Fig. 2

Subcellular distribution of Vangl2 variants, and differential effects of transient overexpression on CE movements and neuronal migration. (A) Two panels for each injection condition, with upper panel showing myod1 in situ of 12–14 hpf embryo, and lower panel showing anti-Myc immunostaining (red) of ectodermal cells in 10 hpf embryo, counterstained with DAPI (blue) to stain nuclei. Uninjected (Uninj.) and GFP RNA injected controls shown in leftmost panels. Following injection of Vangl2 FL RNA, the recombinant protein localizes to the plasma membrane (white arrowhead). The corresponding myod1 in situ shows that convergence and extension (CE) movements of paraxial mesoderm are reduced, resulting in thin, elongated somites. Expression of some variants generated proteins that localized largely to the cytoplasm (black arrowheads; N, PBMΔ, P2X2) or the membrane (white arrowheads; FL, TM-C, DTM) or both compartments (N-PH), while others exhibited significant nuclear localization (arrows; C, Lyn-C). N-TM overexpressing embryos showed punctate cytoplasmic localization, resembling vesicles (white arrowhead). (B) The ability of various Vangl2 constructs to generate CE defects was variable. CE movement defects were scored by examining live embryos at 12–15 hpf. Injection dose: 280 pg RNA/embryo for all constructs except Vangl2 TM-C (150 pg/embryo). Some constructs (FL, TM-C, N-PH, Lyn-C) generated CE defects in >50% of injected embryos, whereas others exhibited weaker effects (N-TM, C, DTM) or marginal effects (GFP, PBMΔ, N). Significant differences p < 0.05 and p < 0.001 (Pearson Chi Square test) are indicated by (**) and (***), respectively. NS, not significant. Number of embryos scored in parenthesis for each construct. Data pooled from 3 to 5 experiments. (C) Transient overexpression did not cause defects in FBM neuron migration. See Fig. 4 legend for scoring criteria.