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BACKGROUND: Amphibians possess three kinds of dermal chromatophore: melanophores, iridophores, and xanthophores. Knockout Xenopus tropicalis that lack the pigmentation of melanophores and iridophores have been reported. The identification of the causal genes for xanthophore pigmentation or differentiation could lead to the creation of a see-through frog without three chromatophores. The genes causing xanthophore differentiation mutants are slc2a11b and slc2a15b in Japanese medaka (Oryzias latipes).
RESULTS: To obtain a heritable line of X tropicalis mutants without yellow pigment, we generated slc2a7 and slc2a15a knockout animals because they have the greatest similarity to the O latipes slc2a11b and slc2a15b genes. The slc2a7 knockout frog had a bluish skin and there were no visible yellow pigments in stereo microscope and skin section observations. Furthermore, no pterinosomes, which are characteristic of xanthophores, were observed via transmission electron microscopy in the skin of knockout animals.
CONCLUSIONS: We report the successful generation of a heritable no-yellow-pigment X tropicalis mutant after knock out of the slc2a7 gene. This finding will enable the creation of a see-through frog with no chromatophores.
FIGURE 1. Target sites of the Slc2a7 and Slc2a15a. Comparisons of the A, Xenopus tropicalis Slc2a7 (NP_001120033.1) and Oryzias latipes Slc2a11b (NP_001267781.1) and B, X tropicalis Slc2a15a (XP_017950705.2) and O latipes Slc2a15b (NP_001267781.1) amino acid sequences; conserved amino acids are indicated by yellow boxes. Acidic, basic, and hydrophobic amino acids are represented by red, blue, and green characters, respectively. Transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 (CRISPR/Cas9) target sites are indicated by red and blue horizontal lines, respectively. The vertical black arrows denote the boundaries of exons of X tropicalis sequences. The red arrow in (A) indicates the two-base pair deletion site of O latipes wl mutant. The green arrow in (A) indicates the 207th isoleucine, which would be important for the function (see Discussion section). The gray horizontal line in (B) shows the deletion region of O latipes lf mutant.
FIGURE 2. Phenotype of slc2a7 knockdown Xenopus tropicalis. Skin of slc2a7 knockdown F0 frogs had yellowish and bluish regions. A, The appearance of a slc2a7 knockdown F0 frog generated by the transcription activator-like effector nuclease (TALEN) method. Some areas of skin were bluish (blue arrowhead) compared to the wild-type yellowish color (yellow arrowhead). Scale bars = 5mm. B, The surface view of dorsal skin under a stereo microscope. Scale bars = 1mm. Yellow and blue arrowheads were indicating same positions in A. C, D, High-magnification photographs of (C) yellowish region and (D) bluish region. Scale bars = 0.2mm. Lighting condition and exposure time were same in C and D. Only the brightness was linearly changed after the photography (A-D) in same amount (B-D).
FIGURE 3, Genotyping individual F1 frogs. The genotypes of 32 F1 siblings obtained from mating transcription activator-like effector nuclease (TALEN)-generated slc2a7-knockdown F0 frogs. The target DNA fragment was amplified using genomic DNA obtained from individual F1 frogs and recloned for sequence determination. The phenotypes and genotypes of F1 frogs are indicated on the left. Each F1 frog has a pair of sequence lines separated by horizontal lines. The number of frogs with a pair of the indicated sequences is shown on the right. The wild-type target DNA sequences are indicated at the top of the panel. A pair of red bars denotes the TALEN-binding sites. The gaps resulting from a deletion (–) and the exchanged nucleotides are indicated by yellow boxes. WT, wild-type target sequence.
FIGURE 4. The slc2a7 knockout Xenopus tropicalis F1 frogs had no yellow pigments. A, The phenotypes of representative yellowish, blackish, and bluish slc2a7 knockout F1 frogs (yellow, black, and blue arrowheads). The genotype of yellowish frog is heterozygous and that of blackish and bluish frogs are compound heterozygous or homozygous. Scale bar = 10 mm. B, Expression level of slc2a7 was decreased in the dorsal skin of slc2a7 knockout frogs. Total RNA was extracted from the dorsal skin of wild-type (W) and slc2a7 knockout (K) adult X tropicalis and subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to determine slc2a7, hps6 and tyr mRNA levels. All slc2a7 knockout frogs have out-of-frame mutations in both alleles (compound heterozygous or homozygous). Data are expressed as mean ± SE (n = 6). Expression levels of slc2a7, hps6 and tyr mRNAs are shown as copy numbers relative to 100 000 copies of eef1α1 mRNA. P values were calculated using the Student's t-test. ND, not detected; RT, reverse transcriptase. C-Q, Comparison of wild-type (C, H, M), heterozygous (D, I, N), compound heterozygous (E, J, O), homozygous (F, K, P) slc2a7 knockout frogs and tyr/hps6-WKO frog (G, L, Q). The genotypes of presented heterozygous, compound heterozygous and homozygous knockout frogs were WT/−2, −3/−2 and −2/−2, respectively. C-G, The appearance of the frogs. Scale bars = 5 mm. H-L, The surface view of dorsal skin under a stereo microscope. Scale bars = 50 μm. M-Q, Comparison of cryostat sections of dorsal skin. e, epidermis; g, secretory granules; m, melanophore. Scale bars = 10 μm. Note the yellow pigments of xanthophores were observed in the dorsal skin of wild-type, heterozygous and tyr/hps6-WKO frogs (yellow arrows in H, I, L, M, N, Q) but not observed in that of the compound heterozygous and homozygous knockout frogs (blue arrows in J, K, O, P). Lighting condition and exposure time were same in C-G, H-K and M-Q. The exposure time of L was one third to H-K. Only the brightness was linearly changed after the photography in same amount (H-L).
FIGURE 5. Transmission electron microscopy shows no pterinosome structure in the slc2a7 knockout frog (F1). Cross-sections of the skin of a wild-type frog, A, C, with typical pterinosome structures (white arrowheads in C) and the skin of a slc2a7 knockout frog without them, B, D. Low- (A, B) and high- (C, D) magnification electron microscopy photographs. The scale bars are 5 m (A, B) and 1 m (C, D). e, epithelium; m, melanophore; x, xanthophore. The unknown elliptic structures with low electron density are indicated by black arrowheads in D. The boxed areas in panels A and B are magnified in panels C and D, respectively.
