Morphogenesis: Biophysics and Genetics of Dorsal Closure

描述（由申请人提供）：果蝇的背侧闭合是发育和伤口愈合过程中细胞片形态发生的模型系统。我们计划利用生物物理学（激光显微手术）、遗传学、药理学和建模方法来研究闭合过程中驱动形态发生的分子、细胞和紧急特性。以前，我们发现非肌肉肌球蛋白II驱动羊膜和细胞上的收缩性，肌动球蛋白丰富的钱包线，这两个组织驱动大部分的进展走向关闭。此外，我们还表明，组织力是由粘附介导的压缩协调的，前后压缩速率常数之间存在不对称性，压缩速率常数可以在激光扰动的响应中上调。我们还表明，去除一种或另一种力会导致剩余力的上调，并以野生型速率完成闭合。此外，羊膜力和压缩速率常数的上调共同解决了闭合的稳健性和弹性。最后，我们表明，驱动闭合的力的矢量总和比贡献的单个力小两到三个数量级。这表明需要对这些大的力进行调节，以便细胞片不可阻挡地闭合。在这里，我们专注于应用激光手术、药理学和定量建模工具来探索驱动野生型和突变型动物细胞片形态发生的细胞和分子机器的结果。通过将这些方法应用于野生型胚胎和选择的在某一方面或另一方面关闭失败的突变胚胎的分析，我们计划如下。1)我们将研究机械门控通道和/或细胞-基质和细胞-细胞连接感知和响应调节关闭速率的力的假设。2)我们将测量每个组织有助于闭合的力的绝对大小。3)我们将研究微管在调节肌动蛋白关闭功能中的作用。4)我们将建立数学模型，以组织和/或细胞分辨率概括突变、药理学干扰或激光研究胚胎的关闭行为。这些关于果蝇细胞片形态发生的研究将为脊椎动物形态发生和伤口愈合过程中协调细胞形状变化的生物学过程提供细胞和分子基础。这项工作的重点是背部关闭，果蝇黑腹果蝇的一个过程，模拟脊椎动物细胞片的运动。果蝇为多学科研究提供了独特的机会，而且许多参与运动的蛋白质在果蝇和人类之间是高度保守的（有些是bb0 - 90%相同的，许多人类蛋白质可以在实验中拯救果蝇同源物的遗传缺陷）。此外，类似的薄片运动是人类发育早期阶段（例如神经管形成）和伤口愈合的特征。此外，细胞-细胞连接和细胞-基质连接之间的程序性相互作用发生在闭合过程中，对于表皮-间充质转化至关重要，当上皮细胞转移并导致癌症时，表皮-间充质转化被激活。这些对果蝇的研究将提供对运动的分子和细胞基础的洞察，并为形态发生和伤口愈合特征的紧急行为提供一个窗口。

DESCRIPTION (provided by applicant): Drosophila's Dorsal Closure is a model system for cell sheet morphogenesis during development and wound healing. We plan to investigate the molecular, cellular, and emergent properties that drive morphogenesis during closure using biophysical (laser microsurgery), genetic, pharmacological and modeling approaches. Previously, we showed that nonmuscle myosin II drives contractility in the amnioserosa and in the supracellular, actomyosin-rich purse-strings and that both tissues drive the bulk of progress toward closure. In addition we showed that the tissue forces are coordinated by adhesion- mediated zipping, that there is an asymmetry between the anterior and posterior zipping rate constants, and that the zipping rate constant can be upregulated in response to laser perturbation. We also showed that the removal of one or another force leads to upregulation of the forces that remain and closure proceeds to completion at wild type rates. Furthermore, the upregulation of the amnioserosa force and the zipping rate constant together address the robustness and resiliency of closure. Finally, we showed that the vector sum of the forces that drive closure is two to three orders of magnitude smaller than the individual forces that contribute. This indicates that regulation of these large forces is required so that cell sheets move inexorably to closure. Here we focus on applying laser-surgical, pharmacological and quantitative-modeling tools to explore the emergent properties that are the consequence of the cellular and molecular machines that drive cell sheet morphogenesis in wild type and mutant animals. By applying these methods to the analysis of wild type embryos an selected mutant embryos that fail in on or another aspect of closure, we plan the following. 1) We will investigate the hypotheses that mechanically gated channels and/or cell-matrix and cell-cell junctions sense and respond to forces to regulate the rate of closure. 2) We will measure the absolute magnitude of the forces each tissue contributes to closure. 3)We will investigate the role of microtubules in regulating actin function for closure. 4) We will formulate mathematical models that recapitulate at tissue and/or cellular resolution the behavior of closure in mutant, pharmacologically perturbed or laser investigated embryos. These studies on cell sheet morphogenesis in Drosophila will provide insight into the cellular and molecular basis for the biological processes that coordinate cell shape changes in vertebrate morphogenesis and wound healing. PUBLIC HEALTH RELEVANCE This work focuses on dorsal closure, a process in the fruit fly Drosophila melanogaster that models cell sheet movements in vertebrates. Drosophila offers unique opportunities for multidisciplinary approaches and many of the proteins involved in movement are highly conserved between flies and humans (some are > 90% identical and many human proteins can experimentally rescue genetic defects in their fly orthologs). In addition, comparable sheet movements characterize early stages of human development (for example, neural tube formation) and wound healing. Moreover, the programmed interplay between cell-cell junctions and cell-matrix junctions that occurs during closure is crucial for the epidermal-mesenchymal transition that is activated when epithelial cells become metastatic and cause cancer. These studies in Drosophila will provide insight into the molecular and cellular basis of motility and provide a window onto the emergent behaviors that characterize morphogenesis and wound healing.