Department of Genetics, Washington University, St. Louis, MO, USA.
Dev Biol. 2011 Feb 15;350(2):405-13. doi: 10.1016/j.ydbio.2010.12.004. Epub 2010 Dec 10.
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.
mitfa 基因编码斑马鱼与小眼相关转录因子（Mitf）的同源物，与其他物种的同源物一样，Mitf 对于神经嵴黑色素细胞的发育是绝对必需的。为了评估 mitfa 在黑色素细胞发育的不同阶段中的作用，我们已经鉴定了 mitfa 的功能减弱等位基因，包括两个对黑色素细胞发育表现为温度敏感的等位基因。分子分析表明，mitf(fh53)ts 是由于一个碱基对的变化导致 DNA 结合域中的天冬酰胺突变为酪氨酸氨基酸，而 mitfa(vc7)ts 等位基因是剪接受体位点的突变，降低了正确剪接转录本的水平。mitfa(vc7)等位基因的剪接本身似乎并不依赖于温度。第三个功能减弱等位基因 mitfa(z25)导致蛋白质的第一个螺旋结构域中的异亮氨酸突变为苯丙氨酸。mitfa(fh53)ts 的温度上升实验表明，mitfa 在黑色素细胞分化的几个阶段都需要，包括早期黑色素母细胞标记物 dct 的表达，再次是从 dct 表达到分化的进展，以及分化后树突形态的维持。mitfa(fh53)ts 突变体在幼虫发育的所有阶段下调时，在 2-3 天内即可恢复黑色素细胞。然而，当早期用 erbB3 抑制剂 AG1478 处理以消除黑色素干细胞（MSCs）时，黑色素细胞的恢复在 48 小时内丢失。这一结果首先表明 MSC 是在限制温度下建立的，并且黑色素母细胞在限制温度下保持大约一天后死亡或丧失恢复能力。
