Regulation of Axial Extension in Vertebrate Embryos

脊椎动物胚胎轴向延伸的调节

• 批准号: 8335601
• 负责人: Paul Michael Skoglund
• 金额: $ 29.01万
• 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 2016-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335601
• 关键词: Actins; Acute; Adhesions; Anencephaly; Anterior; Automobile Driving; Biological; Biological Assay; Biomechanics; Blastopores; Cadherins; 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; 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. 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.

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者先前所做的观察，即驱动青蛙胚胎原肠胚形状变化的细胞收敛和延伸需要皮质肌动蛋白网络。由于这种亚膜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是否在轴向伸展期间仅作为肌动蛋白交联剂起作用。在MII中，肌球蛋白调节轻链（MRLC）是表达收缩性而非交联性所必需的，初步证据表明肌球蛋白II的收缩性是必需的。由于MRLC上Ser19的磷酸化状态控制着MII收缩性的调节，我们通过向胚胎中添加肌球蛋白磷酸酶抑制剂来扰乱Ser19- p事件可能的动态稳态，并通过定量western blotting检测Ser19的磷酸化，发现Ser19- p的急性上调。本实验揭示了肌球蛋白磷酸酶和一个或多个激酶参与调节插层细胞中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的上游调节剂，我们重新检查了插层细胞中的Ca++动力学，发现了Ca++通量的新模式，以及通过假定的Ca++通道蛋白产生这些通量的合理机制，我们证明了原肠胚形成所需的Ca++通道蛋白。我们还确定了第二种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由具有生物力学反馈控制的简单振荡调节电路组成的CE模型，以及检验这一假设的方法。