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Amphibians generally have three types of pigment cells, namely, melanophores (black and brown), xanthophores (yellow and red), and iridophores (iridescent). Single knockout of the tyr, slc2a7, and hps6 genes in Xenopus tropicalis results in the absence of melanophores, xanthophores, and iridophores, respectively. The generation of triple- knockout (3KO) X. tropicalis for these three genes could allow for observation of internal organs without sacrificing the animals, which would be transparent due to the absence of pigments. In this study, we generated 3KO X. tropicalis, which is one of the most widely used model amphibians, through crossing of a slc2a7 single-knockout frog with a tyr and hps6 double-knockout frog, followed by intercrossing of their offspring. The 3KO tadpoles had transparent bodies like the nop mutant and the frogs had translucent bodies. This translucency allowed us to observe the heart, lungs, stomach, liver, and digestive tract through the ventral body skin without surgery. After intravital staining, 3KO X. tropicalis showed much clearer fluorescent signals of mineralized tissues compared with the wild type. These 3KO X. tropicalis provide a useful mutant line for continuous observation of internal organs and fluorescent signals in the body. In particular, such 3KO frogs would revolutionize fluorescence monitoring in transgenic tadpoles and frogs expressing fluorescent proteins.
FIGURE 1. Genealogy of the triple-knockout frog. Triple-heterozygous frogs were generated through crossing of a slc2a7 single-knockout frog with a double-knockout frog for the tyr and hps6 genes. Triple-homozygous knockout frogs were obtained from intercrossing of these triple-heterozygous frogs. Squares and circles represent male and female frogs, respectively. The upper (black), lower left (gray), and lower right (yellow) symbols represent the tyr, hps6, and slc2a7 genes, respectively. Closed, hatched, striped, and open symbols represent the wild type (+/+), chimera, heterozygote (+/), and homozygote of the knockout (/), respectively. Arrows denoting tyr KD, hps6 KD, and slc2a7 KD indicate the timing of genome editing in previous studies (Nakajima et al., 2012; Nakajima et al., 2021; Nakayama et al., 2017).
FIGURE 2. Tadpole phenotypes and survival. (af) Phenotypes of tadpoles obtained through intercrossing of triple-heterozygous knockout frogs at 4days after fertilization. (a, b) Full pigmentation. (c, d) Reduced pigmentation. (e, f) No pigmentation. Scale bars, 1mm (dorsal view: a, c, e) or 0.2mm (left lateral view: b, d, f). (g, h) Survival curve of each phenotype from two independent experiments (g: lot #1, h: lot #2). Fifty tadpoles with full or reduced pigmentation and two sets of 50 tadpoles with no pigmentation were selected using a stereomicroscope at 3days after fertilization. These four groups of tadpoles were maintained under the same conditions and the number of surviving tadpoles was plotted against the number of days since fertilization. Full, reduced, and no pigmentation are represented by closed, gray, and open symbols, respectively.
FIGURE 3. The external appearance of triple-knockout (3KO) frogs. (ad) External appearance of a 3KO tadpole (a, b) and a wild-type tadpole (c, d) at the climax stage. (a, c) Dorsal view. (b, d) Ventral view. Scale bars, 1mm. (e) Comparison of the skin color of tyr, hps6, and slc2a7 knockout frogs, 3KO, and triple-heterozygous frog (3KO hetero). Scale bar, 10mm.
FIGURE 4. Alizarin red S (ARS) staining of triple-knockout (3KO) frogs. (ad) Climax Stage 3KO (a, b) and wild-type (c, d) tadpoles were stained with 0.1% ARS at Nieuwkoop and Faber (NF) stage 64 for 2h. Fluorescence photographs of the dorsal view (a, c) and the ventral view (b, d) were taken on the next day (NF stage 65). Scale bars, 1mm.
FIGURE 5. Observation of internal organs through the ventralskin. Some internal organs could be observed through the ventralskin of a triple-knockout (3KO) juvenile frog (a) and were identified through direct observation (b). Most organs could not be observed through the ventralskin of a wild-type juvenile frog (c) and were instead identified directly (d). dt, digestive tract; he, heart; lu, lung; st, stomach; li, liver. Scale bars, 1mm.
FIGURE 6. Second-generation survival curve of triple-knockout (3KO) tadpoles. Survival curve of two batches of 3KO tadpoles obtained through crossing of 3KO females and males, and one batch obtained through crossing of a ttHhss (tyr /, hps6 +/, and slc2a7 /) female with a 3KO male. The starting numbers of 3KO-1 (closed circle), 3KO-2 (open circle), and ttHhss (closed square) were 47, 50, and 50, respectively, at 3days after fertilization. The ratio of the number of surviving tadpoles to the initial number was plotted against the number of days since fertilization.
