Regulation of Axial Extension in Vertebrate Embryos

脊椎动物胚胎轴向延伸的调节

• 批准号: 8699787
• 负责人: Paul Michael Skoglund
• 金额: $ 29.84万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8699787
• 关键词: Actins; Acute; Adhesions; Anencephaly; Anterior; Automobile Driving; Biological; Biological Assay; Biomechanics; Blastopores; Cadherins; Cell Shape; Cell-Cell Adhesion; Cells; Chemicals; Complex; Congenital Abnormality; Crosslinker; Data; Defect; Dependence; Development; Diagnostic; Dorsal; Embryo; Equilibrium; Event; Failure; Feedback; Filament; Germ Layers; Goals; Health; Homeostasis; Human; Infertility; Intercalated Cell; Lead; Lip structure; MYLK gene; Mediating; Mesoderm Cell; Modeling; Molecular; Morphogenesis; Motor; Myosin ATPase; Myosin Regulatory Light Chains; Myosin Type II; Neural Tube Closure; Neurons; Nonmuscle Myosin Type IIB; Pathway interactions; Pattern; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Probability; Process; Production; Proteins; Rana; Regulation; Research Personnel; Role; Series; Shapes; Signal Transduction; Spinal Dysraphism; Stress; Stretching; System; Takeda brand of pioglitazone hydrochloride; Testing; Therapeutic Intervention; Tissues; Up-Regulation; Western Blotting; Work; base; crosslink; gastrulation; inhibitor/antagonist; intercalation; metaplastic cell transformation; myosin VI; myosin phosphatase; non-muscle myosin; novel; relating to nervous system; research study

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.

描述(申请人提供)：“脊椎动物轴向伸展的调节”的提案旨在扩展这位研究人员先前的观察，即细胞的汇聚和伸展需要皮质肌球蛋白网络，而细胞汇聚和伸展驱动青蛙胚胎在原肠形成时的形状变化。由于这种膜下肌动蛋白网络需要肌球蛋白IIB(MIIB)复合体在这种形态发生中发挥作用，我们的目标是确定这种对肌球蛋白II的依赖是否需要肌动蛋白激活的收缩能力，或者如果MII在轴向伸展过程中仅作为肌动蛋白交联物发挥作用。消耗肌球蛋白调节轻链(MRLC)的初步证据表明，肌球蛋白II的收缩能力是必需的。肌球蛋白调节轻链是MII表达收缩所必需的，但不是交联性活动。由于MII收缩能力的调节是由MRLC上Ser19的磷酸化状态控制的，我们通过在胚胎中添加肌球蛋白磷酸酶抑制剂并通过定量Western blotting检测Ser19的磷酸化来显示Ser19-P的急性上调，从而扰乱了该Ser19-P事件的动态稳态。本实验揭示了肌球蛋白磷酸酶和一个或多个激酶调节细胞内Ser19-P水平的一个活跃的、平衡的过程，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，我们重新研究了插层细胞中的Ca++动态，发现了一种新的Ca++通量模式，以及一种可能的机制，通过我们证明是原肠形成所必需的假定的Ca++通道蛋白来产生这些通量。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的模型，该模型由一个简单的具有生物力学反馈控制的振荡调节电路组成，以及检验这一假设的方法。