Arginylation-Dependent Neural Crest Cell Migration Is Essential for Mouse Development Satoshi Kurosaka1, N. Adrian Leu1, Fangliang Zhang1, Ralph Bunte2, Sougata Saha1, Junling Wang 1 , Caiying Guo 3 , Wei He 4 , Anna Kashina1* 1 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 University Laboratory Animal Resources, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3 Janelia Farm, Ashburn, Virginia, United States of America, 4 Gene Targeting and Transgenic Facility, University of Connecticut Health Center, Farmington, Connecticut, United States of America Abstract Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration–dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1- Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2–3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis. Citation: Kurosaka S, Leu NA, Zhang F, Bunte R, Saha S, et al. (2010) Arginylation-Dependent Neural Crest Cell Migration Is Essential for Mouse Development. PLoS Genet 6(3): e1000878. doi:10.1371/journal.pgen.1000878 Editor: Marianne Bronner-Fraser, California Institute of Technology, United States of America Received January 23, 2009; Accepted February 9, 2010; Published March 12, 2010 Copyright: 2010 Kurosaka 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: This work was supported by NIH grant 5R01HL084419 and awards from W. W. Smith Charitable Trust and Philip Morris Research Management Group to AK. 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: akashina@vet.upenn.edu Introduction Coordinated cell migration during development is crucial for tissue and organ morphogenesis from early gastrulation to adulthood. The largest cell populations that are capable of long- range migration at different developmental stages originate from the neural crest lineage. Neural crest cells are of mesenchymal morphology and migrate from the trunk into different areas of the developing embryo. These cells express a distinct subset of markers, including Wnt1 and others [1–8], at or before the onset of migration. Recent studies of protein arginylation demonstrated an essential role of this poorly understood posttranslational modification in mammalian embryogenesis and suggested that arginylation is a previously unknown major signaling mechanism that regulates multiple physiological pathways. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe cardiovascular defects, including abnormal heart septation, underdeveloped myocardium, and impaired angiogenesis [9]. Remarkably, all these phenotypes resemble the phenotypes seen in the mouse models with knockouts of various genes implicated in cell migration, leading to the hypothesis that the mechanisms underlying cell migration may be the primary targets for regulation by arginylation (see [10] for review). It has been found that a large number of proteins in vivo are arginylated [11–23], including a prominent subset of cytoskeletal targets that play direct mechanistic roles in cell migration. Arginylation of beta actin in cultured fibroblasts regulates lamella formation and the structure of the cell leading edge [24]. Other proteins involved in cell adhesion and migration, such as talin, spectrin, filamin, myosin, etc. are also arginylated in different mouse tissues [23]. All these data suggest that arginylation may be a general mechanism of the regulation of cell movement in different physiological events, however the role of arginylation in directional cell migration in culture and during embryonic development has never been studied before. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced and analyzed a conditional knockout mouse model with Ate1 deletion driven by neural crest- marking Wnt1 promoter (Wnt1-Ate1 mouse line). These mice exhibit perinatal lethality and severe morphogenesis defects resulting from poorly developed neural crest-derived structures, suggesting that Ate1 indeed regulates the migration of neural crest cells that give rise to these structures in embryogenesis. Studies of cell migration patterns in embryos and in culture show that Ate1 knockout results in an overall delay in the migration, likely regulated at the intracellular level, and that Ate1 knockout cells co- PLoS Genetics | www.plosgenetics.org 1 March 2010 | Volume 6 | Issue 3 | e1000878