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

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Abstract
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. Animals display a variety of skin pigment patterns. How these often intricate patterns are formed, however, is the longstanding question. Zebrafish is the only model organism having a pigment pattern, and thus it provides a unique system in which to investigate the mechanism of pattern formation. The striped pigment pattern of zebrafish comprises two types of pigment cells, melanophores (black chromatophores) and xanthophores (yellow chromatophores), and defects in pigment cell differentiation cause abnormal pigment patterns. However, the mechanism(s) underlying the arrangement of pigmented cells during development is unclear. In this paper, the authors cloned and studied the zebrafish mutant gene jaguar/obelix and identified it as inwardly rectifying potassium channel 7.1 (Kir7.1). Although the development of pigment cells is normal in jaguar/obelix fish, they have abnormally wide body stripes; thus, cell positioning is altered, suggesting that the jaguar/obelix functions in the system that determines pigment patterning. The connection between the Kir7.1 channel and the pigment pattern remains unclear, but the mutant melanophores are defective in intracellular aggregation and dispersion of the melanosome (pigment) controlled by the sympathetic neuron, suggesting that the signaling pathway activated by the neuron is also related to pigment pattern formation.