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dvdy24026-sup-0001-suppfig1.tif1289KSupp. Fig. S1. osr2 expression in gut-adjacent mesenchyme. A: Double whole-mount in situ hybridization of a 48-hpf embryo showing gut morphology (agr2; blue-black) and osr2 expression (light red). B: Double in situ hybridization with osr2 probe (blue-black) and fgf24 (light red) showing osr2 expression adjacent to the gut at the approximate position of the future pneumatic duct (arrowhead). Gut morphology from A superimposed in white dashed line in B.
dvdy24026-sup-0002-suppfig2.tif1069KSupp. Fig. S2. Protein sequence alignment of five-finger odd-skipped zinc finger domains. Alignment of five zinc finger domains from Drosophila melanogaster (Dm) bowl (brother of odd with entrails limited; NP_001245861), sob (sister of odd and bowl; AAC47282), Danio rerio (Dr) Osr2A (assembled from ests EH508938, EH535068 and confirmed by sequencing), Homo sapien (Hs) Osr2A (NP_001135934) shows strong conservation of DNA interacting residues in all five zinc fingers (C2H2 domains shaded in grey).
dvdy24026-sup-0003-suppfig3.tif5332KSupp. Fig. S3. Epithelial development and polarity is not affected in osr2 morphants. A: Histological section of a control embryo at the level of the mid-trunk shows cross-sections of the kidney (arrowheads) and gut (arrow) stained with anti-ZO-1, highlighting polarized apical cell–cell junctions. Both osr2 ATG morphants (B) and osr2 exon3 splice morphants (C) also show normal epithelial polarized junctions in the kidney and gut. Blue, DAPI stained nuclei. Representative sections from n=3 for each morpholino.
dvdy24026-sup-0004-suppfig4.tif11837KSupp. Fig. S4. Pronephric kidney patterning and structure is normal in osr2 morphants. A–N: In situ hybridization of wild-type (A, C, E, G, I) and osr2 morphant embryos (B, D, F, H, J) using pronephric nephron markers. pax2a (A–F) is expressed in the entire pronephric nephron, ae2 (G and H) and nbc1 (I and J) are expressed in the proximal nephron, trpm7 (K, L) are expressed in the early distal segment and ret1 (M, N) is expressed in the distal segment of the nephron. Embryonic stages are indicated on the corresponding panels. O, P: a6F immunohistochemistry on wild-type (O) and osr2 morphant (P) showing normal expression of Na+/K+-ATPase in the pronephros.
dvdy24026-sup-0005-suppfig5.tif118KSupp. Fig. S5. pax2a expression is unchanged in osr2 morphants. In situ hybridization of 24-hpf embryos using pax2a probe shows that wild-type uninjected controls (A) show roughly equivalent staining compared to control morphants (B). A slight increase in pax2a signal in translation-blocking (C) and splice-blocking (D) osr2 morphants may be due to greater probe penetration since pax2a signal was also more intense in the neural tube, which is not an osr2-expressing tissue. Arrowheads indicate anterior tubules where osr2 is normally expressed.

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