Pigment Pattern in <em>jaguar/obelix</em> Zebrafish Is Caused by a Kir7.1 Mutation: Implications for the Regulation of Melanosome Movement

Many animals have a variety of pigment patterns, even within a species, and these patterns may be one of the driving forces of speciation. Recent molecular genetic studies on zebrafish have revealed that interaction among pigment cells plays a key role in pattern formation, but the mechanism of pattern formation is unclear. The zebrafish jaguar/obelix mutant has broader stripes than wild-type fish. In this mutant, the development of pigment cells is normal but their distribution is altered, making these fish ideal for studying the process of pigment pattern formation. Here, we utilized a positional cloning method to determine that the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for pigment cell distribution among jaguar/obelix mutant fish. Furthermore, in jaguar/obelix mutant alleles, we identified amino acid changes in the conserved region of Kir7.1, each of which affected K+ channel activity as demonstrated by patch-clamp experiments. Injection of a bacterial artificial chromosome containing the wild-type Kir7.1 genomic sequence rescued the jaguar/obelix phenotype. From these results, we conclude that mutations in Kir7.1 are responsible for jaguar/obelix. We also determined that the ion channel function defect of melanophores expressing mutant Kir7.1 altered the cellular response to external signals. We discovered that mutant melanophores cannot respond correctly to the melanosome dispersion signal derived from the sympathetic neuron and that melanosome aggregation is constitutively activated. In zebrafish and medaka, it is well known that melanosome aggregation and subsequent melanophore death increase when fish are kept under constant light conditions. These observations indicate that melanophores of jaguar/obelix mutant fish have a defect in the signaling pathway downstream of the α2-adrenoceptor. Taken together, our results suggest that the cellular defect of the Kir7.1 mutation is directly responsible for the pattern change in the jaguar/obelix mutant.即便在同一物种内，各类动物亦可呈现出多样的色素斑纹，而这类斑纹或为物种形成的核心驱动力之一。既往针对斑马鱼开展的分子遗传学研究已证实，色素细胞间的相互作用在斑纹形成过程中发挥关键调控作用，但其具体分子机制仍未明确。斑马鱼jaguar/obelix突变体的条纹较野生型更为宽阔。该突变体的色素细胞发育过程正常，但细胞分布发生异常，使其成为研究色素斑纹形成机制的理想动物模型。本研究通过位置克隆技术，证实内向整流钾通道7.1（inwardly rectifying potassium channel 7.1, Kir7.1）基因的异常是导致jaguar/obelix突变体色素细胞分布紊乱的根本原因。此外，我们在jaguar/obelix突变体的等位基因中，发现Kir7.1保守结构域内存在氨基酸突变，且通过膜片钳实验证实，每一处突变均会影响钾通道的离子转运活性。将携带野生型Kir7.1基因组序列的细菌人工染色体注入胚胎后，可有效挽救jaguar/obelix突变体的异常表型。综上，本研究认定Kir7.1基因的突变是导致jaguar/obelix突变体表型异常的直接原因。同时，我们发现表达突变型Kir7.1的黑色素细胞存在离子通道功能缺陷，这一缺陷会改变其对外界信号的细胞应答反应。实验结果显示，突变型黑色素细胞无法对交感神经元释放的黑素体分散信号做出正确应答，且黑素体聚集过程呈现组成型激活状态。在斑马鱼和青鳉中，已有研究证实，当鱼类长期处于恒定光照环境下时，黑素体聚集现象及随后的黑色素细胞死亡概率均会显著升高。上述结果表明，jaguar/obelix突变体的黑色素细胞在α2肾上腺素能受体下游的信号通路中存在功能缺陷。综合以上所有实验结果，本研究提示，Kir7.1基因突变引发的细胞功能缺陷，直接导致了jaguar/obelix突变体的色素斑纹异常。