INTRODUCTION RhoA GTPase and other Rho family proteins are regulators of actin cytoskeletal architecture and components of signal transduction cascades (reviewed by Hall, 1994; Van Aelst and D’Souza-Schorey, 1997). Genetic analysis of RhoA in Drosophila indicated its obligatory role during early developmental processes including gastrulation, head involution, dorsal closure, segmentation and the generation of tissue polarity (Barrett et al., 1997; Magie et al., 1999; Strutt et al., 1997). RhoA is also important for vertebrate morphogenesis with respect to head formation in Xenopus embryos (Wunnenberg-Stapleton et al., 1999). These observations suggest that RhoA plays an important role in regulating embryonic morphogenetic events through its effects on actin cytoskeleton reorganization which controls cell movement and differentiation. The mammalian Rho-associated kinase family (hereafter referred to as Rho kinases) is comprised of Rho kinase/ROKα/ROCK2 and p160ROCK/ROCK1/ROKβ (Ishizaki et al., 1996; Matsui et al., 1996). These serine/ threonine protein kinases, identified as direct effectors of RhoA, are implicated in the regulation of cytoskeletal organization (Amano et al., 1997; Leung et al., 1996). They regulate myosin light chain (MLC) phosphorylation directly by phosphorylating MLC and by inactivating myosin phosphatase (Kimura et al., 1996). Phosphorylation of MLC induces smooth muscle contraction, formation of stress fibers and focal adhesions. Rho kinases also phosphorylate LIM kinase (Maekawa et al., 1999), ezrin/radixin/moesin proteins and adducin (Fukata et al., 1999), thereby regulating actin dynamics, cell adhesion, membrane ruffling and cell motility. Although there is considerable evidence that Rho kinases mediate many in vivo functions of RhoA, their roles in regulating embryonic morphogenesis remain unknown. We asked whether Rho kinases play a morphogenetic role during the elaboration of embryonic tissues. A pyridine derivative, Y27632, was discovered to specifically inhibit the activity of Rho kinases (Uehata et al., 1997). Subsequently, this compound has been widely used to evaluate the roles of Rho kinases in a variety of systems (Sawada et al., 2000; Itoh et al., 1999; Kuwahara et al., 1999). Our studies demonstrate that Y27632 is a potent teratogen that blocked the migration of precardiac mesoderm and cardiac tube fusion in cultured chick and mouse embryos. Rho kinase inhibition by this compound also blocked brain and somite formation, and induced laterality 2953 Development 128, 2953-2962 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV3439 Rho-associated kinases (Rho kinases), which are downstream effectors of RhoA GTPase, regulate diverse cellular functions including actin cytoskeletal organization. We have demonstrated that Rho kinases also direct the early stages of chick and mouse embryonic morphogenesis. We observed that Rho kinase transcripts were enriched in cardiac mesoderm, lateral plate mesoderm and the neural plate. Treatment of neurulating embryos with Y27632, a specific inhibitor of Rho kinases, blocked migration and fusion of the bilateral heart primordia, formation of the brain and neural tube, caudalward movement of Hensen’s node, and establishment of normal left-right asymmetry. Moreover, Y27632 induced precocious expression of cardiac α-actin, an early marker of cardiomyocyte differentiation, coincident with the upregulated expression of serum response factor and GATA4. In addition, specific antisense oligonucleotides significantly diminished Rho kinase mRNA levels and replicated many of the teratologies induced by Y27632. Thus, our study reveals new biological functions for Rho kinases in regulating major morphogenetic events during early chick and mouse development. Key words: Rho kinase, Y27632, Cardia bifida, Cardiomyocyte differentiation, Chick SUMMARY Rho kinases play an obligatory role in vertebrate embryonic organogenesis Lei Wei 1,2,4, *, Wilmer Roberts 1, *, Lu Wang 2,4 , Miho Yamada 1 , Shuxing Zhang 1 , Zhiyong Zhao 5 , Scott A. Rivkees 5 , Robert J. Schwartz 1,2,3,4,‡ and Kyoko Imanaka-Yoshida 6 1 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA 2 Cardiovascular Sciences Section, Baylor College of Medicine, Houston, TX 77030, USA 3 Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX 77030, USA 4 Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA 5 Yale Child Health Research Center, Yale University School of Medicine, New Haven, CN, USA 6 Department of Pathology, Mie University, School of Medicine, Tsu, Mie, Japan *These authors contributed equally to this work ‡ Author for correspondence (e-mail: schwartz@bcm.tmc.edu) Accepted 12 May 2001