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Fig. 7 Loss of mid-suture mesenchyme and misregulated osteogenesis in twist1b; tcf12 mutants. A Dot plot shows enrichment of twist1b and tcf12 expression in the regulatory mesenchyme cluster. B Double in situ analysis of adult coronal suture for twist1b and angptl1b. Dotted lines outline the frontal (F) and parietal (P) bones. In situs were performed in biological triplicate. Arrowheads mark double positive cells within the suture mesenchyme. Asterisk indicates autofluorescence from blood vessel. C Confocal images show grem1a:nlsEOS suture mesenchyme expression relative to the Alizarin Red+ frontal (F) and parietal (P) bones of control (twist1b+/−;tcf12+/−) and twist1b−/−;tcf12−/− mutant fish. Asterisk marks fused region of coronal suture, and boxes denote magnified regions. D Quantification of width of grem1a:nlsEOS domain in controls and in mutants sides with patent or partially fused coronal sutures. N = 8 control coronal sutures, N = 3 mutant patent coronal sutures, N = 5 partially fused coronal sutures, N = 4 control sagittal sutures, N = 5 mutant sagittal sutures. E Serial confocal imaging of individual control (twist1b+/−; tcf12+/−) and twist1b−/−; tcf12−/− mutant fish. The same fish were imaged before conversion of osteoblast:nlsEOS at approximately 8 mm, converted at 9 mm and imaged at 11 mm, and then converted again at 13 mm and imaged at 15 mm. Arrowhead marks the aberrant bone front, and arrow marks the fused region of the coronal suture. F Quantification of osteoblast addition at bone fronts and coronal sutures. N = 12 calvarial bones per genotype imaged from 9–11 mm, N = 6 patent coronal sutures per genotype imaged from 13–15 mm. Error bars report standard error of the mean, and statistics were performed using unpaired t tests. G Confocal images show fli1a:GFP vessels relative to the Alizarin Red+ frontal (F) and parietal (P) bones of control (twist1b+/−;tcf12+/−) and twist1b−/−;tcf12−/− mutant fish. Asterisk marks fused region of coronal suture, and boxes denote magnified regions. N = 5 controls and N = 8 mutants. H Quantification of density of fli1a:GFP vasculature in controls and mutants at patent or fused coronal sutures. Scalebars: 100 μM. P-values were calculated using a two-tailed non-parametric Student’s t tests. Error bars represent S.E.M. Source data are provided as a Source Data file.