Figure 6. Sutures of sp7 mutants show abnormal architecture and gene expression.

A–D) Tissue morphology was assessed by hematoxylin–eosin staining of sagittal sections of the skull to show the coronal suture. In WT (A), suture formation is divided into three progressive phases as the osteogenic fronts (*) approach and overlap: initiation (~4 weeks), approximation (~5 weeks), and overlapping (>6 weeks). In sp7 mutants (B), initiation and approximation phases are similar to WT, but overlapping does not occur, and osteogenic fronts have a rounded shape. During the same period, the frontal bone above the epiphyseal bar cartilage (eb) increases in thickness (C), but in sp7 mutants (D) the frontal bones are thinner (brackets in C, D), and extra sutures are common (arrows). E–I) Tissue sections from transgenic fish were analyzed by immunohistochemistry for GFP, mCherry, and phosphohistone H3. In WT fish (E), expression of the Runx2:egfp transgene is confined to cells at the osteogenic front, while sp7:mcherry expression is maintained in cells along the bone; both transgenes are expressed at the osteogenic fronts (asterisks). Immature osteoblasts expressing Runx2 (GFP+) are observed at the osteogenic fronts of WT fish (F) while in mutants these cells are not limited to the fronts (G). Proliferation was analyzed in WT (H) and sp7 mutant (I) fish. Phosphohistone H3 + cells were more frequently observed at the osteogenic fronts in mutants, and these corresponded to the GFP+ early osteoblasts (merge). J, K) Expression of msxd (J) at the coronal suture and scales (K) was examined by in situ hybridization in WT and sp7−/−; expression was significantly increased in mesenchymal cells of mutants. L, M) To examine the level of BMP signaling, sections were analyzed with an antibody to phosphorylated Smad 1/4/5. Numbers of pSmad+ nuclei in and around the suture were much greater in sp7 mutants (M). Scale bars for all panels are 50 μm. Abbreviations: epiphyseal bar cartilage (eb), frontal bone (f), parietal bone (p), scale (s).