Development of the Zebrafish Pronephros: Cellular and Genetic Analysis of Nephron Tubulogenesis and Segment Patterning

Kidney disease affects 300 million people worldwide, representing the 12th leading cause of death. Understanding how nephrons, the simplest functional unit of the kidney, develop will lend insight into the processes that go awry during disease. The zebrafish pronephros provides an excellent in vivo system for studying the mechanisms of vertebrate nephrogenesis. When and how renal progenitors undergo tubulogenesis to form nephrons is poorly understood but is theorized to involve a mesenchymal-to-epithelial transition (MET). We determined the precise timing of these events in pronephros tubulogenesis, including the temporal and spatial expression of tight junction genes. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed knockdown studies to assess the roles of the related factors atypical protein kinase C iota and zeta (prkcι, prkcζ). We found that prkcι and prkcζserve partially redundant functions to establish polarity in the nephron tubule. Surprisingly, tubule cells in prkcι/ζ morphants displayed ectopic expression of the transcription factor pax2a and the podocyte-associated genes wt1a, wt1b, and podxl, suggesting that prkcι/ζ are needed to maintain renal epithelial identity. We identified cell polarity as serving a specific role in maintaining epithelial integrity and not a pleiotropic effect caused by loss of cardiac contractility and/or fluid flow. pax2a knockdown was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model whereby the redundant activities of prkcι and prkcζ are essential to maintain proper epithelial cell type identity through inhibition of pax2a expression. Pronephros segment patterning was initially intact in prkcι/ζ morphant embryos suggesting that the processes of epithelialization and nephron patterning occur independent of one another. To better understand the conserved patterning of the nephron a forward genetic screen was performed. Mutants with disrupted pronephros patterning were identified by whole mount in situ hybridization (WISH) analysis. One mutant exhibited an aberrant distal early tubule segment. Dysfunction of the analogous human nephron segment, termed the thick ascending limb (TAL), is associated with Gitelman and Bartter?s syndrome, kidney stone accumulation, and hypertension. These studies provide a foundation for further analysis of MET during nephrogenesis and have implications for understanding nephron epithelial cells during kidney disease and regeneration.