Figure 3. Abnormalities in formation of the craniofacial bones in RockDN;Wnt1-cre embryos. A,B) In severely affected RockDN;Wnt1- cre embryos at E14.5, the frontonasal bones (stained with alcian blue) are absent (arrow in B, compare to A). Meckel’s cartilage is also reduced in size (arrowhead in B, compare with A). C,D) Bone (red) and cartilage (blue) staining of a mildly affected RockDN;Wnt1-cre embryo at E18.5 (D), shows that the maxilla (arrowhead) and mandibular (arrow) bones are well formed, although the hyoid bone (red arrow) is reduced in size in mutant embryos. E,F) Inferior views of the base of the skull in mildly affected embryos shows that the basisphenoid and the presphenoid bones are hypoplastic in RockDN;Wnt1-cre embryos, whereas the nasal septum is completely missing. Moreover, the maxillary bones are widely separated in mutant embryos (double arrow in F), compared to control littermates (E). bs = basisphenoid; n = nasal septum; ps = presphenoid. Scale bar = 500 mm. doi:10.1371/journal.pone.0037685.g003 phenotypes. At E8.5, control and RockDN embryos were indistin- guishable based on their external appearance. Staining for b-gal showed normal migration of NCC towards pharyngeal arches 1 and 2 (Figure 5 A,C), suggesting that NCC induction and delamination had occurred normally. However, there was a reduction in b-gal expression anterior to the stream of NCC migrating to the 1st pharyngeal arch (Figure 5 A,C). At E9.5– E10.5, the mutant embryos were still not reliably distinguishable from control littermates on the basis of their craniofacial phenotype. However, the b-gal expression was generally more patchy in the craniofacial region of RockDN embryos examined at E9.5–E10.5 (Figure S1 A,B and Figure S3) and there appeared to be fewer b-gal-stained NCC in all embryos examined at this stage (9/9). Although the frontonasal structures appeared hypoplastic in 87% of RockDN mutant embryos by E11.5–E18.5 (Figure S1 C– H), b-gal expression was confluent in the developing facial prominences. It was deficient, however, in the posterior parts of the medial calvarias bones in embryos with the most severe craniofacial phenotype (data not shown) and below the eyes (Figure 5 K,L). In addition to the structural abnormalities already Rho Kinase and Neural Crest Cell Survival described, the thymus was ectopically located in the cervical region (data not shown) and the cranial ganglia were misshapen and reduced in size (arrowheads in Figure 5 E,G,I,J and data not shown). The b-gal staining pattern also revealed abnormalities in NCC distribution during the development of the pharyngeal arches. At E8.5, the streams of NCC migrating towards the pharyngeal arches were similar in both wild type and RockDN mutant embryos (arrows in Figure 5 A,C). However, at E9.5, and persisting at E10.5, the first and second pharyngeal arches were hypoplastic and the b-gal expression in the arches was less intense and patchy (Figure 5 E–J). These data suggest that although the formation, delamination and early migration of NCC appeared to occur normally at E8.5 in the RockDN mutants, NCC numbers declined later in development. Ectopic and exacerbated apoptosis in RockDN NCC Reduced Rock function is associated with increased apoptosis in developing motor neurones [12]. We therefore examined the expression of activated caspase 3 in the developing RockDN embryos, hypothesising that increased levels of apoptosis in the affected tissues might explain the emergent phenotype. NCC migration begins at E8.0 in the cranial region of mouse embryos and is complete by the end of E9.5 [18]. At E8.5, after the first NCC had delaminated from the neural tube, but before the appearance of any craniofacial phenotype, there was little if any activated caspase 3 labelling anywhere in the control embryos. However, a few activated caspase 3-expressing, dying, cells were observed in the frontonasal region and pharyngeal arches of RockDN embryos at this stage (data not shown). By E9.5, and continuing at E10.5, activated caspase 3-positive cells were abundant in the dorsal part of the neural tube in RockDN embryos, localising to the Wnt1-cre domain (Figure 6 A–D). This correlates with the latter stages of NCC delamination from this region but also continues after migration from the cranial neural tube is complete. The mesenchymal cells within pharyngeal arches 1–3 were compact in control embryos by E9.5. The surface ectoderm formed a smooth layer surrounding the inner ectomesenchyme (Figures 5F and 6 E,F). This outer layer appeared uneven in the RockDN mutants and the mesenchymal cells appeared loosely arranged and disorganised (Figures 5H and 6 G,H). Similar observations were made in the frontonasal processes of control and stage matched mutant embryos (arrowheads in Figure 6 I–L). Examination of the craniofacial region of RockDN embryos revealed that more than 25% of NCC were caspase 3-positive in pharyngeal arches 1–3 and the dorsal frontonasal prominences at E9.5 and E10.5 (Figure 6 E– N and data not shown), compared with only occasional caspase 3-expressing cells in comparable sections from control litter- mates (P = 0.019; Figure 6 E–N and data not shown). Thus, this high incidence of cell death likely explains the less compact appearance of the ectomesenchyme of the pharyngeal arches and frontonasal processes. In contrast to the elevated cell death, analysis of cell proliferation in NCC-rich regions of the developing craniofacial region revealed no significant differenc- es between RockDN and control littermates at any stage examined (P = 0.433; Figure 6O and data not shown). These data suggest that the progressive reductions in NCC numbers observed in the craniofacial and pharyngeal regions at E9.5– E10.5, and the hypoplasia of NCC-derived structures observed in these regions later in development, were caused by loss of NCC by apoptosis. PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37685