Rho Kinase and Neural Crest Cell Survival Figure 2. Craniofacial malformations in RockDN;Wnt1-cre embryos. A,B) Severe frontonasal (arrow), maxilla and mandible hypoplasia (arrowhead) and exencephaly (red arrow) in E17.5 RockDN;Wnt1-cre embryo (B), compared with control littermate (A). Note also the abnormally shaped eye and absence of eyelids in the mutant embryo (magnified inserts in A and B). C,F) Lateral views show the abnormal development of the snout in mildly affected RockDN;Wnt1-cre embryos at E18.5. Mild truncations of the frontonasal processes (arrow) and mandible (arrowhead) are apparent. D,G) Clefting of the upper lip and nose are apparent in a frontal view of an E18.5 RockDN;Wnt1-cre embryo (G; same fetus as in F). Double arrows are the same length in D and G highlighting the increased intra-ocular distance in RockDN;Wnt1-cre embryos. E,H,I,J) Measurements were taken from E15.5 heads (11 control embryos, 6 severely affected RockDN;Wnt1-cre mutants and 3 mildly affected RockDN;Wnt1-cre mutants). The distance between the eyes and the size of the angle between the nose and the forehead was measured (double arrow in E,H) and the results are shown in graphs I and J, respectively. In both cases, the measurements in the severely affected mutant embryos were significantly different from the controls (P,0.0001 *). There were no significant differences between controls and the mildly affected mutants for either measurement. Scale bar in A–D,F,G = 500 mm; E,H = 375 mm. doi:10.1371/journal.pone.0037685.g002 Expression of Rock1 and Rock2 in developing craniofacial structures Inter-crossing of ROSA-EYFP mice with Wnt1-cre enabled the co- localisation of Rock1 protein with NCC. At E9.5 Rock1 was expressed in the neural tube and the dorsal root ganglia (Figure 4 A,D). Expression localised to the perimeter of the cells was found in the mass of the NCC within the pharyngeal arches (Figure 4 B,E and Figure S2 A–D) and in the frontonasal processes (Figure 4 C,F). Rock1 immunoreactivity was most abundant, however, at the boundaries of the NCC-derived mesenchyme with the epithelia of the surface ectoderm and the neural ectoderm in the frontonasal processes (arrows and arrowheads in Figure 4 C,F). Rock2 was expressed in similar NCC-rich regions (Figure S2 E–G) confirming that both isoforms were found in regions rich in migrating and post- migratory NCC. By E11.5, both Rock1 and Rock2 proteins were down-regulated in the craniofacial region (data not shown). Reduction of NCC corresponds to defects in cranial NCC derivatives We used the Rosa 26R (b-galactosidase; b-gal) reporter to investigate the extent of migration of NCC and the distribution of their derivatives with regard to the observed craniofacial PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e37685