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Fig. 3 Vertebral phenotypes of b13a and c13a zebrafish mutants. (A) Cleared and stained skeletal preparations of WT and homozygous b13a and c13a mutants showing variation in tail vertebrae number. Scale bars, 1 mm. For (B), (C), and (E), n = b13a+/+:12, b13a+/−:14, b13a−/−:14, c13a+/+:13, c13a+/−:15, and c13a−/−:12. (B) Total number of vertebrae in both mutants. ANOVA revealed a significant increase in vertebrae number [b13a: F2,37 = 78.28, P < 0.0001; c13a: F2,37 = 63.49, P < 0.0001] and multiple comparisons showed a gene-dosage effect for both genes (*P < 0.0001). (C) Vertebral number per body region in both mutants. Poisson regressions revealed significant increases for both mutants in midcaudal [b13a: χ2(2) = 6.23, P = 0.044; c13a: χ2(2) = 30.003, P < 0.0001] and ural vertebrae [b13a: χ2(2) = 41.22, P < 0.0001; c13a: χ2(2) = 6.88, P = 0.032]. Other comparisons were not significant (ns) [precaudal | c13a: χ2(2) = 1.54, P = 0.46; preural | b13a: χ2(2) = 3.08, P = 0.21; preural | c13a: χ2(2) = 0.32, P = 0.85). Post hoc tests were not applied due to the minimal variance or absence of variance in several observations, rendering those tests invalid. The line is at the mean and circle size is proportional to the number of individuals. (D) Caudal fin endoskeleton of WT and homozygous b13a and c13a mutants. The pleurostyle (blue arrowhead) and uroneural (orange arrowhead) are absent in b13a−/− mutants. An elongated neural spine of the preural centrum 1 (purple arrowhead) is present in both mutants. Extra distal radials (green arrowheads) are present in c13a−/− mutants. A cartilaginous articulation of the haemal arch of the preural centrum 3 with its centrum (light blue arrowhead) is present in the b13a−/− mutant, as revealed by Alcian blue staining. Scale bars, 200 μm. (E) Frequencies of pleurostyle and uroneural loss in b13a and c13a mutants. See Fig. 1 for anatomical terminology.