Fig. 2.

Fig. 2. RSC generation process.

(A) Confocal images of Pax2a immunofluorescence in Tg(cdh17:DsRed;eya2:eGFP) zebrafish show that eya2⁺ cells emerge at 46 hpf, and these cells start expressing Pax2a by 84 hpf. cdh17:DsRed labels renal tubular epithelial cells. The asterisk indicates other eya2:eGFP⁺ cell types that have yet to be identified. The yellow dashed lines indicate the intermediate region between the two renal tubules. The line diagram at the top indicates that the images were captured from zebrafish embryos. The red lines represent the renal tubules, while the green box indicates the region and orientation of image acquisition. (B) Combination of eya2-FISH and GFP immunofluorescence in Tg(eya2:eGFP) zebrafish at 48 hpf. (C) Confocal image showing Tg(eya2:eGFP) zebrafish at 50 hpf. Arrowheads indicate RSCs arranged in a cord-like structure. The structure above it is the lateral line. (D) Confocal images of Pax2a immunofluorescence in Tg(cdh17:DsRed;six2a:eGFP) zebrafish at 86 and 90 hpf, demonstrating that six2a is first expressed at 90 hpf. (E) Time-lapse observation of Tg(six2a:eGFP;cdh17:DsRed) zebrafish from 86 to 90.5 hpf. Arrowheads indicate six2a:eGFP⁺ RSCs. (F) Combination of six2a-FISH and GFP immunofluorescence in Tg(eya2:eGFP) zebrafish at 96 hpf. (G) Time-lapse observation of Tg(cdh17:DsRed;six2a:eGFP) zebrafish from 96 to 103.5 hpf. Arrowheads indicate six2a:eGFP⁺ RSC. (H) The cell volume of six2a:eGFP⁺ RSCs in (G) (96 and 103.5 hpf) was measured using ImageJ (n = 12 cells from three fish). (I) Graphical abstract summarizing RSC generation. A subset of eya2⁺ mesenchymal cells begins to express Pax2a at approximately 84 hpf, followed by the expression of six2a, which facilitates the maturation of RSCs. Yellow circles represent cross sections of the pronephric tubules, and green cells denote RSCs. Axial orientation is shown in the top left corner, indicating the dorsal (D)–ventral (V) and left (L)–right (R) axes. Scale bars, 50 μm [(A) to (G)].