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  • Zebrafish stocks. Stocks of obetc271d and obetd15 were obtained from the Tübingen Stock Centre (Tübingen, Germany). jagb230 was obtained from the Johnson lab (St. Louis, Missouri, United States). Genetic mapping and positional cloning. Mapping of the jaguar/obelix locus was performed as described [44]. Fish carrying either the obetc271d or obetd15 mutant on a Tu genetic background were crossed with wild-type fish containing an AB or India genotypic background. F1 heterozygotes were interbred, and a genetic map of their F2 offspring was created using microsatellite markers (http://zebrafish.mgh.harvard.edu/zebrafish/index.htm). We identified six new markers in ctg11503 (ASSEMBLY Zv2) (Sanger Institute, Cambridge, United Kingdom) (http://www.sanger.ac.uk/Projects/D_rerio) for use in further mapping: M21, M32, M48, M190, M234, and M379. The recombinant numbers for F2 offspring were derived from five different parents (four from obetc271d and one from obetd15) and were summed to calculate the crossover frequency. Because some of the parents did not have polymorphic sequence with respect to particular markers, the number of specimens tested was not identical. For gene prediction, we used ENSEMBLE transcript (Sanger Institute). cDNAs of Kir7.1 and neighboring genes were isolated using RNA extracted from tail fins via RT-PCR. To obtain full-length Kir7.1, we used the following primers: 5′-ATGCCTACCACCATGACA-3′ and 5′-ACTTCTTCTACTCCACGC-3′. Microinjection of the BAC construct. Preparation and microinjection of the BAC DNA was done as described [45]. Approximately 100 kb upstream of Kir7.1 in the dkye-98K22 BAC construct was deleted using a Counter-selection BAC Modification kit (Gene Bridges, http://www.genebridges.com), yielding the modified BAC clone 98K22′. The two BAC constructs, 98K22′ and dkey-126F9, were purified using the Qiagen Large-Construct kit (Qiagen, http://www.qiagen.com). The BAC DNAs (2 ng) were injected into fertilized eggs of homozygote fish (jagb230 and obetd15) at the one- or two-cell stage. The injected eggs were maintained under normal breeding conditions as described [45]. BAC integration into chromosomes was confirmed by PCR amplification of a BAC-specific region. Primer sequences were 5′-TTGCAACAATCTCTCAGACG-3′ and 5′-ATCAATGGTTCAGGCTTGT-3′ for 98K22′ and 5′-TGTGAGTCTGATGCTCGTT-3′ and 5′-GAT TTAGGTGACACTATAG-3′ for 126F9. Single-cell RT-PCR. Pigment cells in the tail fins were dissociated with collagenase type III as previously described [46]. Cells were dispersed in culture dishes, and then single melanophore, xanthophore, or dermal cells were isolated using glass capillaries. After genomic DNA was removed, cDNAs were generated by the SuperScript III CellsDirect cDNA Synthesis System (Invitrogen, http://www.invitrogen.com). The cDNA obtained from a single cell was dissolved in 30 μl of H2O, and a 5-μl aliquot was analyzed using RT-PCR to detect Kir7.1 mRNA. As a control, 1 μl was analyzed to detect β-actin mRNA. PCR was carried out with primer sets that straddle an intron: 5′-CCTGGAGACGCAACTCACTA-3′ and 5′-TCGATGCTGAACTCCAGAGC-3′ for Kir7.1 and 5′-AGGGTTACGCTCTTCCCCATGCCATC-3′ and 5′-GCGCTCAGGGGGAGCAATGATCT-3′ for β-actin. PCR amplifications were performed for 45 cycles for Kir7.1 and 30 cycles for β-actin at 95 °C for 30 s, at 50 °C for 30 s, and 72 °C for 30 s, followed by 72 °C for 2 min. Molecular modeling. The structure of the prokaryote inward rectifier K+ channel, KirBac1.1, was retrieved from the Protein Data Bank (PDB 1P7B) [29]. The pore structure homology model of the zebrafish Kir7.1 channel (residues 40 to 178) was created from the template KirBac1.1 structure using DS modeling software version 1.1 (Accelrys, http://www.accelrys.com). The quality of the models was assessed by their stereochemical properties, and root mean standard deviations were calculated by the software. Transient expression of zebrafish Kir7.1 on HEK293 cell membranes. Plasmid construction and transfection into HEK293 cells were performed as described [47]. The coding regions of wild-type and mutant zebrafish Kir7.1 were subcloned into the expression vector pcDNA3 (Invitrogen). Cotransfection of the Kir7.1 plasmid with pCA-GFP, a plasmid expressing GFP, into the embryonic kidney cell line HEK293 was performed using LipofectAMINE PLUS (Life Technologies, http://www.invitrogen.com). Cells expressing GFP (and also possibly zebrafish Kir7.1) were identified by fluorescence microscopy and used for electrophysiology. Electrophysiological measurements. Electrophysiological measurements were carried out as described [47]. For whole-cell recordings, the pipette (internal) solution contained: 150 mM KCl, 5 mM EGTA, 2 mM MgCl2, 3 mM K2ATP, 0.1 mM Na2GTP, and 5 mM HEPES-KOH (pH 7.3). The (external) bathing solution (normal Tyrode's solution) contained 115 mM NaCl, 20 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES-NaOH (pH 7.4). Currents were recorded using the whole-cell configuration of the patch-clamp technique [48]. The tips of patch electrodes were coated with Sylgard (Dow Corning, http://www.dowcorning.com) and fire-polished. The tip resistance was 4 to 5 MΩ when filled with the pipette solution. All recordings were made at a holding potential of 50 mV. All experiments were performed at room temperature (approximately 25 °C). The channel current was recorded using a patch-clamp amplifier (Axon 200B; Axon Instruments, http://www.axon.com), low-pass-filtered at 1 kHz (−3 dB) by an eight-pole Bessel filter, digitized by an AD converter (Digidata; Axon Instruments), and continuously acquired on a computer (Dell) with commercially available software (pCLAMP9; Axon Instruments). Results are presented as mean values, and error bars represent ±SEM. Measurement of melanophore aggregation and dispersion response against background color. Some of the older mutant fish (older than 5 mo) had a wild-type response to background color, particularly among females, and thus we used young adult fish (approximately 5 mo, around 2.5 cm long) to measure melanosome aggregation and dispersion against background color. Prior to analysis, the young fish were kept in the normal breeding environment (black background color) for at least 2 d to create uniform melanophore conditions for aggregation and dispersion measurements. After being photographed in black background conditions, the fish were transferred to a white cup for 3 min and then anesthetized by 0.02% tricaine (see [45]) and photographed. The fish were then transferred to a white cup without tricaine, and the awakened fish were transferred back to a black cup for 3 min, anesthetized, and photographed again. All photographs were taken quickly (approximately 1 min) to avoid melanosome dispersion caused by the heat from the microscope light or stress. Measurement of melanophore response to α2-adrenoceptor agonist and antagonist. To evaluate the aggregation response of the α2-adrenoceptor to epinephrine, endogenous aggregation via the visual nervous system was blocked by maintaining a black background color. Epinephrine (Sigma) was applied to the breeding tank at a final concentration of 1 mM and the aggregation response was assessed for 3 min. Likewise, to measure the α2-adrenoceptor dispersion response to the antagonist yohimbine (Sigma, http://www.signaaldrich.com), a white background color was maintained to block endogenous dispersion. We observed and scanned the response of the melanophore to yohimbine for a longer period (60 min) as the aggregation state was already induced by endogenous neurotransmitters that bind to α2-adrenoceptor tightly and because antagonist (yohimbine) competes with these neurotransmitters to bind the receptor. Yohimbine was added to the breeding tank to a final concentration of 10 μM.
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