Kidney function depends on the nephron, which comprises a blood filter, a tubule that is subdivided into functionally distinct segments, and a collecting duct. How these regions arise during development is poorly understood. The zebrafish pronephros consists of two linear nephrons that develop from the intermediate mesoderm along the length of the trunk. Here we show that, contrary to current dogma, these nephrons possess multiple proximal and distal tubule domains that resemble the organization of the mammalian nephron. We examined whether pronephric segmentation is mediated by retinoic acid (RA) and the caudal (cdx) transcription factors, which are known regulators of segmental identity during development. Inhibition of RA signaling resulted in a loss of the proximal segments and an expansion of the distal segments, while exogenous RA treatment induced proximal segment fates at the expense of distal fates. Loss of cdx function caused abrogation of distal segments, a posterior shift in the position of the pronephros, and alterations in the expression boundaries of raldh2 and cyp26a1, which encode enzymes that synthesize and degrade RA, respectively. These results suggest that the cdx genes act to localize the activity of RA along the axis, thereby determining where the pronephros forms. Consistent with this, the pronephric-positioning defect and the loss of distal tubule fate were rescued in embryos doubly-deficient for cdx and RA. These findings reveal a novel link between the RA and cdx pathways and provide a model for how pronephric nephrons are segmented and positioned along the embryonic axis.

Podocytes help form the glomerular blood filtration barrier in the kidney and their injury or loss leads to renal disease. The Wilms’ tumor suppressor-1 (Wt1) and the FoxC1/2 transcription factors, as well as Notch signaling, have been implicated as important regulators of podocyte fate. It is not known whether these factors work in parallel or sequentially on different gene targets, or as higher-order transcriptional complexes on common genes. Here, we use the zebrafish to demonstrate that embryos treated with morpholinos against wt1a, foxc1a, or the Notch transcriptional mediator rbpj develop fewer podocytes, as determined by wt1b, hey1 and nephrin expression, while embryos deficient in any two of these factors completely lack podocytes. From GST-pull-downs and co-immunoprecipitation experiments we show that Wt1a, Foxc1a, and Rbpj can physically interact with each other, whereas only Rbpj binds to the Notch intracellular domain (NICD). In transactivation assays, combinations of Wt1, FoxC1/2, and NICD synergistically induce the Hey1 promoter, and have additive or repressive effects on the Podocalyxin promoter, depending on dosage. Taken together, these data suggest that Wt1, FoxC1/2, and Notch signaling converge on common target genes where they physically interact to regulate a podocyte-specific gene program. These findings further our understanding of the transcriptional circuitry responsible for podocyte formation and differentiation during kidney development.

The nephron is the functional unit of the kidney and comprises a blood filter lined by podocytes, proximal and distal tubules, and a collecting duct. How these segments arise during development is not understood. The zebrafish pronephros is ideal for studying segmentation, as it is anatomically simple, consisting of two nephrons. We have isolated 15 solute transporter genes that display segment‐restricted expression patterns. Our data indicates that contrary to current belief, the pronephros is organized into 8 segments and contains mostly tubule rather than duct epithelium. We next examined embryos deficient in retinoic acid (RA), a known regulator of nephrogenesis. Treating embryos with DEAB, an RA antagonist, inhibited the formation of podocytes and proximal tubule segments and expanded distal fates. From a time‐course analysis we found that RA is required at multiple stages to pattern the nephron. To explore how RA induces podocytes, we examined the expression of foxc1a, a transcription factor implicated in podocyte biology. Expression of foxc1a was lost in DEAB‐treated embryos, and knockdown of foxc1a in wild‐types led to a downregulation of various podocyte genes including the transcription factors wt1 and mafb, and components of the slit‐diaphragm filtration barrier. Our findings suggest that nephron segmentation is regulated by RA and that a RA‐foxc1a pathway is important for inducing podocytes.