Defining the molecular and cellular bases of tissue compartmentalization

项目摘要 在生长和重塑过程中保持复杂组织结构的一种保守机制是将 使用间隔边界的细胞组，这些间隔边界是多细胞富含肌动蛋白的结构，形成于 相邻单元格。隔室的边界以高度稳定的细胞-细胞界面的对准“电缆”为代表，以及 它们是近40年前在昆虫胚胎中首次被描述的。从那时起，类似的边界结构 在脊椎动物大脑、肠道、肢芽和体节的不同区域之间被发现。一边学习一边学习 表明边界完整性的丧失会导致出生缺陷，如颅额鼻综合征和 癌症转移，表征这些结构的分子基础的努力一直受阻 由于缺乏专门针对边界细胞的遗传工具。最近有报道称，两个细胞表面 蛋白质--富含亮氨酸的重复蛋白Tartan和Teneurin Ten-m--是启动 果蝇神经外胚层的边界形成。这些上游触发因素的识别最终使 有可能回答有关舱室边界的性质和功能的长期存在的问题。在……里面 在这项建议中，我们将使用各种基因技术来改变Tartan和Ten-m在 神经外胚层，以解决该领域的三个重大知识空白。首先，要确定 导致边界形成的膜张力和粘附性，我们将使用基因工程技术来 打破隔室边界，可视化活胚胎中的细胞骨架和连接标记。我们还将 使用基因交换技术来改变边界的位置，以确定它们的存在如何影响整体 组织架构。第二，确定Tartan和Ten-m如何在分子水平上相互作用以触发边界 形成，我们将进行体内结构-功能分析，以确定Tartan如何控制定位 Ten-m以及哪些Ten-m胞外区是形成边界所必需的。第三，刻画 Ten-m下游的效应器蛋白使细胞-细胞界面在边界上具有独特的物理特性 我们将进行补充生化和高分辨率成像分析。要确定 假定为10-m相互作用伙伴，我们将比较免疫沉淀/质谱分析 为房室边界细胞而浓缩或耗尽的胚胎。要直接将 隔室边界的纳米结构，我们将使用膨胀显微镜来物理放大 并分析细胞骨架和连接蛋白在细胞中的分布 形态学。这项工作的成功完成将极大地提高我们对车厢如何 边界形成并发挥作用。我们的发现也将作为一个范例来理解这两个 广泛表达和对发育具有重要意义的家族--富含亮氨酸的重复蛋白和Teneurin--可能 在其他发展环境中相互作用。

Project Summary A conserved mechanism for keeping complex tissues organized during growth and remodeling is to separate groups of cells using compartment boundaries, which are multicellular actin-rich structures formed between adjacent cells. Compartment boundaries are typified by aligned “cables” of highly stable cell-cell interfaces, and they were first described in insect embryos nearly 40 years ago. Since then, similar boundary structures have been identified in vertebrates between different regions of the brain, gut, limb buds, and somites. While studies indicate that the loss of boundary integrity contributes to birth defects such as cranio-fronto-nasal syndrome and cancer metastasis, efforts to characterize the molecular underpinnings of these structures have been stymied by a lack of genetic tools for specifically targeting boundary cells. It was recently reported that two cell-surface proteins––the leucine-rich repeat protein Tartan and the teneurin Ten-m––are the direct spatial cues that initiate boundary formation in the Drosophila neuroectoderm. The identification of these upstream triggers finally makes it possible to answer long-standing questions concerning the nature and function of compartment boundaries. In this proposal, we will use a variety of genetic techniques to alter the expression patterns of Tartan and Ten-m in the neuroectoderm to address three significant knowledge gaps in the field. First, to identify the changes in membrane tension and adhesion that lead to boundary formation, we will use genetic engineering techniques to disrupt compartment boundaries and visualize cytoskeletal and junctional markers in live embryos. We will also use gene-swapping techniques to alter the location of boundaries to determine how their presence affects overall tissue architecture. Second, to determine how Tartan and Ten-m interact at a molecular level to trigger boundary formation, we will perform in vivo structure-function analyses to determine how Tartan controls the localization of Ten-m and which Ten-m extracellular domains are necessary for boundary formation. Third, to characterize the effector proteins downstream of Ten-m that give cell-cell interfaces at boundaries their unique physical properties, we will perform complementary biochemical and high-resolution imaging analyses. To identify putative Ten-m interaction partners, we will compare immunoprecipitation/mass-spectrometry analyses between embryos that have been enriched or depleted for compartment boundary cells. To directly visualize the nanoscale structure of compartment boundaries, we will use expansion microscopy to physically enlarge Drosophila embryos and analyze the distribution of cytoskeletal and junctional proteins that mediate cell morphology. Successful completion of this work will greatly enhance our knowledge of how compartment boundaries are formed and function. Our findings will also serve as a paradigm for understanding how these two widely expressed and developmentally important families––leucine-rich repeat proteins and teneurins––might interact in other developmental contexts.