ErbB signaling is required for larval melanocyte regeneration.

(A) Cartoon of drug treatment timeline for assaying melanocyte ontogeny and regeneration. Arrow indicates when embryos were collected for photos and melanocyte counts, in this case at 168 hpf. (B) Quantitation of average dorsal melanocytes from somites 1–26 for each treatment in (A) for melanocyte ontogeny (gray) and melanocyte regeneration (red). Error bars represent standard deviation, * represents P<0.05 (Student t-test, N = 10). Photos of representative larvae at 168 hpf for ontogeny (C, E, G) and regeneration (D, F, H). WT larvae regenerate nearly completely (compare D to C). WT treated with AG1478 (E, F) and erbb3b mutants (G, H) largely fail to regenerate but have normal ontogenetic number of melanocytes. A few regeneration melanocytes are observed in the head and sporadically in parts of the trunk (black bars in F and H) but are mostly absent throughout the trunk (red bars in F and H).

Temporal shifts of AG1478 reveal stem cell establishment occurs in a rostrocaudal progression.

(A) Cartoon of drug treatment timeline. (B) Quantitation of average regenerated dorsal melanocytes for each treatment in (A) in the trunk (somites 5–12, in gray) and in the tail (somites 16–21, in black). Error bars represent standard deviation, * P<0.05, - P>0.05 (Student t-test, N = 10). Larvae not treated with AG1478 show full regeneration in the trunk and in the tail. When larvae are treated with the full AG1478 treatment, from 9–48 hpf, they fail to regenerate in either the trunk or the tail. Larvae treated early with AG1478 from 9–30 hpf fail to regenerate in the trunk, but have normal regeneration in the tail. Later treatments of AG1478 from 30–48 hpf show more regeneration in the trunk than in the tail. (C) Larva with early treatment of AG1478 from 14–24 hpf showing a regeneration defect in the trunk but with normal regeneration in the head and tail.

Overexpression of kitla after 4 dpf results in proliferation of melanocytes.

(A) Cartoon of the pT2hsp70:kitla expression construct used in heatshock experiments. The heatshock promoter, hsp70 drives kitla expression, allowing for expression of kitla after heatshocking the injected embryos at 37°C. (B) Cartoon of experimental protocol. Following injection of pT2hsp70:kitla, larvae were heatshocked for 1 hour at 4 and 5 dpf in the presence of BrdU (from 4 to 8 dpf). (C) Quantitation of dorsal melanocytes represented as a percentage of wild type, and the percentage of BrdU labeled melanocytes is represented in red for each treatment. Larvae injected with pT2hsp70:kitla and heatshocked develop approximately 30% more melanocytes than uninjected larva. These larvae also show an increased percentage of BrdU labeled melanocytes (∼27%) that is comparable to the number of excess melanocytes. Representative examples of melanocytes in WT (D, F, H) and pT2hsp70:kitla (E, G, I), showing melanocyte nuclei stained with DAPI for WT (F) and pT2hsp70:kitla (G), and BrdU staining for WT (H) and pT2hsp70:kitla (I). Melanocytes considered having BrdU+ nuclei are labeled with arrows and BrdU- nuclei are labeled with arrowheads.

The regeneration specific drug, ICI-118,551, reveals that excess melanocytes in PT2hsp70:kitla larvae arise from the MSC lineage.

(A) Cartoon of drug treatment timeline. (B) Quantitative data for ontogeny and regeneration represented as percentage of WT melanocyte numbers. Error bars show standard deviations, * P<0.05, - P>0.05 (Student′s t-test, N>7). (C) WT larvae treated with ICI-118,551 develop faintly melanized ontogenetic melanocytes, in contrast to (D) failure to develop melanized melanocytes when challenged to regenerate in the presence of ICI-118,551. (E) In situ analysis reveals regeneration proceeds to the dct+ melanoblast stage (arrows) in the presence of ICI-118,551. (F) ICI-118,551 treated PT2hsp70:kitla embryos develop similar numbers of faintly melanized melanocytes as ICI-118,551 treated embryos shown in (C). Differences in iridophore appearance between (C) and (F) are results of slightly different illumination conditions.

kitla melanocyte proliferation is enhanced by AG1478.

(A) Cartoon of the PT2hsp70:kitla construct in transgenic animals. The constitutive cmv promoter drives expression of kitla. (B) Schematic of drug treatment timeline with early treatment of AG1478. (C) Quantitation of average ontogenetic dorsal melanocytes for each treatment in (A) in the trunk (somites 5–12) and in the tail (somites 16–21). Error bars represent standard deviation, * P<0.05, - P>0.05 (Student t-test, N = 10). As reported previously (see Figure 1) WT larvae show no effect in ontogenetic melanocyte number with AG1478 treatment compared to untreated WT. PT2hsp70:kitla animals have significantly more melanocytes in both the trunk and the tail. Treating PT2hsp70:kitla animals with AG1478 early, from 9–30 hpf, produces a significantly greater number of melanocytes in the trunk. In contrast, the tail region of AG1478-treated PT2hsp70:kitla larvae shows a significant, but much smaller increase than the trunk, in the number of excess melanocytes. (D) Trunk dorsal stripes of WT larvae are typically two melanocytes wide. (E) PT2hsp70:kitla, however, have trunk dorsal stripes that are about 4 cells wide. (F) AG1478-treated PT2hsp70:kitla have trunk dorsal stripes ∼6 melanocytes wide.