by loss of the developmentally important transcription factor Six2 [29–31], although in this case associated with reduced proliferation in the craniofacial region, rather than increased cell death [30]. Nevertheless, the consequences of reduced proliferation and excessive cell death in a particular tissue are similar, with an overall reduction in cell numbers relative to normal controls. Thus, a reduction in the numbers of NCC within the developing midface appears to be the critical factor for the development of frontonasal dysplasia. Conclusions Rodent models of craniofacial abnormalities represent a valuable tool for studying, understanding and ultimately prevent- ing human malformations. For example, mutations in ALX3, whose mouse counterpart has been implicated in the development of the midface [27] also cause frontorhiny, a form of midfacial clefting in humans [32]. Our study suggests an important role for Rock and other regulators of the cytoskeleton in maintaining normal craniofacial morphogenesis. In humans, frontonasal dysplasia (OMIM: 136760) is considered to be a sporadic event, supporting the idea that craniofacial malformations have a complex aetiology. Although there are no reports of mutations in ROCK1 or ROCK2 in patients with craniofacial malformations, loss of both chromosome 18q and 2p (where ROCK1 and ROCK2 reside, respectively) result in a spectrum of abnormalities that include hypertelorism, palatal defects and micrognathia and cardiac (OMIM 218340 and 601808). Thus, haploinsufficiency for these genes might contribute to the phenotype of these patients. From our data, and a review of the current literature relating to frontonasal dysplasia and midface clefting resulting from muta- tions in a range of different genes, we suggest that the common link is a reduction in NCC in the developing frontonasal prominences and first pharyngeal arch, in many cases related to increased NCC apoptosis. The key roles that Rho kinase plays in regulating the cytoskeleton and maintaining cell-matrix interac- tions suggests that it may be a common downstream effector in this cell death process. Materials and Methods Ethics Statement Ethical approval of animal work carried out in this project has been authorised by the Newcastle University Ethics Committee and was covered by Project Licence PPL 60/3876 approved by the UK Home Office. Mice and embryos Rock dominant-negative (RockDN) mice [12] (BRC no. 01294) from RIKEN BioResource Center (Tsukuba, Japan) were inter- crossed with the Rosa 26R reporter line [33], ROSA-EYFP line [34], Wnt1-cre line [13] or the PGK-cre line [16]. Mice were maintained according to the Animals (Scientific Procedures) Act 1986, United Kingdom. Genotyping for RockDN positive mice was performed as described [12]. CD1 mice were obtained from Charles River. Stage matched mutant and control embryos were used for all experiments. Quantitative real-time PCR The relative quantities of the CAT gene cassette was measured by quantitative real time PCR. RNA was extracted from four E11.5 control and four mutant pharyngeal arches 1 and 2 using the Trizol reagent kit (Invitrogen) in duplicate. cDNA was produced using 1 mg of RNA with an initial DNase treatment step (Invitrogen DNase kit) followed by reverse transcription Rho Kinase and Neural Crest Cell Survival (Invitrogen SuperScript II RTase kit). Random 15-mers were used (Sigma). Quantitative real-time PCR was performed using a 7900ht fast real-time PCR system (Applied Biosystems) and the SYBR green JumpStart Taq ReadyMix kit (Sigma). All reactions were performed in triplicate. Two housekeeping genes were used for normalisation, b-actin and GAPDH, and were found to be stably expressed in this experimental setting. Relative quantities of gene expression were calculated using the DDCt method. Primer sequences were for b-actin, F: 59-GCTGGTCGTCGACAACG- GCTC-39, R: 59-CAAACATGATCTGGGTCATCTTTTC-39, for GAPDH F:59-GCTGGTCGTCGACAACGGCTC-39, R:59- CAAACATGATCTGGGTCATCTTTTC-39 and for CAT prim- ers see [12]. Head Measurements Heads were collected from E15.5 embryos, digital protographs were taken, and measurements were made using ImageJ software [35]. Histology and skeletal staining b-gal staining of RockDN;Rosa 26R embryos was performed as described previously [36]. For skeletal staining, the superficial muscle layers, eyes and internal organs were removed before fixing in Bouin’s solution. E18.5 embryos were dehydrated in 95% ethanol and washed in acetone before staining for cartilage in alcian blue for eight days. Bone was stained with alizarin red. E14.5 embryos were washed with 70% ethanol/0.1% NH4OH and cartilage stained in 0.05% alcian blue/5% acetic acid. Embryos were imaged in either glycerol or benzyl alcohol/ benzylbenzoate. Immunohistochemistry Immunolabelling was performed on PFA-fixed, paraffin-em- bedded sections using the Rock2 antibody (Santa Cruz Biotech- nology). Cryosections were labelled using antibodies raised against activated Rock1 (abcam), caspase 3 (Cell Signalling), vinculin (Sigma), paxillin (abcam), laminin (Sigma), GFP-FITC (Molecular Probes) and filamentous actin was stained with rhodamine- phalloidin (Sigma). Each experiment was repeated a minimum of three times and included appropriate controls. The mean percentage cell death and cell proliferation was calculated from three matched sections of the first pharyngeal arch from four control and four mutant E9.5 embryos. Statistical analysis Statistical analysis was carried out using SPSS (IBM). The one way ANOVA test and one sample t-test were used as appropriate. Supporting Information Figure S1 Craniofacial phenotype in RockDN;Wnt1-cre embryos. Neural crest cells and their derivatives are stained blue in each case. A,B) The frontonasal region is hypoplastic in RockDN embryos at E9.5. C–F) Clefting of the midface is obvious in RockDN embryos at E11.5 and E13.5, with wide separation of the lateral prominences (white lines in D,F). There is also marked hypertelorism (double arrows in E,F). G–J) In some mildly affected mutants, the facial clefting in RockDN mutants is apparent only as a midline cleft lip and a bifid nasal tip (white arrows in H,J). Hyperteleorism is still apparent in these more mildly affected embryos however (double headed arrows in G,H; arrows are same length in each case). I and J are magnified images of the PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e37685