Johnson et al., 2011 - mitfa is required at multiple stages of melanocyte differentiation but not to establish the melanocyte stem cell. Developmental Biology   350(2):405-413 Full text @ Dev. Biol.

Fig. 1 Temperature sensitivity of two mitfa hypomorphic alleles: larval phenotypes. A) Wild-type, B) mitfaw2, C) mitfafh53 raised at 24 °C, D) mitfafh53 raised at 32 °C, E) mitfavc7 raised at 24 °C, and F) mitfavc7 raised at 32 °C.

PHENOTYPE:
Fish:
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Observed In:
Stage: Day 5

Fig. 2 Temperature sensitivity of the mitfa fh53 allele: adult phenotypes. A) Wild-type, B) mitfaw2, C) mitfafh53 raised at 29 °C, and D) mitfafh53 raised at 29 °C to adulthood and then shifted to 25 °C for one month, showing recovery of melanocytes.

PHENOTYPE:
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Condition:
Observed In:
Stage: Adult

Fig. 4 The vc7 allele affects spicing of mitfa. A) Electropherogram of sequence obtained from mitfavc7 allele. B) Location of vc7 mutation in genomic sequence. C) Semi-quantitative RT/PCR shows that missplicing is not temperature-sensitive. vc7 results in transcripts that (a) include intron 6 and (c) skip exon 6, in addition to correctly-spliced transcript (b). M, 200 basepair ladder; m, 100 basepair ladder. D) Reading frame is preserved in aberrantly-spliced transcripts a and c.

Fig. 5 mitfa is required at multiple steps in melanocyte development. A) mitfa expression persists at restrictive temperature. Wild-type and mitfafh53 larvae are shown. B) No dct is expressed in neural crest cells when held at restrictive temperature, but retinal expression of dct is not affected. Wild-type, mitfaw2, mitfafh53, and mitfavc7 larvae are shown. Embryos were treated with 0.2 mM PTU to suppress melanin synthesis. All larvae are at the equivalent of 82 h at standard temperature.

Fig. 6 Embryos raised at permissive temperature to dct+ stage, when shifted show no melanized cells. A) Wild-type embryo showing dct expression in retinal pigment epithelium (RPE) and neural crest melanoblasts, B) wild-type embryos 20 h after upshift (stage equivalent to 45 h at standard temperature) showing differentiated melanocytes, C) mitfafh53 embryo showing dct expression at time of temperature upshift, and D) mitfafh53 embryos 20 h after upshift do not display any melanized cells save for RPE. In A and C, embryos were treated with 0.2 mM PTU to suppress melanin synthesis.

Fig. 7 Loss of dendricity in melanocytes at restrictive temperature. Wild-type and mitfafh53 embryos were raised at permissive temperature for 72 h, photographed (A,C) then shifted to 32 °C for 48 h (B,D).

PHENOTYPE:
Fish:
Conditions:
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Stage Range: Long-pec to Day 5

Fig. 8 mitfa is not required in the stem cell, but is required for survival of direct-developing melanocyte progenitors. A) Graph showing dorsal melanocyte number in mitfafh53 homozygous larvae held at 32 °C for the indicated times and then shifted to permissive temperature, and melanocytes counted three days later. B) Homozygous mitfa fh53 larvae held at 32 °C in the presence of DMSO (top) or AG1478 (bottom) then downshifted to permissive temperature.

Fig. 9 in situ hybridization for mitfa in wild-type (A,B) and mitfafh53 (C,D) larvae held at restrictive temperature with or without AG1478 treatment from 9 to 48 hour development. AG1478 treatment reduces the number of mitfa-expressing cells in both wild-type and mutant embryos. Larvae are at the equivalent of the 65 hour stage at standard temperature. Embryos were treated with 0.2 mM PTU to suppress melanin synthesis.

Acknowledgments:
ZFIN wishes to thank the journal Developmental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Biology, 350(2), Johnson, S.L., Nguyen, A.N., and Lister, J.A., mitfa is required at multiple stages of melanocyte differentiation but not to establish the melanocyte stem cell, 405-413, Copyright (2011) with permission from Elsevier. Full text @ Dev. Biol.