Regulation of Axial Extension in Vertebrate Embryos

描述（由申请人提供）：“脊椎动物轴向延伸的调节”提案旨在扩展该研究人员之前的观察结果，即细胞会聚和延伸需要皮质肌动球蛋白网络，从而驱动青蛙胚胎在原肠胚形成时的形状变化。由于这种膜下肌动蛋白网络需要肌球蛋白 IIB (MIIB) 复合物在这种形态发生中发挥作用，我们的目的是确定这种 MII 依赖性是否需要肌动蛋白激活的收缩性，或者 MII 在轴向伸展过程中是否仅作为肌动蛋白交联剂发挥作用。初步证据表明，MII 中表达收缩活性而非交联活性所需的肌球蛋白调节轻链 (MRLC) 被耗尽，表明肌球蛋白 II 的收缩性是必需的。由于 MII 收缩性的调节是由 MRLC 上 Ser19 的磷酸化状态控制的，因此我们通过向胚胎中添加肌球蛋白磷酸酶抑制剂，并通过定量蛋白质印迹法检测 Ser19 磷酸化来扰乱该 Ser19-P 事件可能的动态稳态，以揭示 Ser19-P 的急性上调。该实验揭示了在闰细胞中存在一个涉及肌球蛋白磷酸酶和一种或多种激酶来调节Ser19-P水平的活跃、平衡的过程，并且进一步的急性药物实验表明相关的激酶是MLCK。由于 Ca 是 MLCK 的上游调节因子，因此我们重新检查了嵌入细胞中的 Ca 动力学，发现了一种新的 Ca 通量模式，以及通过我们证明原肠胚形成所需的假定 Ca 通道蛋白产生这些通量的合理机制。我们还确定了第二个运动蛋白在调节皮质肌动蛋白网络中的作用，使我们能够提出一个 CE 模型，该模型由具有生物力学反馈控制的简单振荡调节电路组成，以及检验该假设的方法。 公共健康相关性：“脊椎动物轴向延伸的调节”提案旨在揭示嵌入中胚层细胞调节力产生的近端分子机制，从而驱动原肠胚形成过程中发生的胚胎形状变化。这些研究是在青蛙胚胎中进行的，但可能与人类健康相关，因为这种中胚层形态发生的部分失败会导致随后神经管闭合失败，从而导致脊柱裂（后部闭合失败）或无脑畸形（前部闭合），而这种形态发生的完全失败则与正常发育不相容。最终的目标是，了解轴向延伸的调节方式至少将导致在人类不孕症情况下的诊断应用，并且在这些情况下可以考虑治疗干预的合理可能性。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.