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.

Nephron proximal tubule patterning and corpuscles of Stannius formation are regulated by the sim1a transcription factor and retinoic acid in zebrafish

“…1A, C) (Wingert, et al, 2007; Wingert and Davidson, 2011; Li, et al, 2014). The renal progenitors undergo a MET by the 20-22 somite stage (ss) that gives rise to two epithelial tubules (Gerlach and Wingert, 2014) which become joined at their proximal ends by connecting to a single glomerulus at the midline and link distally to the cloaca, forming a common exit portal for urine (Fig. 1A) (Drummond, et al, 1998).…”

“…The pronephros is anatomically simple, as it consists of two nephrons (Drummond, et al, 1998). These pronephric nephrons emerge from bilateral fields of renal progenitors that derive from the IM during somitogenesis, and then undergo a mesenchymal to epithelial transition (MET) to form tubular structures (Gerlach and Wingert, 2014). Each pronephric nephron contains the following segments: a blood filter comprised of podocytes (P), neck (N), proximal convoluted and straight tubules (PCT, PST), distal early and late tubules (DE, DL), and a pronephric duct (PD) (Figure 1A) (Wingert, et al, 2007).…”

Microbead Implantation in the Zebrafish Embryo

“…Thus, microbead transplantation can be a viable method to explore other tissues, such as the intermediate mesoderm (IM) which gives rise to the kidney. Specifically, it would aid in renal developmental studies, to investigate how different molecules affect nephron segmentation 42 and tubulogenesis 43,44 , processes that are only superficially understood at present. Furthermore, microbead implantation has begun to be used to study regeneration processes in zebrafish 29] and could be adapted for use with any number of organ regeneration models, such as following laser ablation of embryonic tissues like the nephron 45 or in conjunction with methods that have been formulated to perform research with the corresponding adult structures [46][47][48][49] .…”

Insights into kidney stem cell development and regeneration using zebrafish

“…Analogous to other vertebrates, these precursors in zebrafish are arranged in bilateral fields [34] . These renal progenitors undergo convergence and extension movements during late somitogenesis to form a pronephros composed of two nephrons, which involves a mesenchymal to epithelial transition to form the tubule and duct regions of the nephron [49,50] . The nephrons become fully functional after morphogenesis of the blood filter during the first few days of development [51][52][53][54][55][56][57] .…”