Regulation of Planar Cell Polarity in Migrating Cells

描述（由申请人提供）：平面细胞极性（PCP）描述了组织平面内细胞的极化，是胚胎形态发生的基本特征。脊椎动物PCP的破坏与多种疾病有关，包括神经管闭合缺陷、纤毛病、耳聋和潜在的癌症。所有PCP研究领域都面临两个主要问题。首先，将核心PCP蛋白的信号转化为细胞极性变化的下游效应物是什么？其次，在平面组织中，细胞间如何传递信息以建立和维持极性？广泛表征PCP在苍蝇

DESCRIPTION (provided by applicant): Planar cell polarity (PCP) describes the polarization of cells within the plane of a tissue and is an essential feature of embryonic morphogenesis. Disruption of vertebrate PCP is associated with multiple disorders including neural tube closure defects, ciliopathies, deafness, and potentially cancer. Two major questions face all fields of PCP research. First, what are the downstream effectors that transform signals from core PCP proteins into changes in cell polarity? Second, how is information communicated between cells to establish and maintain polarity in a planar tissue? Extensive characterization of PCP in the fly wing epithelium demonstrates those antagonistic interactions between Van Gogh and Frizzled (Fz)/Disheveled (Dsh) signaling function to polarize the formation of actin-rich hairs. By contrast despite a decade of speculation that asymmetric expression of these core PCP proteins polarizes the formation of membrane protrusions during gastrulation cell movements, the actual molecular mechanisms remain unclear. The goal of this proposal is to provide a new experimental paradigm explaining how PCP signaling coordinates the directed migration of polarized gastrula cell populations. Our central hypothesis is that Vang-like 2 (Vangl2) and Fz/Dsh signaling differentially impact cell-matrix interactions and cell-cell adhesion, respectivel, to control polarized membrane protrusive activity. Specific Aims: First, we will establish the mechanism whereby Vangl2 regulates Mmp14 activity, ECM proteolysis, and PCP. We will use a combination of in vivo molecular and genetic manipulations, cell transplantation methods, and confocal/time-lapse imaging to identify domains of Vangl2 expression and analyze membrane protrusion dynamics. We will use in vitro biochemical assays to identify signaling mechanisms connecting Vangl2 and integrin alpha5beta1 function. In the second aim, we will determine the contribution of cadherin-mediated adhesion to Fz/Dsh-dependent regulation of ECM assembly and PCP. We will use molecular and genetic methods, transplantations, and microscopic imaging to identify Fz, Dsh, and cadherin expression domains, manipulate cell adhesion, and analyze membrane protrusion dynamics. We will use biochemical methods to identify mechanisms whereby Fz/Dsh signaling regulates cell surface N-cadherin expression during gastrulation. In the third aim, we will dissect the abilities of Vangl2 and Fz/Dsh signaling to coordinate PCP across a field of migrating gastrula cells. Here, we will use a powerful combination of mosaic analyses and confocal imaging to quantify non-autonomous effects on membrane protrusion orientation, cell polarity, and PCP protein localization. Using these methods, we will identify both short- and long-range field effects and determine for the first time how changes in ECM structure and cell cohesion impact PCP during the collective movement of gastrula cells. Completion of this grant will significantly advance our understanding of vertebrate PCP. Since PCP occurs in the context of an ECM microenvironment and cell-cell adhesion, our data should have broad implications for other developmental and disease processes.