Temperature sensitivity of two mitfa hypomorphic alleles: larval phenotypes. A) Wild-type, B) mitfa^w2, C) mitfa^fh53 raised at 24 °C, D) mitfa^fh53 raised at 32 °C, E) mitfa^vc7 raised at 24 °C, and F) mitfa^vc7 raised at 32 °C.

Temperature sensitivity of the mitfa fh53 allele: adult phenotypes. A) Wild-type, B) mitfa^w2, C) mitfa^fh53 raised at 29 °C, and D) mitfa^fh53 raised at 29 °C to adulthood and then shifted to 25 °C for one month, showing recovery of melanocytes.

The vc7 allele affects spicing of mitfa. A) Electropherogram of sequence obtained from mitfa^vc7 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.

mitfa is required at multiple steps in melanocyte development. A) mitfa expression persists at restrictive temperature. Wild-type and mitfa^fh53 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, mitfa^w2, mitfa^fh53, and mitfa^vc7 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.

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) mitfa^fh53 embryo showing dct expression at time of temperature upshift, and D) mitfa^fh53 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.

Loss of dendricity in melanocytes at restrictive temperature. Wild-type and mitfa^fh53 embryos were raised at permissive temperature for 72 h, photographed (A,C) then shifted to 32 °C for 48 h (B,D).

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 mitfa^fh53 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.

in situ hybridization for mitfa in wild-type (A,B) and mitfa^fh53 (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.