Fig 5

Kcnk5b enhanced depolarization is required for enhanced fin bud growth.

(A, B) DiSBAC₂(3) fluorescence measurements of the ectoderm (A) and mesenchyme cells (B) of developing pectoral fin buds at 32, 42, 48, and 56 hpf. (C–F) Confocal images of developing fin buds displaying the intensities fluorescence as photon counts per pixel at 32 hpf (C), 42 hpf (D), 48 hpf (E), and 56 hpf (F). Colors represent the range of counted photons per pixel. Blue representing the lowest level of counted photons, and red representing their highest counts (up to 750 photons or more). Images representing high photon count (C’, D’, E’, F’) and low photon count (C”, D”, E”, F”). The total exposure range was set at 1,500 counts (1.5). (G) Distribution of counted photons from DiSBAC₂(3) in the confocal plane of the representative fin bud at 32 hpf. (H) Distribution of counted photons from DiSBAC₂(3) in the confocal plane of the representative fin bud at 32 hpf. (I) Assessment of DiSBAC₂(3) fluorescence intensity as counted photons for fin buds expressing mCherry or kcnk5b-mCherry. (J) Assessment of DiSBAC₂(3) fluorescence intensity of mCherry-expressing or kcnk5b-mCherry fin buds at 32 hpf after treating for 4 h as indicated: ethanol (Et), DMSO, 10 μm vinpocetine (vin), 40 μm dibucaine (dib). (K) Representative image of pectoral fin bud of non-transgenic 48 hpf AB fish after heat shock at 32 hpf and start of treatment at 36 hpf with the drug solvents Ethanol (Et) and DMSO. (L) Representative image of pectoral fin buds of 48 hpf AB fish heat shocked at 32 hpf and start of treatment with 10 μm Vinpocetine (Vin) and 40 μm Dibucane (Dib) at 36 hpf. (M) Representative image of pectoral fin buds of 48 hpf transgenic Tg[hsp70:kcnk5b-GFP] after heat shock at 32 hpf and start of treatment with EtOH and DMSO at 36 hpf. (N) Representative image of pectoral fin buds of 48 hpf transgenic Tg[hsp70:kcnk5b-GFP] after heat shock at 32 hpf and start of treatment with 10 μm Vin and 40 μm Dib at 36 hpf. (O) Assessment of pectoral fin bud growth at 48 hpf expressing either AB and kcnk5b-GFP in the indicated treatment groups. Experiments were repeated 3 or more time (N ≥ 3). Each repeat contained 6 or more embryos; one fin bud was measured per embryo. For the DiSBAC₂(3) fluorescence measurements, 6 independent points were measured from different 4 locations in each fin bud, distal, anterior, posterior, and proximal, and then averaged to represent a data point (A, B, I, J). For the fin bud size measurements, we measured 1 fin bud and eye per embryo. We measured at least 15 embryos per repeat, and each measurement is 1 data point (O). P values represent statistical analysis by Student’s two-tailed t test. P values >0.05 are designated as “not significant” (NS). Scale bars equal 100 μm. Numerical data used in this figure are included in S5 Data. hpf, hours post fertilization.