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Fig. 4
Altered bone growth dynamics precede craniosynostosis in mutant zebrafish.
(A) Dorsal views of the developing skull bones in the same wild-type and mutant individuals across four developmental stages. Live fish were stained with Calcein Green at 6, 7, and 8 mm and Alizarin Red at 9 mm. For the right sides, arrows show initiation of the frontal bone at 6 mm and dashed lines show the frontal (left) and parietal (right) fronts at successive later stages. (B) Individual wild-type and tcf12; twist1b mutant fish were stained with Calcein Green at 10.25 ± 0.25 mm, recovered, and then stained again with Alizarin Red and imaged at 14 ± 0.5 mm. These same fish were then grown to 20 mm, at which stage they were fixed and stained again with Alizarin Red to assess suture patency. White dotted lines indicate bone generated by 10.25 mm. Arrowheads indicate missing coronal suture. Scale bars, 1 mm. (C) Quantification of calvarial bone growth. Bone produced by 10.25 ± 0.25 mm was calculated based on the area (µm) stained with Calcein Green (white outlines in B). At 10.25 mm, compared to control frontal bones (n = 12), tcf12; twist1b mutant frontal bones that developed synostosis later (n = 11) and those that did not (n = 11) showed similar increases in bone formation. Bone shape was assessed by overlaying tracings of posterior frontal bones and parietal bones for wild types and mutants. Specific growth directionality in the anterior-posterior, medial-lateral, and diagonal axes were measured and quantified (green arrows). (D) Bone growth from 10.25 ± 0.25 mm to 14 ± 0.5 mm was analyzed in respect to prospective suture zones. Growth in the metopic (yellow) and sagittal (blue) zones did not differ significantly in controls versus tcf12; twist1b mutants, which correlated with no defects in these sutures in mutants. In contrast, growth in the coronal zone was reduced in tcf12; twist1b mutants, with a more pronounced decrease in mutant sides that later developed synostosis. p values were determined by Student’s t-tests; error bars represent standard error of the mean.