Rho Kinase and Neural Crest Cell Survival Figure 5. b-gal staining of NCC (blue) in RockDN;Wnt1-cre embryos. A–D) At E8.5, the numbers of NCCs migrating towards pharyngeal arches 1 and 2 (arrows in A,C) and within pharyngeal arch 1 (B and D) are similar. There is a reduction in NCCs populating the region anterior to the developing eye (asterisks in A,C). E–H) At E9.5, there is increased distance between the frontonasal regions and the first pharyngeal arch (double arrows in E,G). There is a reduction in the numbers of NCC in the head (asterisks in G, compare to E) and the first pharyngeal arch (F,H). The surface ectoderm (arrowheads in F,H) is thickened and uneven in the mutant (compare H with F). I,J) At E10.5, the hypoplasia of the frontal region is more obvious in severely affected RockDN;Wnt1-cre embryos and the pharyngeal arches are frequently hypoplastic (numbered 1, 2 and * (posterior pharyngeal arches 3–6)). The cranial ganglia are misshapen and smaller in size in RockDN;Wnt1-cre embryos (white arrowheads in E,G,I,J). K,L) By E13.5, abnormalities in the distribution of NCC are seen even in the more mildly affected embryos, with reduced b-gal staining observed in the midline of the forming calvarias bones (arrows) and below the eye (white arrowheads). The frontonasal region is mildly truncated. pa1 = pharyngeal arch 1; 1 = pharyngeal arch 1; 2 = pharyngeal arch 2. Scale bar in A,C = 200 mm; B,D = 2 mm; E,G = 160 mm; F,H = 40 mm; I–L = 600 mm. doi:10.1371/journal.pone.0037685.g005 embryos, vinculin was lost at the ectomesenchyme : neural ectoderm boundary but was expressed more broadly in the NCC- derived ectomesenchyme, in both the first pharyngeal arch (Figure 7 I,J,M,N) and in the frontonasal region (Figure 7 K,L,O,P). Ectopic vinculin expression was also found in the surface ectoderm of the frontonasal region and pharyngeal arches (Figure 7 J,L). Co-staining for vinculin and activated-caspase 3 showed some localisation within the same areas, suggesting a possible link between disruption of the actin cytoskeleton, cell- substrate adhesion and cell death of the RockDN-expressing NCC. Staining for paxillin, another component of focal adhesions, confirmed these observations (Figure 8 A–H). Paxillin was localised to the ectomesenchyme : neural ectoderm boundary and individual cell membranes in control embryos (Figure 8 A,C). However, staining was lost from the ectomesenchyme : neural ectoderm boundary in RockDN embryos and was observed in punctate foci in the ectomesenchyme (Figure 8 F,H) in a similar distribution to activated-caspase 3 immunoreactivity, supporting the idea that cell death was occurring at sites of abnormal focal adhesion formation. Immuno-staining for laminin confirmed that the interactions with the extracellular matrix were abnormal, as basal lamina localisation was lost from the ectomesenchyme : neural ectoderm boundary in the frontonasal processes and was abnormally distributed throughout the surface ectoderm in both the frontonasal processes and the pharyngeal arches (Figure 8 I–L and data not shown). Discussion We have shown a cell-autonomous requirement for Rock function within NCC in the development of the frontal aspect of the face, and that in the absence of this, there is increased cell death within the forming facial protuberances leading to hypoplasia and midline facial clefting. Cell-extracellular matrix adhesion, and specifically the distribution of focal adhesion proteins and the basement membrane protein laminin, is abnormal in the ectomesenchyme from RockDN mutants. Rock is known to play important roles in modulating the cytoskeleton [4,5] and the induction of stress fibres by the RhoA-Rock pathway is required for the formation of focal adhesions [21]. Thus, the cell death resulting from Rock inhibition is likely to be a secondary consequence of cytoskeletal disorganisation, as the NCC lose adhesion to the surrounding extracellular matrix, round up, and die. Similarly, reduced neuronal survival and apoptotic cell death are seen when Rock function is abrogated in developing motor neurons [12]. These authors suggested that anoikis, cell death as a result of failing to interact with the surrounding matrix, occurs in the absence of Rock; we also favour this as the mechanism leading to high levels of cell death in the RockDN;Wnt1-cre embryos described here. Although the role of focal adhesions has been well explored in the two-dimensional environment of the culture dish, little is known about the role of focal adhesions within living tissues. In cells in three-dimensional matrices, focal adhesions cannot be PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e37685