Fig. 8

Deletion of BMP antagonists alters bone growth and promotes ectopic bone formation. A Schematic of mutant alleles generated using CRISPR/Cas9. Red arrow indicates site of mutation relative to protein-coding regions (green boxes). B Confocal imaging of Calcein-stained calvaria show impaired suture formation in grem1a−/−; nog2−/−; nog3+/− zebrafish compared to controls. Dotted lines represent regions of the orthogonal sections at the coronal suture shown below. Frontal (F) and parietal (P) bone. C Quantification of non-overlapping regions in the calvaria. N = 3 for controls, N = 5 for mutants. D Representative imaging of individual control and mutant zebrafish after repeated staining and imaging using Calcein green and Alizarin red stains. Arrowheads indicate non-overlapping bones at the coronal suture. E, F Quantification of area of bone growth between 11–13 mm and 13–15 mm at frontal and parietal bones. N = 7 control frontal bones, N = 8 mutant frontal bones at 11–13 mm. N = 6 control frontal bones, N = 10 mutant frontal bones at 13–15 mm. N = 7 control parietal bones, N = 10 mutant parietal bones at 11–13 mm. N = 6 control parietal bones, N = 10 mutant parietal bones at 13–15 mm. G Confocal imaging of supernumerary bones (numbered) at the sagittal suture (ss) between parietal bones in control and mutant zebrafish. H Quantification of supernumerary bones at the sagittal suture. N = 8 for control and mutant fish. I Confocal imaging of control and mutant coronal sutures reveal ectopic bone islands at the mutant coronal suture. Arrowheads mark ectopic bone islands between frontal and parietal bones. J Quantification of ectopic bone islands at the coronal suture. N = 8 for control and mutant fish. K Immunofluorescence for pSmad1/5 at the coronal suture of control and mutant zebrafish. Dotted line outlines bones. White box indicates quantified area for the coronal suture and red box indicates quantified area for parietal bone. L Quantification of fraction of pSmad1/5+ nuclei at coronal sutures or along the parietal bone. N = 3 for control and mutant fish. 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