RESEARCH ARTICLE Migrating cells mediate long-range WNT signaling Olivier Serralbo and Christophe Marcelle* ABSTRACT In amniotes, it is widely accepted that WNTs secreted by the dorsal neural tube form a concentration gradient that regulates early somite patterning and myotome organization. Here we demonstrate in the chicken embryo that WNT protein is not secreted to act at a distance, but rather loaded onto migrating neural crest cells that deliver it to somites. Inhibiting neural crest migration or ablating their population has a profound impact on the WNT response in somites. Furthermore, we show that a central player in the efficient delivery of WNT to somites is the heparan sulfate proteoglycan GPC4, expressed by neural crest. Together, our data describe a novel mode of signaling whereby WNT proteins hitch a ride on migratory neural crest cells to pattern the somites at a distance from its source. KEY WORDS: Somite, WNT, Dermomyotome, Chick embryo INTRODUCTION Somites are blocks of cells sequentially generated through the epithelialization of the segmental plate (Christ and Ordahl, 1995; Scaal and Christ, 2004). Shortly after their formation, somites differentiate along their dorsoventral axis into a ventral mesenchyme, the sclerotome and a dorsal epithelium termed the dermomyotome. The dermomyotome is then patterned along its mediolateral axis into a medial compartment termed the dorsomedial lip (DML), a central dermomyotome and a ventrolateral lip (VLL), each with distinct function and fate. The DML is the most extensively studied dermomyotome subdomain. It plays a crucial role during the initial stage of muscle morphogenesis, during which the primitive muscle (the primary myotome) expands solely from the generation of muscle cells (myocytes) originating from the DML (Denetclaw et al., 2001; Gros et al., 2004; Kahane et al., 1998; Venters and Ordahl, 2002). This production of myocytes at the DML is dependent upon the transient activation of NOTCH signaling in selected epithelial progenitors present in this structure (Rios et al., 2011). Finally, the DML also plays a crucial role as a myotome-organizing center, as it expresses a secreted factor, WNT11, which acts as a directional cue for the elongation of early myocytes in the anteroposterior axis of the chick embryo. WNT11 mediates this effect through the evolutionarily conserved planar cell polarity (PCP) pathway (Gros et al., 2009). The signals crucial for DML formation have been identified. Using WNT11 as a specific molecular marker of the DML, it was shown in the chick embryo that its expression is dependent upon WNT1 and/or WNT3a expressed by the dorsal neural tube, which trigger WNT11 expression through a WNT/β-catenin-dependent pathway (Gros et al., 2009; Marcelle et al., 1997). These results were confirmed and extended in mouse, where it was demonstrated that WNT1 and WNT3a exert their inducing activity on Wnt11 redundantly (Ikeya and Takada, 1998). In chick and mouse embryos, the distance between the WNT1/3a source and the DML is considerable (∼120 μm and ∼90 μm, respectively; Fig. 1A-E,K-O). How WNT can act at such a distance from its source to pattern the medial somite compartment is unknown. A widely accepted view in the field is that WNTs form a concentration gradient from the dorsal neural tube that acts to pattern the somites at a distance (Capdevila et al., 1998; Fan et al., 1997; Marcelle et al., 1997; Munsterberg et al., 1995). However, the existence of this gradient has never been demonstrated experimentally. Importantly, WNT proteins are highly hydrophobic because of post-translational modifications by lipid adducts on highly conserved amino acid residues (Port and Basler, 2010; Takada et al., 2006; Willert et al., 2003) and as such are notoriously poorly mobile. These modifications attach WNTs to the lipid bilayers of membranes, restricting them to the secreting cells. In a number of cellular contexts, however, WNTs are known to act at a considerable distance from their source. This is accomplished through several molecular and cellular mechanisms. Heparan sulfate proteoglycans (HSPGs) of the glypican family, such as Dally and Dally-like protein (Dlp), allow the transfer of WNT from cell to cell, acting as a co-receptor for the ligand, thus allowing the formation of a gradient of WNT activity at a distance from the secreting cells (Yan and Lin, 2009). WNTs can also travel on membranous particles, called argosomes (Panáková et al., 2005), or within exosome-like vesicles (Korkut et al., 2009). Finally, although not formally demonstrated for WNTs, it is also possible that WNTs are transported at a distance similarly to Sonic hedgehog and the TGFβ family member Decapentaplegic, in which the receiving cells grow long cytoplasmic extensions that directly contact morphogen- secreting cells (Hsiung et al., 2005; Sanders et al., 2013). Here we investigated the mode of signaling of WNTs from the dorsal neural tube to the DML, and uncovered a novel mechanism for long-range WNT signaling, whereby WNT expressed in the dorsal neural tube is loaded onto migrating cells (the neural crest cells) that physically transport the signal to the receiving cells of the DML. We also demonstrate the central role that the Dally-like molecule GPC4 plays in the transfer of the WNT signal from the presenting to the receiving cells. RESULTS WNT1 decorates the cell surface of migrating neural crest cells Epithelial cells of the DML display long filopodia at their basal end that extend mediodorsally towards the dorsal neural tube (Rios et al., 2010, 2012). Although they may participate in the capture of signals from the environment, their length (∼9-12 μm; our unpublished observations) is insufficient to reach the source of WNT in the roof plate. We therefore investigated alternative ways of WNT transfer from the dorsal neural tube to the DML. The expression patterns of WNT1 and WNT3a have been extensively characterized during mouse and chicken embryonic Received 6 January 2014; Accepted 13 March 2014 EMBL Australia, Australian Regenerative Medicine Institute (ARMI), Monash University, Building 75, Clayton, Victoria 3800, Australia. *Author for correspondence (christophe.marcelle@monash.edu) 2057 © 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2057-2063 doi:10.1242/dev.107656 DEVELOPMENT