RESEARCH ARTICLE 1467 Development 139, 1467-1475 (2012) doi:10.1242/dev.076083 © 2012. Published by The Company of Biologists Ltd INTRODUCTION The adult body of most animals shows stereotypic left-right (L-R) asymmetry. In the early stages of animal embryogenesis, the morphology is bilaterally symmetrical, but later the symmetry becomes broken along the L-R axis (Hirokawa et al., 2006; Raya and Belmonte, 2006). Left-sided expression of the nodal gene precedes the onset of morphological L-R asymmetry in chick and mouse embryos (Levin et al., 1995; Collignon et al., 1996; Lowe et al., 1996). Involvement of the nodal protein, a member of the transforming growth factor beta (TGF) superfamily, in polarization of the L-R axis is conserved among many vertebrates, including Xenopus and zebrafish (Burdine and Schier, 2000; Whitman and Mercola, 2001). The earliest event in the L-R determination process in mouse embryos is nodal flow, which is a leftward flow of fluid in the node. Cells of the node have monocilia, which are motile and generate a leftward flow of liquid that acts as a braking mechanism for acquisition of L-R symmetry in the mouse (Nonaka et al., 1998). The cilia tilt posteriorly and rotate, thereby generating a directed flow (Nonaka et al., 2005; Okada et al., 2005). The planar cell polarity (PCP) pathway and hydrodynamic forces are involved in this tilting of the nodal cilia (Guirao et al., 2010; Borovina et al., 2010). Through this system, the L-R symmetry of the embryo is broken using the polarity of the anterior-posterior (A-P) axis and a stereotypic direction of rotation of the motile monocilia. This leftward flow in a cavity within the embryo is also known to occur in other vertebrates (Okada et al., 2005). However, in Xenopus and zebrafish, some reports have indicated that specification of L-R asymmetry depends on a very early asymmetric signal, such as differential ion flux created by H + /K + -ATPase activity (Levin et al., 2002; Qiu et al., 2005; Kawakami et al., 2005). The morphology of ascidians, which are the closest relatives to vertebrates, shows L-R asymmetry in both the larval and adult stages (Hirano and Nishida, 2000; Boorman and Shimeld, 2002). In the tadpole larvae of Halocynthia roretzi, morphological L-R asymmetry is represented in two ways. First, the larval tail always bends towards the left side within the limited perivitelline space. Second, the brain structure is remarkably asymmetric, the brain vesicle and sensory pigment cells being located on the right side of the midline (Morokuma et al., 2002; Taniguchi and Nishida, 2004). It has been shown that ascidian genes nodal and Pitx, which encodes a transcription factor downstream of nodal, are expressed on the left side of the embryo at the neurula and initial tailbud stages, although the expression is restricted to the epidermis and is not evident in the mesoderm, unlike in vertebrates (Morokuma et al., 2002; Shimeld and Levin, 2006; Yoshida and Saiga, 2008). There is no cavity within ascidian embryos in which liquid flow is generated by ciliary movements. In the present study we first investigated the involvement of nodal signaling in the formation of morphological L-R asymmetry in Halocynthia roretzi, and then attempted to clarify the initial process through which bilateral symmetry is broken. Prior to nodal expression, neurulae rotate with stereotypic orientation along the A-P axis within the vitelline membrane and the rotation always stops when the left side of the neurula is oriented downwards. We term this phenomenon ‘neurula rotation’. We demonstrate that neurula rotation, which is likely to be driven by epidermal monocilia, specifies the future L-R axis and suggest that contact between the vitelline membrane and left-side epidermal cells triggers nodal expression in the left-side epidermis. Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043, Osaka, Japan. *Author for correspondence (knishide@bio.sci.osaka-u.ac.jp) Accepted 15 February 2012 SUMMARY Tadpole larvae of the ascidian Halocynthia roretzi show morphological left-right asymmetry. The tail invariably bends towards the left side within the vitelline membrane. The structure of the larval brain is remarkably asymmetric. nodal, a conserved gene that shows left-sided expression, is also expressed on the left side in H. roretzi but in the epidermis unlike in vertebrates. We show that nodal signaling at the late neurula stage is required for stereotypic morphological left-right asymmetry at later stages. We uncover a novel mechanism to break embryonic symmetry, in which rotation of whole embryos provides the initial cue for left- sided expression of nodal. Two hours prior to the onset of nodal expression, the neurula embryo rotates along the anterior- posterior axis in a counterclockwise direction when seen in posterior view, and then this rotation stops when the left side of the embryo is oriented downwards. It is likely that epidermis monocilia, which appear at the neurula rotation stage, generate the driving force for the rotation. When the embryo lies on the left side, protrusion of the neural fold physically prevents it from rotating further. Experiments in which neurula rotation is perturbed by various means, including centrifugation and sandwiching between glass, indicate that contact of the left epidermis with the vitelline membrane as a consequence of neurula rotation promotes nodal expression in the left epidermis. We suggest that chemical, and not mechanical, signals from the vitelline membrane promote nodal expression. Neurula rotation is also conserved in other ascidian species. KEY WORDS: Left-right asymmetry, nodal, Brain asymmetry, Neurula rotation, Ascidians Neurula rotation determines left-right asymmetry in ascidian tadpole larvae Kazuhiko Nishide*, Michio Mugitani, Gaku Kumano and Hiroki Nishida DEVELOPMENT