The roy orbison mutant phenotype, one of the two strains found in the transparent casper strain. During embryogenesis, roy lacks nearly all reflective iridophores, but melanophore development is relatively normal. In contrast, as adults, roy has a severe defect in both iridophores and melanophores. casper is largely similar to roy except that it also has a mutation in mitfa (nacre), leading to complete loss of all pigmented melanocytes.

(A) Injection of full-length mpv17 mRNA leads to rescue of the roy orbison mutant phenotype, as demonstrated by the appearance of iridophores both in the eye and throughout the body of the fish (roy n = 0/20 fish with eye iridophores, roy+mRNA injection, n = 14/20 fish with eye iridophores). (B) ATG morpholino knockdown of mpv17 in the AB fish leads to a strong phenocopy of the roy phenotype, with loss of eye and body iridophores (AB = 71/79 fish with eye iridophores, AB+morpholino injection, n = 8/67 fish with eye iridophores). (C) Quantification of eye iridophores after morpholino injection to mpv17 shows a significant increase in # of fish with absent iridophores (*, p<0.05, paired t-test).

A) Schematic of the zebrafish mpv17 locus, with coding sequence (CDS) and total mRNA-coding region including introns, red triangles depict sgRNA locations ccB and ccC in coding exons. (B,B′) Brightfield imaging of 5 dpf zebrafish embryos to visualize iridophores, anterior to the left; scale bars represent 500 µm. Wildtype (wt) control showing prominent iridophores surrounding the eye lens and as individual cells along the body (solid arrowheads). (C,C′) Representative Cas9 RNP-injected, somatic-mutant embryo (crispant) where the ORF of mpv17 was targeted with sgRNA ccB in coding exon 1; note diminished iridophores in eye (red arrowhead) and severe reduction of body iridophores. (D) Phenotype distribution in mpv17^ccB crispants versus wt controls. (E,E′) Mutant mpv17 alleles isolated in the F1 generation injected with sgRNA ccB (left) and ccC (right) and recovered in complementation analysis; genomic reference sequence depicted on top, black vertical bar indicates Cas9 cleavage site, blue box depicts PAM. (F, F’) Wildtype (wt) forms iridophores in eye and along the body (red arrowhead) and properly inflates the swimbladder (red asterisks). (G-H) Homozygous mpv17^Δ5 and trans-heterozygous mpv17^Δ1/Δ5 embryos show severe reduction of iridophores. (I,J) The same phenotype occurs in embryos trans-heterozygous for mpv17^Δ5 and roy^a9 (I, I′) or mpv17^Δ5 and tra^b6 (J,J′) as confirmed by >genotyping for mpv17^Δ5 (n = 4). (K,K′) Trans-heterozygous embryos for roy^a9 and tra^b6 also show severe iridophore reduction, establishing non-complementation between roy^a9 and tra^b6 as mutant alleles of mpv17. (L) 3 month old wildtype with iridophores in the eye and along the body and melanocytes form normally into longitudinal stripes. (M) Trans-heterozygous mpv17^Δ1/Δ5 fish completely lack eye iridophores and most iridophores along the body axis. Melanocytes in the stripes are heavily reduced. Scale bars represent 5 mm. White asterisks in whole embryo views mark failed inflation of the swimbladder in all observed mpv17 mutant combinations.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.