title: Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta.
abstract: The zebrafish pronephros provides an excellent in vivo system to study the mechanisms of vertebrate nephron development. When and how renal progenitors in the zebrafish embryo undergo tubulogenesis to form nephrons is poorly understood, but is known to involve a mesenchymal to epithelial transition (MET) and the acquisition of polarity. Here, we determined the precise timing of these events in pronephros tubulogenesis. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed gene 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 pronephros tubule epithelium polarity. Further, the loss of prkcι or the combined knockdown of prkcι/ζ disrupted proximal tubule morphogenesis and podocyte migration due to cardiac defects that prevented normal fluid flow to the kidney. 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. Knockdown of genes essential for cardiac contractility and vascular flow to the kidney, such as tnnt2a, or elimination of pronephros fluid output through knockdown of the intraflagellar transport gene ift88, was not associated with ectopic pronephros gene expression, thus suggesting a unique role for prkcι/ζ in maintaining tubule epithelial identity separate from the consequence of disruptions to renal fluid flow. Interestingly, knockdown of pax2a, but not wt1a, was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model in which the redundant activities of prkcι and prkcζ are essential to establish tubule epithelial polarity and also serve to maintain proper epithelial cell type identity in the tubule by inhibiting pax2a expression. These studies provide a valuable foundation for further analysis of MET during nephrogenesis, and have implications for understanding the pathways that affect nephron epithelial cells during kidney disease and regeneration.
authors.affiliation: Department of Biological Sciences and Center for Zebrafish Research, University of Notre Dame, 100 Galvin Life Sciences, Notre Dame, IN 46556, USA.
authors.forename: Gary F
authors.initials: GF
authors.lastname: Gerlach
authors.affiliation: Department of Biological Sciences and Center for Zebrafish Research, University of Notre Dame, 100 Galvin Life Sciences, Notre Dame, IN 46556, USA. Electronic address: rwingert@nd.edu.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Wingert
mesh_terms: D000818:Animals; D016764:Cell Polarity / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D003911:Dextrans / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000378:metabolism*; D018613:Microscopy, Confocal; D038081:Organogenesis / Q000502:physiology*; D051762:PAX2 Transcription Factor / Q000378:metabolism; D060910:Pronephros / Q000196:embryology*; D011493:Protein Kinase C / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology*; D029961:Zebrafish Proteins / Q000378:metabolism; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta
chemical_list: D017931:DNA Primers; D003911:Dextrans; D007527:Isoenzymes; D051762:PAX2 Transcription Factor; D011493:Protein Kinase C; D029961:Zebrafish Proteins; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta; C497320:pax2a protein, zebrafish
keywords: Atypical protein kinase C; Mesenchymal to epithelial transition; Nephrogenesis; Pax2a; Polarity; Pronephros; Tubulogenesis; Vertebrate; kidney