的作用， 在这个过程中的力量。内胚层是三个主要的胚层之一，最终产生 胃肠道和呼吸道上皮细胞以及甲状腺和胸腺等其他器官。期间 在发育过程中，内胚层细胞经历细胞表型的根本变化。在原肠胚形成期间，它们 高度迁移，但彼此之间的协调程度最低，我们称之为“单细胞迁移”。最终 然而，它们分化成在起重要屏障作用连贯组织内的成熟上皮细胞 在体内发挥作用。这个关键的过渡期，从动态的，单细胞迁移到上皮片层 形成，是特别容易在斑马鱼胚胎，由于其光学透明性和顺从性， 长期显微镜在初步的实验中，我们发现这个过渡期伴随着 质膜起泡的变化是由细胞内的物理力量，即膜到 皮质附着力（MCA）和皮质张力。在本建议中，我们将定量描述这些 物理力量，并确定其意义的内胚层发展有两个具体的目标。上 目的，我们将采取互补的体外和体内方法，以定量确定是否和什么 MCA和收缩力在内胚层细胞从单细胞转化为单细胞的过程中发生变化， 向席状构造迁移。在第二个目标中，我们将确定在体内的意义， 通过实验操纵分化中的起泡频率， 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，建立定量的实验 管道，可以扩展到其他组织和未来的研究。

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.