Rho Kinase and Neural Crest Cell Survival Figure 4. Expression of Rock1 in the developing head. A–F) D–F are magnified regions from A–C, respectively. At E9.5, Rock1 protein (red) is expressed in the neural tube and the dorsal root ganglia, where it colocalises with Wnt1-cre+ve NCC (green, detected by GFP antibody, arrows in A,D), and in the pharyngeal tissue (B,E). In the frontonasal processes (C,F) Rock1 is expressed at the boarders of NCC rich ectomesenchyme and the surface ectoderm (arrows) and neural ectoderm (arrowheads), which are devoid of NCC. G) The position of the transverse sections shown in A–C are illustrated on a cartoon of an E9.5 embryo. Line i represents the position through the neural tube (A) and pharyngeal arch 1 (B) and line ii is the level of the fronotnasal processes (C). drg = dorsal root ganglia; fnp = frontonasal processes; mes = mesenchyme; ne = neural ectoderm; nt = neural tube; pa1 = pharyngeal arch 1; se = surface ectoderm. Scale bar in A–C = 45 mm. doi:10.1371/journal.pone.0037685.g004 Abnormal NCC-matrix interactions are associated with NCC death in craniofacial tissues of RockDN;Wnt1-cre embryos As Rock is a key regulator of the actin cytoskeleton, and disruption of the actin cytoskeleton has been linked to induction of cell death in a variety of contexts [19] we carried out phalloidin staining for filamentous actin in sections taken from E9.5 control and RockDN embryos, as this was the stage when cell death was at its peak. In control embryos, filamentous actin was found in a marked cortical distribution, lining the perimeter of the cell, in the NCC-derived ectomesenchyme of the pharyngeal arches (Figure 7 A,E) and frontonasal region (Figure 7 C,G), as well as the non- NCC-derived neural ectoderm of these regions. Cortical phalloidin staining was markedly reduced in the mesenchyme of the pharyngeal arches (Figure 7 B,F) and frontonasal processes (Figure 7 D,H) of stage-matched RockDN embryos, but was found in the same pattern as the controls in the neural ectoderm, which is not derived from NCC (Figure 7 G,H). Moreover, there were foci of intense phalloidin staining scattered throughout the ectomesenchyme of the pharyngeal arches and frontonasal process of mutant embryos, which were rarely seen in control embryos (blue arrow in Figure 7 F,H). These foci of rounded up, intensely phalloidin-stained cells were interspersed with activated caspase 3-expressing cells (white arrow in Figure 7 F,H), with occasional phalloidin-intense cells also expressing activated caspase 3 (arrowheads in Figure 7 F,H). Thus, disruption of the actin cytoskeleton is associated with cell death in the craniofacial region. In cell culture, vinculin localises to integrin-mediated focal adhesion complexes, linking the internal cytoskeleton of the cell to the substrate on which it sits, and also, at lower levels, in cadherin- mediated cell-cell junctions (reviewed in [20]). As detachment of cells from the matrix has been observed in RockDN-expressing motor neurones [12], we looked for evidence of disruption of cell- matrix adhesion, as a possible cause of the cell death we observe in the craniofacial region of RockDN mutants. Whereas in cultured NCC, vinculin localised to distinct foci at the periphery of the cells as expected (data not shown), this was not the case in the three dimensional environment of the developing embryo. In control embryos, vinculin was expressed strongly in discrete regions of the tissue, associated with boundaries between different cell types, such as the boundary between the NCC-derived ectomesenchyme and the neural ectoderm (Figure 7 K,O). Lower levels were also found at the periphery of individual cells, likely reflecting its association with cadherin-based cell-cell junctions. In matched RockDN PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e37685