RESUMO
The mitfa gene encodes a zebrafish ortholog of the microphthalmia-associated transcription factor (Mitf) which, like its counterparts in other species, is absolutely required for development of neural crest melanocytes. In order to evaluate mitfa's role in different stages of melanocyte development, we have identified hypomorphic alleles of mitfa, including two alleles that are temperature-sensitive for melanocyte development. Molecular analysis revealed that the mitf(fh53)ts results from a single base pair change producing an asparagine to tyrosine amino acid substitution in the DNA-binding domain, and the mitfa(vc7)ts allele is a mutation in a splice donor site that reduces the level of correctly-spliced transcripts. Splicing in the mitfa(vc7) allele does not itself appear to be temperature-dependent. A third, hypomorphic allele, mitfa(z25) results in an isoleucine to phenylalanine substitution in the first helix domain of the protein. Temperature upshift experiments with mitfa(fh53)ts show that mitfa is required at several stages of melanocyte differentiation, including for expression of the early melanoblast marker dct, again for progression from dct expression to differentiation, and again for maintenance of dendritic form following differentiation. mitfa(fh53)ts mutants recover melanocytes within 2-3days when downshifted at all stages of larval development. However, when melanocyte stem cells (MSCs) are ablated by early treatment with the erbB3 inhibitor AG1478, melanocyte recovery is lost by 48 h. This result indicates first that the MSC is established at the restrictive temperature, and that melanoblasts die or lose the ability to recover after being held at the restrictive temperature for approximately one day.