Bill Chaudhry1 Abstract Neural Crest Cell Survival Is Dependent on Rho Kinase and Is Required for Development of the Mid Face in Mouse Embryos Helen M. Phillips1, Tania Papoutsi1, Helena Soenen1, Patricia Ybot-Gonzalez2, Deborah J. Henderson1*, 1 Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Departamento de Pediatria, Hospital Infantil Virgen del Rocio, Seville, Spain Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell- matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia. Citation: Phillips HM, Papoutsi T, Soenen H, Ybot-Gonzalez P, Henderson DJ, et al. (2012) Neural Crest Cell Survival Is Dependent on Rho Kinase and Is Required for Development of the Mid Face in Mouse Embryos. PLoS ONE 7(5): e37685. doi:10.1371/journal.pone.0037685 Editor: Patrick Callaerts, VIB & Katholieke Universiteit Leuven, Belgium Received August 10, 2011; Accepted April 24, 2012; Published May 21, 2012 Copyright: 2012 Phillips et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding provided by British Heart Foundation (grants BS/05/003 and RG/07/007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: deborah.henderson@ncl.ac.uk Introduction The vertebrate face is largely formed from neural crest-derived mesenchyme, covered in ectoderm, with a small endodermal contribution (reviewed in [1]). Although the surface ectoderm and pharyngeal endoderm have important signalling roles in the patterning of the forming craniofacial region, it is the cranial neural crest cells (NCC) that form most of the bone and cartilage within the head, the teeth and the cranial ganglia. The rostral NCC make extensive contributions to the frontonasal skeleton and the calvaria of the skull, whereas the more caudal cranial NCC migrate into the pharyngeal arches where they form the mandible and maxilla, the middle ear and the hyoid bone (reviewed in [2]]. The forming craniofacial region is initially defined by a series of swellings or prominences. The medial frontonasal prominence forms the forehead, middle of the nose, philtrum of the upper lip and primary palate. Three paired prominences, derived from the first pharyngeal arch, form the lateral regions of the mid and lower face, associated with the maxilla, the mandible and the secondary palate [1]. In order for these initially featureless prominences to give rise to their complex derivatives, processes of growth, expansion and fusion have to be orchestrated. Interactions between NCC, the surface ectoderm, mesoderm and endoderm are all crucial for the normal development of the developing face (reviewed in [3]]. Studies in animal models, particularly mouse and chick, have highlighted the importance of specific signalling cascades, including the sonic hedgehog, fibroblast growth factor and bone morphogenetic protein pathways, in the development of the craniofacial region [1,2]. The two Rho kinase isoforms (Rock 1 and 2) are highly conserved serine/threonine kinases that play essential roles in fundamental cellular processes such as contraction, adhesion, migration, apoptosis and proliferation. Almost all of the analyses of Rock function have been carried out in cell culture. In these in vitro experiments, within the two-dimensional environment of the culture dish, Rock is found at the rear of migrating cells and regulates stress fibre and focal adhesion assembly, facilitating cell body and trailing edge retraction [4,5]. Rock1 and Rock2 show similar expression patterns in the developing embryo [6] and both the Rock1 and Rock2 single knockout mice exhibit omphalocele and open eyelids at birth [7,8], whereas only the Rock2 mice develop defects in development of the placenta and intra-uterine growth retardation [7], suggesting that the two genes work co- operatively in the development of these structures and have at least some non-redundant functions. The data showing that Rock1/ Rock2 double heterozygotes have the same embryonic phenotype as do the single homozygotes for Rock1 or Rock2 on some genetic backgrounds [7–9], suggests that the overall level of Rock protein (Rock1 or Rock2) may be critical. Rock1/Rock2 double mice have not been described in the literature. However, chemical inhibition of Rock (using Y27632) in neurulating chicken and mouse embryos results in a variety of malformations, including brain abnormalities and neural tube defects [6]. The broad and overlapping expression of Rock1 and Rock2 in the developing PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37685