Fig. 3: Increased expression of csf1 in male pectoral fins by cis-regulatory mutations.

a, b RNA-seq of the pectoral fins. Minus-Average (MA) plots with the average gene expression levels on the X axis and log₂ fold-change in expression between the sexes in O. woworae (a) or between O. woworae males and O. asinua males (b) on the Y axis. Each dot represents a single gene with differentially expressed genes (DEGs) colored (FDR < 0.01). The gene names of DEGs within the red pectoral fin QTLs are also shown. c Venn diagram indicating overlap among inter-sex DEGs in O. woworae, interspecies DEGs in males, and genes within 95% Bayesian credible intervals of all red pectoral fin QTLs on LG7. Five genes were identified as strong candidate genes. d Comparison of csf1 expression levels among males and females of O. woworae and O. asinua, showing the highest expression in O. woworae males (n = 4 for each). Mean ± SD are shown. A dot indicates each individual. Expression levels of the other four candidates are shown in Supplementary Fig. 4. e qPCR analysis comparing the relative expression levels of csf1 in the seven different fin parts of O. woworae (red) and O. asinua (blue) males (n = 8 for each). The expression levels were normalized by the expression of an internal control gene, rpl13a. f GFP fluorescence in the pectoral (left) and caudal fins (right) in males (upper) and females (lower) of GFP knock-in O. woworae (csf1^Olhs:GFP). Expression in the lens is caused by the basal activity of the heat-shock promoter. g Microscopic images of pectoral fins in bright-field (left) and GFP fluorescent observation (right). Images are representative of three experiments. Scale bars indicate 200 µm. h Expression levels of csf1 in the pectoral fin of the F₂ family (n = 8 for each). The X axis indicates the genotype at the red pectoral fin QTL on LG7 (C/C, homozygous for the O. celebensis allele; W/W, homozygous for the O. woworae allele; C/W, heterozygote). Different letters above the boxes indicate significantly different groups (P < 0.05, post hoc test with the Tukey’s honest significant difference method; see Supplementary Table 4 for the statistical results). i Allele-specific expression analysis of csf1 in pectoral fins of the heterozygous fish (n = 8 for each). The ratio of the O. woworae allele to the O. celebensis allele was significantly higher in cDNA compared with that in genomic DNA. The P values are calculated separately for each sex using two-sided Wilcoxon’s signed rank test (male, V = 36, P = 0.007812; female, V = 28, P = 0.01562; *P < 0.05 and **P < 0.01). h, i the blue and brown colors indicate males and females, respectively. j Administration of methyltestosterone (MT) to O. woworae females increased csf1 expression in the pectoral fin compared with the control fish administered dimethyl sulfoxide (DMSO) (n = 12 in each group). The P value is calculated using two-sided Welch’s t test (t_12.233 = −5.4148, P = 0.00015, ***P < 0.001). The gray and green colors indicate fish with DMSO and fish with MT, respectively. e, h–j Each circle represents each individual, the center line indicates the median, box limits indicate the upper and lower quartiles, the whiskers indicate 1.5× interquartile range, and the points are outliers. Source data are provided as a Source Data file.