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Fig. S1. Melanocyte counting. All melanocyte counts took place on a defined head region, which extended from halfway between the eyes down to ears (yellow dotted line). Scale bar: 100 microns.
Fig. S2. Pigmented embryonic melanocytes of trunk and tail are positive for the proliferative antigen phospho-Histone H3 (p-H3). p-H3-positive nucleus on dorsal yolk of 36 hpf embryo. p-H3-positive melanocytes were found at low frequencies in 28-36 hpf embryos sampled (2.0±2.7 cells per embryo, n=30). p-H3-expressing cells were found widely distributed throughout the embryo, and are likely to contribute to all of the embryonic melanocyte stripes.
Fig. S3. Tyrp1-GFP expression precedes melanocyte pigmentation. (A) Dorsal view of the head region of a zebrafish embryo expressing GFP from the tyrp1 promoter during melanocyte development. (B) Detail of the differentiating melanocyte (yellow boxes from A). One melanocyte (a) is followed from its differentiation from an unpigmented cell expressing GFP (32.7 hpf), through onset of pigmentation with enhanced dendricity (33.3 hpf), to the development of a mature melanocyte (70 hpf).
Fig. S4. Division of pigmented cells. (A-C) Examples of division of pigmented cells during regeneration (A,B) after nitrofuran treatment and wash out, and (C) after shifting a mitfvc7 temperature-sensitive mutant from 32.0°C to the permissive temperature of ∼23.5°C as described in Fig. 4.
Fig. S5. Schematic representation of quantitative analysis of melanocyte development in individual mitfavc7 temperature-sensitive fish. Grey symbols represent melanocytes that have developed from undifferentiated precursor cells. Grey squares indicate a melanocyte that has developed from an undifferentiated precursor cell and does not divide. Other grey symbols represent melanocytes that have developed from an undifferentiated precursor, but divided; their progeny are shown as a symbol of the same shape but coloured yellow. Thick grey lines indicate length of each individual time-lapse movie for each embryo. Only fish that were imaged for at least 108 hpf were included in analysis (Fig. 5). mitfavc7 embryos were grown for ∼20 hours at 28.5°C to ensure health of the embryo, and then shifted to (A) 23-24°C, (B) 25°C, (C) 25.5°C and (D) 26-26.5°C.
Fig. S6. Rescue of melanocytes in the nacre mutant line. The nacre (mitfa loss-of-function allele) does not develop neural crest melanocytes. Microinjection of single-cell embryos with human MITF DNA (M:11, n=14) rescues the development of some neural crest derived melanocytes by 3 dpf. The number of rescued melanocytes is increased by expression of the human melanoma allele 4TΔ2B (M:85, n=23) compared with the wild-type human MITF (P<0.001; ANOVA; 95% CI 74.05 (50.90, 97.20).
Movie 1. Melanocytes developing in the head region of the zebrafish (approximately day 1-4 of development) shown in Fig. 1A. The best plane of focus has been selected for each frame.
Movie 2. Melanocytes developing in the head region of an albino zebrafish expressing GFP from the tyrp1 promoter shown in Fig. 2B. All planes of focus (z-stacks) are compressed into one image (z-projection) for each frame.
Movie 3. Regenerating melanocytes in the head region of a nitrofuran-treated embryo shown in Fig. S4 in the supplementary material. The bright-field image (left) and GFP fluorescence in sox10-expressing cells (right; Carney et al., 2006). The best plane of focus has been selected for each frame.
Additional reference
Carney, T. J., Dutton, K. A., Greenhill, E., Delfino-Mach, M., Dufourcq, P., Blader, P. and Kelsh, R. N. (2006). A direct role for Sox10 in specification of neural crest-derived sensory neurons. Development 133, 4619-4630.
