Characterization of podocyte development in zebrafish embryos. A) Schematic of pronephric development in zebrafish. B) Micrographs of in situ hybridizations show the expression patterns of early (wt1a, wt1b, mafba, hey1, lhx1a, pax2a) and mature (nephrin, podocin, podocalyxin, integrinα3) podocyte markers from 15 somites through 48 h of development. C) Analysis of podocyte development in relation to the surrounding tissues. Micrographs of in situ hybridizations compare podocyte (wt1b⁺, pax2a⁺ at 24 hpf), neck (pax2a⁺, wt1b^-), first proximal tubule segment (slc20a1a⁺) and interrenal (nr5a1a⁺) development at 24 hpf and 36 hpf. Cdh17⁺ cells represent all non-podocyte renal epithelia which includes both neck and tubule fates.

Podocytes arise from intermediate mesoderm adjacent to somite 3. A) Schematic of a flat-mounted 8-somite embryo showing the region of intermediate mesoderm (blue line) next to the somites (ovals) that was analyzed for expression of podocyte transcription factors or associated pathway components. Red box represents the regions shown in B) and C). B) In situ hybridization showing the expression patterns of pax2a, wt1a, foxc1a and the notch ligands jag1b and jag2a. Overlapping expression of all factors is seen in the region next to somite 3, marked by the asterisk. C) Micrographs showing overlapping expression of wt1a, wt1b and foxc1a with pax2a at the level of somite 3. Arrow points to overlapping expression of wt1b and pax2a in the anteriormost pax2a region (presumptive podocyte progenitors). D) DIC images of tissue adjacent to somite 3 analyzed by laser ablation. The top image highlights the region of intermediate mesoderm in blue prior to ablation. The image on the bottom shows the region after ablation, where the cells have lysed. E) In situ hybridization of ablated embryos at 10 somites (pax2a), 36 hpf (wt1b and cdh17) and 48 hpf (nephrin). Ablations were performed at the level of somites 1–2 or somite 3. Arrows point to remaining areas of pax2a and wt1b expression on the ablated side of 10 somite and 36 hpf embryos, respectively. The asterisk in the bottom-right panel marks the lack of nephrin expression in the ablated half of the embryo.

Analysis of podocyte, neck and interrenal development by in situ hybridization following morpholino knockdown of wt1a, foxc1a and/or rbpj. Each transcription factor was knocked down either by itself or in combination with each of the other factors as indicated. The expression analysis was carried out at 15 somites, 24 hpf, 36 hpf and 48 hpf. wt1b⁺ = podocytes; cdh17⁺ = non-podocyte renal epithelia; slc20a1a⁺ = first proximal tubule segment; nephrin⁺ = mature podocyte; podocalyxin⁺ = mature podocyte; nr5a1a⁺ = interrenal cells; pax2a⁺ = podocytes and neck segment (15 somite), neck segment (48 hpf). Analysis of hey1 expression by in situ hybridization was performed at 15 somites under single and double knockdown conditions. Arrows point to podocyte expression of hey1.