Rho Kinase and Neural Crest Cell Survival Figure 6. Ectopic and excessive cell death in E9.5 RockDN;Wnt1-cre embryos. A–D are sections through the neural tube (line i in M), E–H through the pharyngeal arch 1 (line i in M) and I–L through the frontonasal processes (line ii in M). A,C,E,G,I,K show caspase 3-expressing cells (red) and Wnt1-cre+ve NCCs (green). B,D,F,H,J,L are the same sections but only showing the caspase 3-expressing cells. A–D) Whereas only very occasional activated caspase 3-expressing, dying, cells (red) are seen in the neural epithelium in control embryos, there are many dying cells observed in the dorsal part of the neural tube, from which the NCC emerge, in RockDN;Wnt1-cre mutant embryos (arrows in C and D). E–L) Very few activated caspase3-expressing cells are observed in the NCC-derived ectomesenchyme of pharyngeal arch 1 (E,F) and the frontonasal region (I,J) in control embryos. In contrast, many dying cells are seen in corresponding regions from RockDN;Wnt1-cre mutants (G,H,K,L). The surface ectoderm in the mutant is more irregular, compared to controls (arrowheads in E,G,I,K) and the inner NCC-derived ectomesenchyme is loosely arranged with gaps between the cells (arrows in G,K). N,O) The mean apoptotic and mitotic indexes were calculated for NCC within E9.5 pharyngeal arches. There is a significant increase in cell death in the mutant samples compared to controls (P = 0.019; * in N). There is no significant difference in cell proliferation between the two samples (P = 0.433; O). Scale bar = 50 mm. doi:10.1371/journal.pone.0037685.g006 detected as punctate aggregates at the cell surface [22]. In the developing craniofacial region of normal mouse embryos, we mainly detected vinculin localised to the boundaries between different cell types, for example the region between the ectomesenchyme and ectoderm of the developing frontonasal process. These boundaries are the sites of basement membranes, which are specialisations of the extracellular matrix that provide structural support for the surrounding tissues, and where cell- adhesion to the matrix is crucial [23]. The basement membrane component laminin, was found in a similar distribution to vinculin at these tissue boundaries in the craniofacial region of control embryos. Laminin and vinculin were lost from the ectomesench- yme : neural ectoderm boundary in the frontonasal processes, but were markedly elevated in the surface ectoderm of both this region and the pharyngeal arches. Thus, the relationship between the ectomesenchyme and neighbouring tissue types appears to be disrupted. This potential role for Rock1 at the basement membrane is supported by a recent publication [24] that has shown that Rock1 regulates basement membrane placement at the basal surface of the epithelium of the developing salivary gland, playing a role in establishing polarity within the tissue. In this scenario, Rock inhibition resulted in accumulation of basement membrane proteins throughout the epithelium [24]. Similarly, we see excessive vinculin and laminin within the surface ectoderm, PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e37685