Figure 1.
Adult Pigment Patterns in jaguar/obelix Mutant Zebrafish
Pigment patterns of whole body (top), trunk (middle), and anal fin (bottom) in wild-type (WT), heterozygous (obetd15/+), and homozygous (obetd15/obetd15) fish. Pigment patterns in all alleles of jaguar/obelix (jagb230, obetc271d, and obetd15) mutants are almost identical (unpublished data).
Figure 2.
Positional Cloning and Sequence Alignment of the jaguar/obelix Gene
(A) Line diagram depicting positional cloning of the jaguar/obelix. The Kir7.1 locus is depicted by the small black box in the upper line; enlargement of the region bounded by the markers M32 and M21 is indicated by dashed lines. Open boxes with vertical lines represent the positions and intron-exon structures of putative genes predicted from the ENSEMBL transcript. Microsatellite markers and original markers are shown above the lines, and recombination rates are indicated below the lines. M48, which is located in the intron of Kir7.1, was perfectly linked to the mutant phenotype (blue vertical bar), and when we used the amino acid substitutions of Kir7.1 as the marker, they were also linked perfectly (red vertical bars). BAC clones (126F9, 98K22, and 246M18) covering this region are represented by gray lines.
(B) Sequence alignment of Kir7.1 from zebrafish, human, and mouse. Positions of mutations identified in jaguar/obelix alleles are indicated by black circles. M1 and M2, transmembrane region; P, P-region (also shown in Figure 4A).
Figure 3.
Rescue of the jaguar/obelix Phenotype by BAC Injection and Expression of Kir7.1 mRNA
(A) Line diagram depicting the genomic locations of the putative Kir7.1 gene and BAC clones used for the rescue experiments. We used two BAC clones (98K22′ and 126F9, represented by gray lines) for microinjection. Numbers below the thick black line indicate the positions of the BACs and Kir7.1 on LG15. Open boxes with vertical lines represent the positions and intron-exon structures of putative genes predicted from the ENSEMBL transcript.
(B) BAC rescue of pigment pattern in zebrafish mutants. Fertilized eggs from homozygous mutant fish (jagb230 and obetd15) were used in the phenotype rescue experiment. For the injected fish that survived to adulthood, representative pigment patterns of whole body (top), trunk (middle), and anal fin (bottom) are shown. The left panels depict patterns resulting from phenotype rescue using BAC clone 98K22′. The middle panels depict patterns resulting from phenotype rescue using BAC clone 126F9. The right panels depict patterns of noninjected fish. All mutant fish (jagb230 and obetd15) rescued by BAC injection had a partial stripe patterns.
(C) PCR analysis to confirm BAC integration in the rescued fin. Agarose gel analysis of PCR products derived from a BAC-specific sequence in nonrescued fins (lanes 2 and 3) and rescued fins (lanes 4 and 5). DNA fragments derived from the BAC clones were detected in all rescued fish (n = 6), but no PCR fragment was obtained from DNA of nonrescued fish (n = 6). Molecular mass markers are indicated (m).
(D) Expression of Kir7.1 mRNA as detected by single-cell RT-PCR. Agarose gel analysis of RT-PCR products derived from individual melanophore (M), xanthophore (X), or fin dermal cells (F). Molecular mass markers are indicated (m).
Figure 4.
Functional Analysis of Kir7.1 Mutations
(A) Positions of amino acid substitutions in the three alleles of jaguar/obelix with respect to wild-type. obetc271d (L130F) and jagb230 (T128M) have amino acid substitutions in the highly conserved P-domain (orange box), and obetd15 (F168L) has mutations in the transmembrane region (green box).
(B) Positions of the mutated amino acids in the 3D structure of zebrafish Kir7.1. The three-dimensional structure of Kir7.1 (residues 40 to 178) was deduced from the published X-ray crystal structure of KirBac1.1. Mutated residues (T128, L130, and F168) are highlighted as Corey-Pouling-Keltun (CPK) space-filling structures.
(C–F) The electrophysiological properties of mutant zebrafish Kir7.1 expressed exogenously on HEK293 cell membranes. The voltage-clamp protocol and typical elicited currents on HEK293 cell membranes exogenously expressing zebrafish wild-type (WT) Kir7.1 are shown in (C). Elicited currents on HEK293 cell membranes for the mutant obetc271d are shown in (D). Current inhibition in the presence of 1 mM Ba2+ is shown in (C) and (D), lower panel, indicating that the detected current is from potassium ions. Horizontal scale bar, 200 ms; vertical scale bar, 500 pA; arrowheads, zero-current levels.
(E) Current (I)–voltage (V) relationship; wild-type (WT) (closed circles) and obetc271d (open circles) (±SEM, n = 7, respectively).
(F) Current (I)–voltage (V) relationship; obetc271d (circles, ±SEM, n = 7), obetd15 (squares, ±SEM, n = 7), and jagb230 (triangles, ±SEM, n = 6). All mutant Kir7.1 show no functional current.
Figure 5.
Melanophore Aggregation-Dispersion Response to the Change of Background Color
Melanosome aggregation-dispersion in response to a rapid succession of background color change was measured. The upper panels show the responses of melanophores in the trunk of wild-type (WT) fish, and the lower panels show that of mutant fish (obetc271d). The left panels depict the usual state (black background), the middle panels depict melanophores after being sustained for 3 min in a white (light) background, and the right panels depict melanophores after 3 min in a black (dark) background. The wild-type melanophores responded normally to the change of background color (n = 6), whereas mutant fish (jagb230 and obetc271d) melanophores strongly responded to the change of background color from black to white but did not respond to the change from white to black (n = 9 for all).
Figure 6.
Melanophore Response to an α2-Adrenoceptor Agonist or Antagonist
(A) Melanophore response to the agonist epinephrine. Fish were placed in black backgrounds for the duration of the experiment to block the endogenous aggregation signal from sympathetic nerves. The upper panels show the responses of melanophores in the trunk of wild-type fish (WT), and the lower panels show that of mutant fish (obetc271d). Melanophores of wild-type (n = 6) and mutant fish (jagb230 and obetc271d) (n = 12, for all) showed melanosome aggregation when epinephrine was added in the breading water, although the extent of aggregation in the mutant melanophores appeared to be more pronounced than in the wild-type.
(B) Melanophore response to the antagonist yohimbine. All fish were kept in an environment with a white background for several minutes (approximately 10 min) prior to addition of antagonist (left panels) and for the duration of the experiment to block endogenous dispersion. The upper panels show the responses of melanophores in the trunk of wild-type (WT) fish, and the lower panels show that of mutant fish (obetc271d). Melanosomes of wild-type fish dispersed after adding the antagonist, because the antagonist blocks the endogenous aggregation signal (middle and right panels) (n = 7). However, in mutant fish (jag b230 and obe tc271d), many of the melanophores remained aggregated for a long time (longer than 60 min) (n = 12 for all) (unpublished data).