Active N-Ras induced convergence and extension cell-movement defects and craniofacial defects. (A) In situ hybridization was performed using embryos at the eight-somite stage (∼12 hpf) using krox20 and myod-specific probes: non-injected control (NIC) embryos or embryos injected with synthetic RNA encoding wild-type (WT) N-Ras or mutants, as indicated, were used. Dorsal views of flat-mounted embryos are depicted, anterior to the top. Scale bar: 250 μm. (B) The ratio of the width of the krox20 staining and the length from the first to the eighth somite was determined using ImageJ software and is depicted here. Error bars indicate s.e.m.; Student’s t-tests (two-tailed, assuming unequal variance) were performed between mutant and wild-type N-Ras-injected groups; *P<0.005. Total numbers of embryos that were used for the analyses are indicated (n). (C) Embryos from the Tg(–4.9sox10:EGFP)^ba2 transgenic line, in which neural crest cells are marked, were injected at the one-cell stage with RNA encoding N-RAS-I24N. The ceratohyal is marked by GFP in the transgenic embryos at 4 dpf and lines (red) were drawn demarcating the ceratohyal, facilitating analysis of the angle in the ceratohyal, which is a direct marker for the width and bluntness of the heads of these embryos. Representative images of a non-injected control (NIC) and a N-RAS-I24N-injected embryo are depicted. Scale bar: 100 μm. (D) Quantification of the angle of the ceratohyal. The number of individuals used (n) is indicated. A Student’s t-test was performed; asterisk indicates significant difference, P<0.005.