The proposal "Regulation of Vertebrate Axial Extension" aims to expand on the observation previously made by this investigator that a cortical acto-myosin network is required for the cellular convergence and extension that drives the shape change of frog embryos at gastrulation. Because this sub-membranous actin network requires the myosin IIB (MIIB) complex to function in this morphogenesis we aim to determine whether this MII dependence requires actin-activated contractility, or if MII functions solely as a actin crosslinker during axial extension. Preliminary evidence depleting the myosin regulatory light chain (MRLC), required for expression of contractile but not crosslinking activity in MII, indicates that myosin II contractility is required. Because regulation of MII contractility is controlled by the phosphorylation state of Ser19 on MRLC, we perturbed a possible dynamic homeostasis of this Ser19-P event by adding an inhibitor of myosin phosphatase to embryos, and assaying for Ser19 phosphorylation by quantitative western blotting to reveal an acute upregulation of Ser19-P. This experiment reveals that there is an active, balanced process involving myosin phosphatase and a kinase or kinases to regulate Ser19-P levels in intercalating cells, and further acute drug experiments indicate that the relevant kinase is MLCK. Because Ca ++ is an upstream regulator of MLCK, we reexamined Ca++ dynamics in intercalating cells, finding a novel pattern of Ca++ fluxes, as well as a plausible mechanism for generating these fluxes through a putative Ca++ channel protein that we show to be required for gastrulation. We also identify a role for a second motor protein in regulating the cortical actin network, allowing us to propose a model for CE consisting of a simple oscillating regulatory circuit with biomechanical feedback control, as well as the means to test this hypothesis.
The proposal Regulation of Vertebrate Axial Extension aims to uncover the proximal molecular mechanism by which intercalating mesodermal cells regulate force production that drives the change in embryo shape that occurs during gastrulation. These studies are performed in frog embryos but are likely to have relevance to human health both because partial failure of this mesodermal morphogenesis leads to subsequent failure of neural tube closure to cause spina bifida (failure of posterior closure) or anencephaly (anterior closure) while complete failure of this morphogenesis is incompatible with normal development. The eventual aim is that an understanding how axial extension is regulated will at least lead to diagnostic applications in human infertility situations, with a reasonable probability that therapeutic interventions could be contemplated in these situations.