|
Rapid functional evaluation of novel BMD associated genes in zebrafish identifies a role of novel gene atp6v1c1 in skull development and a role of prkar1a in bone growth. a Schematic of skull formation in zebrafish. Four ossification centres (grey) are formed by condensation of osteoblasts in the periphery of the skull at around 3 weeks post-fertilization (wpf). Ossification planes grow towards the centre of the skull forming the frontal (F) and parietal (P) bones, completely covering the brain at 7wpf and forming the metopic (m), coronal (c) and sagittal (s) sutures. b Schematics of the zebrafish crispant experiments. Fish carrying osteoblast reporter line Tg(Ola.Sp7:NLS-gfp) were crossed, and embryos at one cell stage were injected with the CRISPR/Cas9 system targeting respective genes. Observations were carried out in vivo during skull/suture formation (5-7wpf). Scale bars = 500 um. c Live imaging of skulls and ex vivo Alizarin Red Staining of control, and zic1, atp6v1c1 (a + b) and prkar1a (a + b) crispants. Ectopic sutures are indicated with green arrowheads in zic1 and atp6v1c1 (a + b) crispants. Sutures were outlined with a dashed line in Alizarin Red pictures. Cavities in the skull are indicated with magenta arrowheads in prkar1a (a + b) crispant. Scale bars = 500 um. d 3D renders micro-computed topographies (µCTs) images of the skull. Images were colour-coded to show BMD variation. e–h Site-specific BMD measurements of fish of similar standard size. Data are mean ± SD. All P-values are indicated. Graphs were generated in Prism 8. e, g Comparison of zic1 (n = 6), atp6v1c1 (a + b) (n = 3) crispants and controls (n = 6). Standard size 1.8 cm. One-way ordinary ANOVA, multiple comparisons test f, h Comparison of prkar1a (a + b) crispants (n = 3) and controls (n = 3). Standard size 1.6 cm. Nonparametric, Two-tailed, T-tests.
|
