Control of epithelial cell layer spreading in zebrafish

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Epithelial cell layer spreading is a common and fundamental process in various developmental and disease-related events, such as dorsal closure in Drosophila and wound healing. In the previous funding period, we have dissected the force-generating mechanisms driving epithelial cell layer spreading during zebrafish gastrulation. Specifically, we showed that spreading of the enveloping cell layer (EVL) over the yolk cell during epiboly is triggered by contraction of the actomyosin cortex forming a band-like structure within the yolk syncytial layer (YSL), a thin cytoplasmic layer on the yolk cell surface close to the margin of the EVL. We could further show that this actomyosin band pulls on the margin of the EVL by two distinct motor activities - a cable-constriction motor and a flow-friction motor. The cable-constriction motor operates via circumferential contraction of the actomyosin band, which couples to the spherical geometry of the yolk sac to pull on the EVL margin, much like a purse string. The flow-friction motor operates via retrograde actomyosin flows within the YSL. These are resisted by friction to adjacent structures and thus directly pull the EVL margin towards the vegetal pole. While these findings provided novel insight into the mechanisms by which the YSL actomyosin band drives EVL epiboly movements, important questions remain as to the initiation of actomyosin band formation within the YSL and the mechanical linkage between EVL and YSL allowing the YSL to pull on the EVL. To address these questions, we have begun to analyze how the EVL at its margin establishes contacts with the underlying YSL, and how this contact formation triggers the band-like accumulation of actomyosin within the YSL. Based on our preliminary observations, we hypothesize that microtubules (MTs) within the YSL promote tight junction (TJ) formation between the YSL and EVL, and that proper formation of these junctions in turn directs actomyosin band assembly within the YSL required for EVL epiboly movements. We will address this hypothesis by an interdisciplinary approach employing methods and tools from developmental biology, cell biology, biophysics and theoretical modeling. We aim to elucidate the interplay between MTs, TJs and the actomyosin network during EVL cell layer spreading. We expect that this approach will provide insight into the mechanisms of force-generation, transduction and mechansensation during epithelial cell layer spreading.