A novel epithelial cell migration drives nephron repatterning and convolution

描述（由申请人提供）： 人类肾单位被细分为节段，每个节段在整个肾功能中起着不同的作用。这些片段在它们的位置、结构和分子概况上不同。关于决定节段边界的确切定位的因素以及决定各种肾单位节段的形态的力，如小管卷曲，知之甚少。我们发现，在斑马鱼原肾上皮细胞从事一致的迁移到肾小球，这取决于管腔流体的流动。这种迁移导致确定肾单位节段边界的位置和近端肾的回旋。在拟议的研究中，我们将探索这种新现象的机制，并为细胞迁移的研究建立新的工具。在Aim 1中，我们将通过结合荧光抗体检测和共聚焦显微镜的实时荧光成像，所有这些都在高级图像分析的框架内，研究原肾迁移的亚细胞机制。在目标2中，我们将扩展我们的发现，即在干扰Notch信号传导的斑马鱼mindbomb突变体中，前肾迁移被消除，小管未能形成近端卷积。我们将使用吗啉代寡核苷酸，化学抑制剂和诱导显性负转基因方法的组合，以确定确切的分子底物和Notch信号的作用模式，这种表型。在目标3中，我们将探讨前肾迁移的特定分子机制。我们发现，Pi3K抑制剂LY294002干扰肾细胞迁移涉及Pi3K和可能的Cdc42。我们将通过表达诱导型显性负性结构破坏PI3K和Cdc42的功能，并研究这两种分子在前肾迁移中的作用。最后，我们将使用一个有针对性的小分子化学库来筛选已知的抑制剂对原肾迁移的影响。总之，我们提出对极化上皮内细胞迁移的新过程进行深入研究，该过程将器官功能与器官形态发生联系起来。 公共卫生相关性：这项研究将大大提高我们对肾单位形态发生的认识，并帮助我们开发未来的再生技术。此外，它将作出开创性的贡献，上皮细胞迁移的研究，目前知之甚少。这一过程在癌症生物学中也有潜在的意义，例如癌症侵袭和原位病变的扩散。

DESCRIPTION (provided by applicant): The human nephron is subdivided into segments, each playing a distinct role in overall kidney function. The segments differ in their position, architecture, and molecular profile. Little is known about factors determining the exact positioning of segment boundaries as well as the forces determining the morphology of various nephron segments, such as tubule convolution. We discovered that in zebrafish pronephros epithelial cells are engaged in a concerted migration towards the glomerulus that depends on the flow of luminal fluid. This migration results in establishing the position of nephron segment boundaries and in convolution of the proximal kidney. In the proposed study, we will explore the mechanism of this novel phenomenon and establish new tools for the study of cell migration. In Aim1, we will investigate the subcellular mechanisms of pronephric migration by combining live fluorescent imaging with fluorescent antibody assays and confocal microscopy, all within the framework of advanced image analysis. In Aim 2, we will expand on our finding that in zebrafish mindbomb mutant which interferes with Notch signaling, the pronephric migration is eliminated and the tubule fails to form the proximal convolution. We will use a combination of morpholino oligonucleotide, chemical inhibitor and inducible dominant negative transgenic approaches to define the exact molecular substrate and the mode of action of Notch signaling underlying this phenotype. In Aim 3, we will probe specific molecular mechanisms underlying the pronephric migration. We found that Pi3K inhibitor LY294002 interferes with kidney cell migration implicating Pi3K and possibly Cdc42. We will disrupt the function of PI3K and Cdc42 by expressing inducible dominant negative constructs and examine the role of these two molecules in pronephric migration. Lastly, we will use a targeted small molecule chemical library to screen for effects of known inhibitors on pronephric migration. In summary, we propose an in-depth investigation of a novel process of cell migration within polarized epithelium, which links organ function with organ morphogenesis. PUBLIC HEALTH RELEVANCE: This study will significantly advance our knowledge of nephron morphogenesis and help us develop future regenerative technologies. In addition, it will make a seminal contribution to the study of epithelial migration of which very little is currently known. This process also has potential implications in cancer biology, such as cancer invasion and spread of in-situ lesions.