Determining the role of cellular forces in endoderm differentiation and development

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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.