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K+-leak channels enhance the growth of pectoral fin buds. (A, B) Brightfield images of a post-heat-shocked 48 hpf non-transgenic embryo (A) or post-heat-shocked 48 hpf hsp70:kcnk5b-GFP transgenic embryo (B). The area of the fin bud (black-dotted line). (C) Measurements of pectoral fin bud areas of heat-shocked groups of non-transgenic (AB), Tg[hsp70:GFP], Tg[hsp70:kcnk5b-GFP], and Tg[hsp70:kcnk10-GFP]. The non-transgenic and Tg[hsp70:kcnk5b-GFP] embryos are siblings. Each measured bud was standardized to the eye area (red-dotted circles in A,B) in the same embryo. (D–S) Expression of the indicated morphogens. (D, G, J, M) Lateral views of whole-mount in situs of heat-shock control non-transgenic embryos at 48 hpf for fgf8a (D), fgf10a (G),shha (at 52 hpf) (J), aldh1a2 (M) and dorsal view of aldh1a2 (O) or lateral views of heat-shocked Tg[hsp70:kcnk5b-GFP] embryos at 48 hpf for fgf8a (E), fgf10a (H), shha (at 52 hpf) (K), aldh1a2 (N), and dorsal view of aldh1a2 (P). White arrows indicate proximal expression of aldh1a2. qRT-PCR measurements from fin buds for fgf8a (F), fgf10a (I), shha (L), and aldh1a2 (Q). Lateral views of in situs of heat-shocked non-transgenic fin bud for bmp4 (R) and msx2b (U) and of heat-shocked Tg[hsp70:kcnk5b-GFP] embryos at 48 hpf for bmp4 (S) and msx2b (V). qRT-PCR measurements from fin buds for bmp4 (T), msx2b (W) the in situ experiments were repeated 3 or more time (N ≥ 3). Each in situ repeat contains 6–12 embryos per replicate. For the fin bud size measurements, we measured 1 fin bud and eye per embryo. We measured at least 4 embryos per experimental repeat. For the qRT-PCRs, we collected 80 fin bud samples per isolation. We assessed gene expression with 3 or more isolations. Each isolation was assessed in duplicate or triplicate. Each measured value is represented as a data point. P values represent statistical analysis by Student’s two-tailed t test. P values ≥0.05 are designated as “not significant” (NS). The scale bars are 0.5 mm (A, B), 100 μm (D–P, R–S,U–T). Numerical data used in this figure are included in S2 Data. hpf, hours post fertilization.
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