Rho Kinase and Neural Crest Cell Survival Figure 8. Disruption of focal adhesions and extracellular matrix in E9.5 RockDN;Wnt1-cre embryos. A,B,E,F are sections of the first pharyngeal arch (line i in O) and C,D,G,H,I–N are from the frontonasal processes (line ii in O). A–H) E–H are magnified regions shown in the boxes on A–D, respectively. Paxillin has a cortical distribution in the first pharyngeal arch in the control embryos (A,E) and also marks the boundary between the neural ectoderm and NCC-derived ectomesenchyme (arrow in C,G) in the frontonasal processes. This boundary staining is lost in the mutant (arrow in D,H) and there are intense paxillin positive foci in the pharyngeal arch and frontonasal processes (arrowheads in B,F,D,H), confirming loss of cell-substrate adhesion. I–L) Laminin was lost from the ectomesenchyme-neural ectoderm boundary (compare arrowhead in J with L) and was abnormally distributed in the surface ectoderm in the frontonasal processes in the RockDN;Wnt1-cre embryos (compare arrow in I with K). The H&E staining of the same sections are shown in M and N, allowing visualisation of the different cellular layers. fnp = frontonasal process; pa1 = first pharyngeal arch; ne = neural ectoderm; se = surface ectoderm; mes = mesenchyme. Scale bar in A–D,I,K = 50 mm; M,N = 40 mm. doi:10.1371/journal.pone.0037685.g008 Both phalloidin, which binds filamentous actin, and paxillin, another component of focal adhesions, were found in condensed foci in the ectomesenchyme of RockDN mutants, which were intermingled with dying cells. Thus, increased cell death was associated with disruption of the cytoskeleton and abnormal distribution of focal-adhesion proteins. The observation that only a small number of phalloidin/paxillin-rich condensed foci co- expressed activated-caspase 3 likely relates to the role the latter plays in the process of cell death, regulating chromatin conden- sation and DNA fragmentation [25]. Thus, activated-caspase 3 would only be expected in the ectomesenchymal cells in the latter stages of cell death. We propose that in contrast, the disruption of focal adhesion and cytoskeletal proteins, and the consequent detachment from the surrounding matrix, is the cause of the cell death, and thus would be apparent within the tissue for much longer. Rac1 has also been implicated in playing an essential role in regulating cell-matrix adhesion in NCC [26] and Rac1f/f;Wnt1- cre embryos develop similar craniofacial abnormalities as the RockDN;Wnt1-cre embryos. Excessive cell death, in combination with disrupted NCC-matrix adhesion was observed in the in the frontonasal region at E11.5 in these embryos, once the midfacial clefting was apparent, although the authors did not look earlier, before the defects arose. Together, these data suggest that it is the defect in the interaction of NCC with the surrounding matrix, with the multiple effects that this has on the tissue, that is the crucial factor in the development of the craniofacial malformations observed. Frontonasal dysplasia has been described as a consequence of loss of expression of certain transcription factors, including Alx3 and Alx4. Although alone, Alx3 mice have no phenotype, severe midfacial clefting and truncation of the snout is observed when the mice are inter crossed with Alx4 mice (Alx3+/2;Alx42/2 and Alx32/2;Alx42/2; [27]). Excessive cell death was observed in the frontonasal processes at E10.0, before the appearance of the defects, as we have observed in RockDN;Wnt1-cre mice. Similarly, embryos for the related transcription factor Cart1, when combined with loss of Alx4 (Alx42/2;Cart1+/2 and Alx42/2;Cart12/2), also display a similar phenotype, with severe midfacial clefting and open eyes at birth [28], as we see in the RockDN;Wnt1-cre embryos. Brachyrrhine (3H1 Br/+) mice display frontonasal dysplasia, thought to be caused PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e37685