embryo, together with the normal development of the majority of the embryo in the single knockouts for Rock1 or Rock2 [7–9] suggests there may be some degree of functional redundancy. Further studies have implicated Rock in delamination of NCC in quail embryos [10] and in NCC migration in Xenopus and zebrafish [11] although this has not been demonstrated in mammalian embryos. In this study we utilised transgenic mice that allowed cre- mediated tissue-specific expression of dominant-negative (DN) Rock (RockDN); [12] to explore its role in NCC using Wnt1-cre [13] mice. In the RockDN mice, the Rho kinase RB/BH (TT) fragment [14] is expressed under the control of the cytomegalovirus promoter specifically blocking the function of both Rock1 and Rock2 isoforms. The point mutations introduced into the Rock RB/BH (TT) interact with endogenous Rock and abolish Rho binding activity, but have no effect on the activity of related kinases [15]. Thus, expression of Rock RB/BH (TT) can be used to specifically block Rho kinase function in vivo. Use of Cre-loxP technology means that the RockDN protein can be expressed in the cell type of choice. Thus the use of these mice can bypass any early lethality that might result from the loss of both Rock isoforms during early development, but also overcome potential functional redundancy. The RockDN;Wnt1-cre embryos developed severe NCC-related defects affecting the craniofacial region including hypoplasia of structures derived from the frontonasal prominence and first pharyngeal arch. Results Specific knock-down of Rock in NCC causes severe craniofacial malformation The role of Rock isoforms in migrating NCC, and their derivatives, was investigated using mice expressing a RockDN construct [12] under the control of the Wnt1-cre driver [13], Figure 1A. The RockDN protein RB/PH (TT) [12,14] is unable to bind to Rho and specifically blocks Rock function [15]. Female mice heterozygous for the RockDN construct and male mice heterozygous for the Wnt1-cre construct were mated (Figure 1A), with resulting litters containing embryos of the genotypes: RockDN+;Wnt1-cre+ (hereafter referred to as RockDN), RockDN+;Wnt1-cre2, RockDN2;Wnt1-cre+ and RockDN2;Wnt1-cre2; the latter three genotypes were phenotypically indistinguishable and acted as controls. To assess the efficiency of cre recombination we evaluated the levels of the CAT gene cassette transcripts in pharyngeal arches 1 and 2 of embryonic heads taken from RockDN+;Wnt1-cre+ mutants and RockDN+;Wnt1-cre2 controls at E11.5, as this tissue was rich in NCC (Figure S1 C,D). Quantitative real time PCR revealed a significant 13 fold reduction in levels of the CAT cassette in the mutant heads (P = 0.006), compared with transgenic embryos where cre was not present; a reduction to almost undetectable levels (Figure 1B). Efficient removal of the CAT cassette is consistent with expression of dominant-negative Rock in the majority of NCC. Notably, expression of RockDN in all cells of the embryo, by intercrossing RockDN mice with PGK-cre mice [16] suggested that ubiquitously blocking Rock function results in embryonic death before E9.5 (data not shown). We reviewed craniofacial development in the RockDN;Wnt1-cre embryos. By late gestation, RockDN embryos showed craniofacial malformations affecting the fronto-medial aspect of the head, although these varied in severity. The predominant feature was mid-facial clefting in combination with hypoplasia of the frontonasal region (Figure 2). Those with the severest phenotype (47/54, from E11.5 to E18.5) had marked reductions of the Rho Kinase and Neural Crest Cell Survival Figure 1. Conditional expression of RockDN in NCC. A) Strategy for expressing RockDN construct in NCC. B) Quantitative real time PCR for the CAT gene cassette, using RNA extracted from pharyngeal arches 1 and 2, from E11.5 mutant RockDN+;Wnt1-cre+ and control embryos. There is a statistically significant (P,0.05 *), 13 fold decrease, in the expression of the CAT box in the mutant sample, as calculated using the one-way Anova test. doi:10.1371/journal.pone.0037685.g001 frontonasal region, maxilla and mandible (Figure 2 A,B). Facial clefting was apparent in 50/54 embryos (Figure 2G and Figure S1 C–H), although in three cases this was apparent externally only as a bifid nose (Figure S1J). At E15.5, the distance between the eyes and the width of the frontonasal processes was larger in mutant embryos compared to control littermates (P = 4.20261026, Figure 2I and data not shown), consistent with hypertelorism and broader skulls in the mutants. There was no significant difference between the heights of the heads (data not shown). The snout was also upturned in the RockDN embryos at E15.5, shown by a decreased angle between the snout and the forehead when compared to control littermates (P = 3.20861028; Figure 2 E,H,J). There was no significant difference between the two groups for any measurement at E11.5, despite mid-facial clefting being apparent at that stage (data not shown). At E17.5–E18.5, two out of four RockDN embryos had failed to form eyelids (Figure 2B), as seen in mice for either Rock1 or Rock2 [7–9]. This was not unexpected, as the eyelids form from NCC-derived mesenchyme [17]. Some abnormalities may have occurred as secondary events. The hypertelorism observed throughout gestation is likely to be secondary to the mid-facial clefting as this produces a broader skull. In addition, two out of four RockDN embryos examined at E17.5–E18.5 had exencephaly (Figure 2B). As all embryos collected earlier in gestation had closed cranial neural tubes, this does not suggest a primary involvement of Rock in neural tube closure, but rather suggests secondary opening, likely as a consequence of a defect in the NCC-derived calvarias bones. Cartilage staining of E14.5 embryos revealed that the nasal capsular cartilage is missing and the Meckel’s cartilage appears shortened in the RockDN embryos (Figure 3 A,B). Bone and cartilage staining of more mildly affected E18.5 embryos demonstrated that the majority of the cranium was well formed although there were subtle abnormalities in some NCC-derived bones (Figure 3 C–F); specifically there was mis-shaping and hypoplasia of the basisphenoid bone (Figure 3 E,F). The bones of the nasal septum and the maxillary bones failed to meet in the midline (double arrow in Figure 3F) and the hyoid bone was hypoplastic (red arrow in Figure 3 C,D). Thus, expression of the RockDN construct in NCC resulted in defects in the craniofacial structures derived from the frontonasal prominence and the first pharyngeal arches. Severe abnormalities of the outflow region of the heart were also observed in all RockDN;Wnt1-cre embryos by E11.5; these are described elsewhere (Phillips et al., manuscript in preparation). PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37685