Figures for Johansson et al., 2020

Figure Caption/Comments:

Figure 2

Inhibition of Prl3 Leads to Premature Melanoblast Expansion during Regeneration

(A) mitfa in situ hybridization of DMSO and B4-Rh-treated mitfavc7 zebrafish. Red box: zoomed region. Arrows: mitfa+ melanoblasts. LS, lateral stripe; NT, neural tube. Percentage area covered by mitfa-positive staining per embryo quantified (∗∗∗∗p < 0.0001, t = 4.670, df = 35, unpaired, two-tailed t test). Line and error bars, mean ± SEM (DMSO, n = 23; B4-Rh, n = 14 embryos).

(B) sox10 (DMSO, n = 21; B4-Rh, n = 16) and foxd3 (DMSO, n = 10; B4-Rh, n = 10) RNA in situ hybridization of treated mitfavc7 zebrafish.

(C) Confocal imaging of the MSC lineage during treatments specified. Tg(mitfa:GFP) marks regenerating melanoblasts (green arrows). Tg(nbt:dsRED) marks neural tube and axons (red arrows). n = 3 zebrafish per condition. NT, neural tube; PN, peripheral nerves; LS, lateral stripe. Maximum projection. Stack height: 30 μm. Scale bar: 20 μm.

(D) Quantification of area of GFP expression (pixels2) on individual peripheral nerves of 84 hpf Tg(mitfa:GFP, nbt:dsRED) zebrafish embryos, 12-h post-washout treatment specified. B4-Rhod, DMSO, NFN1, and combination, n = 3 embryos; B4-Rhod control, n = 2 embryos. Line and error bars, mean ± SEM ∗∗p = 0.0039, ∗∗∗∗p = < 0.0001, ANOVA using Tukey’s analysis.

(E) Lateral stripe melanocytes following specified treatments. AG.: AG1478. B4-Rhod: B4-Rhodanine. Red arrows: missing melanocytes; faint melanocytes from opposite side of the body are also visible. Quantification of melanocytes on the lateral stripe of embryos at 96 hpf, p = 0.0364; ∗∗∗p = 0.0003 and 0.0006 for DMSO versus AG. and AG. versus AG. + B4-Rhod, respectively, ANOVA using Tukey’s analysis.

(F) Melanocyte regeneration in mitfavc7 mutant embryos following specified treatments. AG.: AG1478. B4-Rhod or B4-Rh: B4-Rhodanine. Quantification of dorsal stripe melanocytes per embryo 40-h post-washout and mitfa activation, ∗∗p = 0.0013, 0.0023 for DMSO versus B4-Rhod and B4-Rhod versus AG + B4-Rhod, respectively; ∗∗∗∗p < 0.0001, ANOVA using Tukey’s analysis.

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