Identification of fish-specific genes. (a) Schematic diagram of phylogenetic relationships between fish and tetrapods. Numbers in parentheses for each lineage indicate the number of species used in the BlastP analysis. In the skeleton images, blue and green areas show endochondral (endoskeleton) and membrane bone (fin ray), respectively. (b) Schematic diagram representing fish-specific genes. Rectangles indicate genes in the genome (indicated by lines), and dotted rectangles indicate genes that were not present in the genome of the lineage. Gene B, but not genes A, C, and D, represents a fish-specific gene like those we sought in this study. (c) Flowchart showing the process of identifying fish-specific genes. Numbers in parentheses represent the number of genes in zebrafish at each step of the process. (d–f) Examples of fish-specific gene phylogenetic trees. Green indicates fish and blue indicates tetrapods. Bold lines represent clades of fish-specific genes. Genes that did not have gene names in the Ensembl database were assigned gene names as follows: ENSCMIP00000038549, CMI_and1; ENSCMIP00000004194, CMI_and3; ENSCMIP00000004396, CMI_mych; ENSCMIP00000019689, CMI_anos1b. CMI, Callorhinchus milii; DAR, Danio rerio; GAL, Gallus gallus; HOM, Homo sapiens; LAC, Latimeria chalumnae; LOC, Lepisosteus oculatus; MUS, Mus musculus; ORL, Oryzias latipes; TRU, Takifugu rubripes.

RNA-seq analysis of developing pectoral fins. (a) Excised pectoral fins at 48 hpf (left) and 14 dpf (right). Fin mesenchyme in 48 hpf samples was labeled with EGFP (gt1116A), which confirmed that the entire fin bud region was excised. Scale bars: 100 μm. (b) Heatmap showing clusters based on expression fluctuation, representing expression of 12,699 clustered genes out of 25,432 protein-coding genes. The vertical axis indicates developmental stage. (c, d) The numbers of all zebrafish genes (c) and fish-specific genes (d) that were included in each cluster. The color of the bar corresponds to that of each cluster in (b). “no_cluster” indicates genes that were not assigned to any clusters.

Expression levels of 33 candidate genes during pectoral fin development. The line graph shows the expression levels (TPM) at each developmental stage (32 hpf, 40 hpf, 48 hpf, 5 dpf, 14 dpf, 28 dpf, 42 dpf, and the adult stage). Red lines show the TPM value of 10. Error bars indicate standard deviation.

Loss-of-function analysis of candidate genes by the CRISPR/Cas9 system. (a) Dorsal view of 2 dpf (50–54 hpf, upper photos) and 5 dpf (122–126 hpf, lower photos) zebrafish after the candidate gene was knocked out (crispant). The gt1116A strain (trapping the prdm16 gene) was used to visualize the pectoral fin mesenchyme, and the pectoral fins are indicated by arrows in the control (upper left). EGFP fluorescence and bright-field images are merged in all photos. fgf24 F0 zebrafish did not form pectoral fin buds (arrowheads). Scale bars: 250 μm. (b) AR-stained image of the pectoral fin of an and1/and2 double-knockout juvenile. AR fluorescence image and bright-field images are merged. Scale bars: 250 μm

Functional analysis of qkia. (a) Expression pattern of qkia in the developing zebrafish embryo detected by in situ hybridization. Asterisks indicate qkia expression in the zebrafish myotome at 48 hpf (left) and the pectoral fin muscles at 48 hpf and 96 hpf (middle and right). (b) Whole body view of control and qkia knockout zebrafish embryos at 5 dpf. (c) Trunk muscle shown by fluorescence immunohistochemistry with F310 at 27 hpf and 5 dpf. Lateral views; the anterior side is on the left. (d) Fluorescence immunohistochemistry of 5 dpf F0 pectoral fins with F310 to label fast muscle (upper) and 14 dpf pectoral fins with phalloidin to label F-actin (lower). The 5 dpf control image is flipped horizontally. (e) Sequence of the mutated site in a qkia mutant line. Blue indicates sgRNA target sequences and red indicates the inserted sequence. (f) Fluorescence immunohistochemistry of a 5 dpf control and an F2 qkia mutant pectoral fin with F310. Scale bars: (a) leftmost panel, 500 μm; (a) middle and rightmost panels, 200 μm; (b), 1.0 mm; (c, d, f), 50 μm.