2935 RESEARCH ARTICLE INTRODUCTION Vertebrate muscles develop from somites, transient epithelial structures which, in response to environmental signals, dissociate to form the dermomyotome (DM) and sclerotome (Scl) (Christ and Ordahl, 1995). Although the DM contributes to most myotomal fibers (Christ et al., 1978; Cinnamon et al., 2006; Cinnamon et al., 1999; Gros et al., 2004; Huang and Christ, 2000; Kahane et al., 1998b; Kahane et al., 2002) and to mitotic muscle progenitors (Ben-Yair and Kalcheim, 2005; Gros et al., 2005; Kahane et al., 2001; Kassar-Duchosoy et al., 2005; Relaix et al., 2005), it is not the source of the myotomal founder cells. The earliest muscle progenitors, termed ‘myotomal pioneers’, arise along the medial portion of the epithelial somite (Kahane et al., 1998a), where many of them are already post-mitotic and express MyoD, Myf5 and desmin. Upon dissociation, these progenitors bend underneath the forming dorsomedial lip (DML) of the DM, become mesenchymal and engage in a typical directional pattern of migration towards the rostral pole of each somite. This process is then followed by a rostral-to-caudal (R-C) and medial-to-lateral (M-L) order of fiber differentiation (Kahane et al., 2002; Kalcheim et al., 1999). Whereas medial pioneers encompass the entire M-L extent of cervical and limb-level segments, at flank regions they are complemented laterally by a population of myoblasts emerging from the lateral epithelial somite. In addition to being the first skeletal muscle cells in the embryonic somite, a recent study showed that in the absence of medial pioneers, the whole myotome is mispatterned (Kahane et al., 2007). Hence, the significance of myotomal pioneers resides in their capacity to organize further somitic waves that contribute to the developing muscle. Despite our knowledge of their cellular dynamics, the molecular mechanisms directing their unique rostralward migration and differentiation are unknown. The Roundabout (Robo) receptor and its ligand Slit were first identified in Drosophila as guidance molecules for CNS axons (Kidd et al., 1998) and for muscle progenitors (Kidd et al., 1999; Kramer et al., 2001). Their vertebrate homologs play distinct roles across multiple systems (Brose and Tessier-Lavigne, 2000; Legg et al., 2008; Shiau et al., 2008; Wang et al., 2003). Robo receptors are single-pass transmembrane receptors. Robos 1-3 are mainly expressed in the nervous system (Morlot et al., 2007; Wong et al., 2001), whereas Robo 4 is specific to endothelium (Shibata et al., 2008). Slits 1-3 are multidomain leucine-rich repeat (LRR) molecules. Slit-Robo interactions are mediated by the second LRR domain of Slits and the two N-terminal Ig domains of Robos (Morlot et al., 2007). Robo members regulate Rho GTPases to affect cell motility and axonal guidance (Patel and Van Vactor, 2002). Whereas attraction is primarily mediated by Cdc42 and Rac, repulsion is executed through Rho GTPases, in particular RhoA (Dickson, 2001; Kozma et al., 1997). RhoA signals through Rock1 to enhance actomyosin contractility. It has been implicated in myosin activation and retraction of the back of the cell (Ridley, 2004; Wong et al., 2006), and has also been found to be active at the leading edge during migration (Kardash et al., 2010; Kurokawa et al., 2005; Pertz et al., 2006). The present study is the first to document that regulation of directional migration and differentiation of pioneer myoblasts is intrinsic to the somite. In search for somite-derived signals, we found that Robo2 is expressed by pioneer myoblasts already at the epithelial stage whereas Slit1 is present in the DM and is abundant in the caudal half of the dissociating Scl. Loss of Robo2 function impaired the caudorostral migration of pioneers as well as myofiber formation. Similar results were obtained when Scl-derived, but not DM-derived Slit1, was abrogated. Loss of RhoA activity phenocopied the above effects and activation of endogenous Rho partially rescued the normal phenotype in pioneers lacking Robo2 activity. In all cases, myogenic specification was unaffected. By Development 138, 2935-2945 (2011) doi:10.1242/dev.065714 © 2011. Published by The Company of Biologists Ltd Department of Medical Neurobiology, IMRIC and ELSC, Hebrew University-Hadassah Medical School, Jerusalem 91120–P.O.Box 12272, Israel. *Author for correspondence (kalcheim@cc.huji.ac.il) Accepted 27 April 2011 SUMMARY Pioneer myoblasts generate the first myotomal fibers and act as a scaffold to pattern further myotome development. From their origin in the medial epithelial somite, they dissociate and migrate towards the rostral edge of each somite, from which differentiation proceeds in both rostral-to-caudal and medial-to-lateral directions. The mechanisms underlying formation of this unique wave of pioneer myofibers remain unknown. We show that rostrocaudal or mediolateral somite inversions in avian embryos do not alter the original directions of pioneer myoblast migration and differentiation into fibers, demonstrating that regulation of pioneer patterning is somite-intrinsic. Furthermore, pioneer myoblasts express Robo2 downstream of MyoD and Myf5, whereas the dermomyotome and caudal sclerotome express Slit1. Loss of Robo2 or of sclerotome-derived Slit1 function perturbed both directional cell migration and fiber formation, and their effects were mediated through RhoA. Although myoblast specification was not affected, expression of the intermediate filament desmin was reduced. Hence, Slit1 and Robo2, via RhoA, act to pattern formation of the pioneer myotome through the regulation of cytoskeletal assembly. KEY WORDS: Avian embryo, Desmin, Dermomyotome, MyoD, Myf5, Myotome, Myofiber patterning, Rho GTPases, Somite Sclerotome-derived Slit1 drives directional migration and differentiation of Robo2-expressing pioneer myoblasts Osnat Halperin-Barlev and Chaya Kalcheim* DEVELOPMENT