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.
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.
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