Figure 3.

Gene structure and phenotypic characteristics of runx2b^+/+ and runx2b^−/−fish. (A) runx2b gene structure and RUNT domain characteristics. CRISPR-Cas9 target sequences are marked as blue letters in the DNA sequences. Deleted nucleotides are shown by ‘-’ and inserted sequences are marked as ‘*’ in runx2b^−/− zebrafish. (B) The runx2b protein structure. The β-sheet domain changed in runx2b^−/− zebrafish. The red area indicates the missing or changed amino acids in runx2b^−/− zebrafish compared with those in runx2b^+/+ zebrafish. The α-helix is indicated in green; β-sheet is indicated in yellow; random coil is indicated in blue. (C) Micro-CT scan with a resolution under 6 μm showing overall skeletal structures. Regions of vertebrae 9–14 and 19–23 were scanned with micro-CT under 4 μm resolution to show details of the IBs. (D and E) Alizarin red S staining of the abdominal and caudal region. IBs (arrowheads) are lost in runx2b^−/− zebrafish. (F) Masson's trichrome staining of transverse paraffin sections. The general tissue organization is maintained in runx2b^−/− zebrafish compared to that in runx2b^+/+ zebrafish. (G) Transverse paraffin sections of the area normally containing IBs (arrowheads) stained with Alizarin red S, indicated the loss of mineralized IBs in runx2b^−/− zebrafish. (H and I) IBs phenotype of runx2b F₀ generation mutants in blunt snout bream. The IBs are partially lost in the tail and back parts of the runx2b F₀ generation mutants. The black arrowhead indicates mineralized IBs, while yellow arrowhead indicates incomplete mineralized IBs.