Rho Kinase and Neural Crest Cell Survival Figure 7. Disruption of the actin cytoskeleton and vinculin-containing focal contacts in E9.5 RockDN;Wnt1-cre embryos. A,B,E,F,I,J,M,N (line i in Q) are sections from first pharyngeal arch and C,D,G,H,K,L,O,P (line ii in Q) are from the frontonasal processes. A–H show phalloidin (red) and caspase 3 (green) immunofluorescence, with E–H being magnified regions as shown by the boxes in A–D, respectively. I–L show vinculin (red) and caspase 3 (green) dual immunofluorescence, with M–P being magnified regions as shown by boxes in I–L. The dotted lines in C,D,G,H,K,L,O,P indicate the boundary between the inner ectomesenchyme and the neural ectoderm and the surface ectoderm. A–H) Filamentous actin, labelled with phalloidin (red) outlines the cells in NCC-derived ectomesenchyme and neural ectoderm in the first pharyngeal arch (A,E) and frontonasal processes (C,G) of control embryos. Cortical phalloidin staining is lost in the ectomesenchyme from RockDN;Wnt1-cre mutants (F,H) but is maintained in the neural ectoderm (compare G with H). In addition, intense phalloidin-labelled foci are observed throughout the ectomesenchyme of the RockDN;Wnt1- cre mutants (dense red foci, blue arrow in F,H). Green caspase 3-positive cells are interspersed (white arrow) and overlapping with the phalloidin- intense cells (arrowheads in F,H). I–L) Vinculin and caspase 3 staining. In the pharyngeal arch the vinculin staining is not restricted to the centre of the arch in the mutant (compare J with I). In the frontonasal processes vinculin outlines the boundary between the surface ectoderm and the neural ectoderm with the inner NCC-derived ectomesenchyme (dotted lines in K,L). This discrete vinculin staining is lost in the RockDN;Wnt1-cre mutants (compare P with O). cas3 = activated caspase-3; mes = mesenchyme; ne = neural ectoderm; phall = phalloidin; se = surface ectoderm; vin = vinculin. Scale bar = 50 mm. doi:10.1371/journal.pone.0037685.g007 but also within the ectomesenchyme, of the pharyngeal arches and the frontonasal processes, suggesting Rock1 may be playing a similar roles in restricting basement membrane to the basal surface of the epithelium in the developing craniofacial region. The knockdown of Rock only in the ectomesenchyme (NCC) in our mutant, suggests that this effect can be non-cell autonomous. PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e37685