Fig. 7

Loss of function of another PCP gene vangl2 leads to severe 3D curvature defect, anterior CVMs and increased notochord cell apoptosis. (A) Representative images of 2 dpf embryos injected with increasing doses of a morpholino (MO) targeting vangl2. WT, uninjected wild type. (B) The body length of vangl2 morphants is significantly reduced compared with non-injected fish at all three doses. Uninjected (WT), n=155; 0.5 ng, n=109; 1 ng, n=251; 2 ng, n=228. (C) ptk7a and ptk7audm400/+ embryos showed significant body length truncation following injection of low dose (0.5 ng) vangl2-MO. Uninjected ptk7a, n=49; uninjected ptk7audm400/+, n=37; MO-injected ptk7a, n=38; MO-injected ptk7audm400/+, n=48. (D) Lateral views of an uninjected WT fish and of a vangl2 morphant (0.5 ng MO) showing a scoliosis-like phenotype. (E) Calcein-stained images of WT and of vangl2 morphants (1 ng MO) at 22 dpf. Vangl2 morphants show a hemi vertebrae (asterisk) and fused V5-V6 vertebrae (arrow). Scale bars: 200 µm. (F) Representative µCT images of vangl2 morphants (0.5 ng) and of non-injected WT adult fish from lateral view and dorsal view (inset). Scale bars: 2 mm. (G) Reconstructed 3D structure with Imaris surface module from µCT scans (ventral and lateral views are shown in upper and lower panels, respectively). Dashed line indicates the separation between the first two vertebrae. V1 in vangl2 morphants displays a wedge vertebra. Scale bars: 300 µm. (H) Representative images of the notochords of uninjected WT fish and of vangl2 morphants (2 ng) stained with NucView530 Caspase-3 live staining at 19 hpf. Examples of apoptotic cells are indicated by arrows. Dashed lines delineate the notochord. Scale bars: 20 µm. (I) Quantification of apoptotic cells in the anterior part of the notochord from s1 to s6 or s8 at 19 hpf. The number of apoptotic cells is significantly increased in vangl2 morphants (2 ng MO; n=6) compared with uninjected WT embryos (n=6). (J) Representative multiphoton images of cell apoptosis staining of the anterior notochord of vangl2m209 mutants and their siblings at 19 hpf. Examples of apoptotic cells are indicated by arrows. Dashed lines delineate the notochord. Scale bars: 20 µm. (K) Quantification of apoptotic cells in the anterior part s1 to s6 or s10 of the notochord at 19 hpf in vangl2m209 embryos (n=6) and their vangl2m209/+ siblings (n=14). (L) Quantification of the apoptotic cells from s10 to the end of tail at 20 hpf between vangl2m209 mutants (n=11) and their vangl2+(n=2) and vangl2m209/+ (n=4) siblings. The number of apoptotic cells is significantly increased in vangl2m209 mutants compared with their siblings at anterior and posterior regions of the notochord. For B,C, P-values were calculated using two-tailed Welch's t-test. n.s., not significant. For I,K,L, P-values were calculated using two-tailed Mann?Whitney U-test.