Fig. 4

Fig. 4

Some Unpigmented mitfa-Expressing Cells Enter the Cell Cycle upon Melanocyte Ablation

(A) Flank of an adult zebrafish expressing miniCoopR-mitfa:nlsEGFP. Neocuproine treatment was performed for 24 hr, and hours after onset of treatment are indicated. An unpigmented mitfa-expressing cell undergoing mitosis is shown (yellow arrowheads). neo, neocuproine; BF, brightfield. Scale bar, 100 µM.

(B) Flank of an adult zebrafish coexpressing miniCoopR-ubi:mCherry-zCdt1 and mitfa:nlsEGFP. Differentiated melanocytes (white arrowheads) and unpigmented mitfa-expressing cells (yellow arrowheads) were in the G0/G1 phase of the cell cycle. n = 100 melanocytes and n = 142 unpigmented mitfa-expressing cells from a total of three fish; representative images are shown. Fish were treated with epinephrine prior to imaging. mC, miniCoopR; mCh, mCherry; BF, brightfield. Scale bar, 100 µM.

(C) Flank of an adult zebrafish coexpressing miniCoopR-ubi:mAG-zGeminin and mitfa:nlsmCherry before (left) and after (right) neocuproine treatment. Prior to neocuproine treatment differentiated melanocytes (white arrowheads) and unpigmented mitfa-expressing cells (yellow arrowheads) were not in the S/G2/M phase of the cell cycle. n = 300 melanocytes and n = 900 unpigmented mitfa-expressing cells from a total of three fish; representative images are shown. After neocuproine treatment unpigmented mitfa-expressing cells entered the S/G2/M phase of the cell cycle. The nucleus of an unpigmented AG-zGeminin-positive mitfa-expressing cell in the S/G2/M phase after neocuproine treatment is shown (yellow arrowheads). Fish were treated with epinephrine prior to imaging. mC, miniCoopR; mCh, mCherry; BF, brightfield; Gem, Geminin; neo, neocuproine. Scale bar, 100 µM.

(D) Quantification of AG-zGeminin-positive unpigmented mitfa-expressing cells. n = 150 cells from each of three fish for neocuproine-treated group, n = 300 cells from each of three fish for w/o neocuproine control group. Data are shown as mean percent positive per fish ± SEM; p values calculated by Student’s t test, p < 0.05. neo, neocuproine.

(E) Timeline of the experiment. Adult wild-type zebrafish were treated with neocuproine for 24 hr to ablate differentiated melanocytes and were injected with EdU every other day (arrowheads). Control animals were injected with EdU but were not treated with neocuproine. Fish were sacrificed at day 10 and were scored for EdU incorporation. neo, neocuproine.

(F) Representative images of control (top row) or neocuproine-treated (bottom row) unpigmented mitfa-expressing cells after melanocyte regeneration. neo, neocuproine; BF, brightfield. BF scale bar, 25 µM; Mitfa, EdU scale bar, 10 µM.

(G) Quantification of percent EdU-positive, unpigmented Mitfa-positive cells. n = 103, 83 and 38 cells from each of three fish, respectively, for neo; n = 20, 30, and 15 cells from each of three fish, respectively, for w/o neo. Data are shown as mean percent positive per fish ± SEM; p value calculated by Student’s t test. neo, neocuproine.

See also Figure S4.