FIGURE 4

Expression of human COG4^p.G516R elevates the wnt4 transcript in zebrafish embryos. (A) Quantitative PCR analyses of selective non-canonical Wnt pathway ligands in zebrafish embryos injected with human COG4^WT or COG4^p.G516R mRNA. At 6 hpf, wnt4 expression is significantly upregulated in embryos injected with COG4^p.G516R mRNA, but not in those injected with COG4^WT mRNA. Similar upregulation of wnt4 transcripts is observed at 10 hpf as well. Bar graphs represent average gene expression relative to the housekeeping gene β-actin. The data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison tests was used. ^****p < 0.0001; ns, not significant. (B) Representative images of whole-mount in situ hybridization from control and treated embryos. Whole-mount in situ hybridization analyses demonstrate that wnt4 expression is elevated in embryos injected with COG4^p.G516R mRNA (a″, at 6 hpf; b″ at 12 hpf), but there are no obvious changes in embryos injected with human COG4^WT mRNA (a′ and b′). (b, b’) Arrows point to the restricted wnt4 expression domain in the hindbrain of zebrafish embryos at 12 hpf; (b″) arrows point to expanded expression of wnt4 in the hindbrain region. At 12 hpf, wnt11f2 expression intensity is not significantly changed in embryos injected with COG4^p.G516R mRNA (c″, d″), compared to either control siblings (c, d) or human COG4^WT mRNA-injected embryos (c′, d′). The dotted black color lines landmark the degree of angle between the head and tail, which is much more increased in embryos injected with COG4^p.G516R mRNA, suggesting defects in extension movement during gastrulation. Arrows in c, c′ and d, d″ indicate that wnt11f2 is restricted to the dorsal midline; however, its expression pattern is dispersed in COG4^p.G516R mRNA-injected embryos, suggesting that the convergence movement is impaired (c″, white dotted line and arrow; d″, there is no clear midline expression). Scale bars: 200 μm. Experiments were performed in triplicates with similar results.