The dual role of the extracellular matrix in inter-tissue adhesion and tissue movement

项目摘要 该提案的总体目标是确定细胞外基质(ECM)如何对 活体三维组织的形态发生。细胞外基质在细胞迁移领域有很好的研究； 然而，分析环境与环境管理在其他发展背景下的作用的例子有限。要理解 ECM在开发过程中的作用，一个可以跟踪ECM的三维系统， 需要在发育过程中对其进行量化和体内操作。斑马鱼中细长的神经管是 非常适合在三维组织形态发生过程中检查ECM，由于成像容易， 量化纤维连接蛋白基质重塑的能力，以及提供的大量突变和工具 斑马鱼操纵纤维连接蛋白基质和组织力学。在这个系统中，纤维连接蛋白存在。 在两个组织之间，分裂前的中胚层(PSM)和神经管。纤维连接蛋白就像一种粘合剂，可以将 两个组织在一起，是神经管沿着内侧轴正确会聚所必需的。然而， 神经管也相对于PSM和纤维连接蛋白基质向后移动。当主要整合素 将细胞与纤维连接蛋白基质结合的受体被移除，细胞运动没有显著变化 在神经管中观察到。此外，当纤维连接蛋白被去除时，神经管完全拉长。 总之，这些数据表明，细胞沿纤维连接蛋白基质的迁移不是后部所必需的 神经管的运动。总体而言，这表明神经管通过一个 除了细胞沿纤维连接蛋白基质迁移，同时还保持与 纤维连接蛋白基质。可能发生这种情况的一个模型是通过神经中整合素动力学的变化 管，其中整合素活性和稳定性降低可允许组织水平运动，同时静止 保持附着力。在目标1中，我将在后部研究整合素结合动力学。 神经管的运动。使用包括FRET/FLIM、抗体染色和实时成像在内的技术， 将生成整合素动态的跨比例视图。这些数据将产生新的假设 整合素对神经管运动有贡献，将使用影响神经管后运动的突变来测试 神经管和加强或削弱整合素结合活性的突变。在目标2中，我将决定 神经管细胞与细胞外基质的黏附如何影响它们的细胞运动。这将使用以下工具完成 实时跟踪纤维连接蛋白基质重塑和细胞运动与其与纤维连接蛋白位置的关系 矩阵。该数据将产生关于细胞-细胞和细胞-ECM接触如何影响细胞行为的假设， 将使用影响神经管会聚的已建立的突变和通过改变细胞来进一步测试 粘附性和细胞伸缩性。

Project Summary The overall goal of the proposal is to determine how the extracellular matrix (ECM) contributes to the morphogenesis of a three-dimensional tissue in vivo. The ECM is well studied in the field of cell migration; however, limited examples exist analyzing the role of the ECM in other developmental contexts. To understand the role of the ECM during development, a three-dimensional system in which the ECM can be tracked, quantified, and manipulated in vivo during development is needed. The elongating neural tube in zebrafish is well-suited to examine the ECM during three-dimensional tissue morphogenesis, due to the ease of imaging, the ability to quantify Fibronectin matrix remodeling, and the numerous mutants and tools that are available in zebrafish to manipulate the Fibronectin matrix and tissue mechanics. In this system, Fibronectin is present between two tissues, the presomitic mesoderm (PSM) and neural tube. Fibronectin acts as a glue to hold the two tissues together and is required for proper neural tube convergence along the medio-lateral axis. However, the neural tube also moves posteriorly in relation to the PSM and Fibronectin matrix. When the main integrin receptor that binds cells to the Fibronectin matrix is removed, no significant changes in cell motion are observed in the neural tube. Furthermore, when Fibronectin is removed, the neural tube elongates fully. Together these data indicate that cell migration along the Fibronectin matrix is not required for posterior movement of the neural tube. Overall, this indicates that the neural tube moves past the PSM through a mechanism other than cell migration along the Fibronectin matrix while also maintaining adhesion to the Fibronectin matrix. One model for how this might occur is through changes in integrin dynamics in the neural tube, in which decreases in integrin activation and stability could allow for tissue level motion while still maintaining adhesion. In aim 1, I will investigate integrin binding dynamics in the context of posterior motion of the neural tube. Using techniques, including FRET/FLIM, antibody staining, and live imaging, a cross-scale view of integrin dynamics will be generated. These data will generate new hypotheses for how integrins contribute to neural tube motion which will be tested using mutations that affect posterior motion of the neural tube and mutations that strengthen or weaken integrin binding activity. In aim 2, I will determine how adhesion of neural tube cells to the ECM influences their cell motion. This will be completed using live tracking of Fibronectin matrix remodeling and cell motion in relation to their position to the Fibronectin matrix. This data will generate hypotheses for how cell-cell and cell-ECM contacts influence cell behavior, which will be further tested using established mutations that affect neural tube convergence and by altering cell adhesion and cell contractility.