A novel epithelial cell migration drives nephron repatterning and convolution

描述(由申请人提供)： 人类的肾单位被细分为不同的部分，每个部分在整个肾脏功能中扮演着不同的角色。这些片段在它们的位置、结构和分子图谱上有所不同。决定肾段边界的确切位置的因素，以及决定不同肾单位段形态的力量，如肾小管卷曲，目前知之甚少。我们发现，在斑马鱼的原肾上皮细胞参与了一种依赖于腔内液体流动的向肾小球的协调迁移。这种移行导致确定肾单位段边界的位置，并导致近端肾的回旋。在这项拟议的研究中，我们将探索这一新现象的机制，并建立新的工具来研究细胞迁移。在AIM1中，我们将在高级图像分析的框架内，通过结合实时荧光成像、荧光抗体分析和共聚焦显微镜来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，在干扰Notch信号的斑马鱼思维炸弹突变体中，前肾迁移被消除，小管无法形成近端卷曲。我们将结合使用吗啉寡核苷酸、化学抑制剂和可诱导显性负性转基因方法来确定这种表型背后的确切分子底物和Notch信号的作用方式。在目标3中，我们将探索原肾迁移的特定分子机制。我们发现PI3K抑制剂LY294002干扰了肾细胞的迁移，可能与PI3K和CDC42有关。我们将通过表达可诱导的显性负结构来干扰PI3K和CDC42的功能，并研究这两个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，我们建议深入研究极化上皮内细胞迁移的新过程，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：这项研究将极大地促进我们对肾单位形态发生的了解，并帮助我们开发未来的再生技术。此外，它还将对目前知之甚少的上皮迁移研究做出开创性的贡献。这一过程也对癌症生物学有潜在的影响，例如癌症的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.