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INTRODUCTIONThe embryonic myotome is the source of all epaxial and hypaxialskeletal muscles. The mechanisms underlying myotomedevelopment were recently reevaluated (Brent and Tabin, 2002;Buckingham, 2001; Hollway and Currie, 2003; Kalcheim and Ben-Yair, 2005). Studies in avian embryos showed that the dorsomediallip (DML) of the dermomyotome (DM) and, later, the ventrolaterallip (VLL) contribute to myotome growth in medial and lateralorientations, respectively (Denetclaw et al., 2001; Denetclaw et al.,1997; Denetclaw and Ordahl, 2000; Ordahl et al., 2001). Our studiesdistinguished at least two separable waves that account for theformation of the postmitotic myotome. A first wave of pioneermyoblasts constitutes the medial region of the epithelial somite withcells expressing Myod and Myf5. Upon sclerotome dissociation,pioneers delaminate, migrate towards the rostral somitic domain,and, from this region, generate myofibers in rostrocaudal andmediolateral directions (Kahane et al., 1998b; Kahane et al., 2002).Following the establishment of this primary structure, the advent ofa second wave of myoblasts was shown, which emanates from allfour lips of the DM, the contribution of the DML and VLL beingrestricted to the epaxial and hypaxial domains, respectively, and thatof the rostral and caudal lips spreading along the entire mediolateralmyotome. This unique mechanism leads to an overall intercalatory

mode of myotome expansion (Cinnamon et al., 2001; Cinnamon etal., 1999; Kahane et al., 1998a; Kahane et al., 2002). Thecontributions of the four DM lips were further confirmed by quail-chick analysis (Huang and Christ, 2000) and by GFP electroporation(Gros et al., 2004).

Subsequent growth of the myotome occurs by addition of cellsthat remain transiently proliferative. We reported that the firstmitotically active muscle progenitors, termed the ‘third wave’,originate in the rostral and caudal lips of the DM (Kahane et al.,2001). Somewhat later, the central DM sheet dissociates and itsprogeny, composed of at least bipotent cells, colonizes both thesubectodermal space to generate dermis, and the myotome to giverise to the majority of mitotic muscle progenitors, a population thatmaintains the expression of DM markers, such as PAX3, PAX7 andFREK (Ben-Yair and Kalcheim, 2005; Ben-Yair et al., 2003). Thefinal fate of these progenitors is varied. Most are likely to withdrawfrom the cell cycle at progressive stages and undergo myogenesis,some may also contribute to muscle fibroblasts, endothelial cells andmuscle satellite cells (Gros et al., 2005; Kassar-Duchossoy et al.,2005; Relaix et al., 2005; Scaal and Christ, 2004). Altogether, anemerging fate map suggests that the entire DM epithelium hasmyogenic potential that is, nevertheless, regionally biased to theimmediate production of myofibers and/or of mitotic muscleprogenitors.

The existence of these discrete somitic microenvironmentsendowed with different and multiple fates raises fundamentalquestions about the mechanisms of lineage segregation in thevarious subdomains. Based on these findings, a functional analysisof genes expressed in somites can now be undertaken in the

Differential effects of N-cadherin-mediated adhesion on thedevelopment of myotomal wavesYuval Cinnamon, Raz Ben-Yair and Chaya Kalcheim*

Myotomal fibers form by a first wave of pioneer myoblasts from the medial epithelial somite, and by a second wave from all fourlips of the dermomyotome. Then, a third wave of mitotic progenitors colonizes the myotome, initially stemming from the extremelips and, later, from the central dermomyotome sheet. In vitro studies have suggested that N-cadherin plays a role in myogenesis,but its role in vivo remains poorly understood. We find that during the growth phase of the dermomyotome sheet, when theorientation of mitotic spindles is parallel to the mediolateral extent of the epithelium, N-cadherin protein is inherited by bothdaughter cells. Prior to dermomyotome dissociation into dermis and muscle progenitors, when mitoses become perpendicularlyoriented, N-cadherin remains associated only with the apical cell located in apposition to the myotome, generating molecularasymmetry between basal and apical progeny. Local gene missexpression confirms that N-cadherin-mediated adhesion is sufficientto promote myotome colonization, whereas its absence drives cells towards the subectodermal domain, hence coupling theasymmetric distribution of N-cadherin to a shift in mitotic orientation and to fate segregation. Site-directed electroporation toadditional, discrete somite regions, further reveals that N-cadherin-mediated adhesion is necessary for maintaining the epithelialconfiguration of all dermomyotome domains while promoting the onset of Myod transcription and the translocation into themyotome of myofibers and/or of Pax-positive progenitors. By contrast, N-cadherin has no effect on migration or differentiation ofthe first wave of myotomal pioneers. Altogether, we show for the first time that the asymmetric localization of N-cadherin duringmitosis indirectly influences fate segregation by differentially driving the allocation of progenitors to muscle versus dermalprimordia, that the adhesive domain of N-cadherin maintains the integrity of the dermomyotome epithelium, which is necessaryfor myogenic specification, and that different molecular mechanisms underlie the establishment of pioneer and later myotomalwaves.

KEY WORDS: Adherens junctions, Asymmetric cell division, Avian embryo, Cell adhesion, Dermis, Dermomyotome, Desmin, Epithelial tomesenchymal transition, Muscle, Myod, Myf5, Pax3, Pax7, Somite

Development 133, 1101-1112 (2006) doi:10.1242/dev.02291

Department of Anatomy and Cell Biology, Hebrew University-Hadassah MedicalSchool, PO Box 12272, Jerusalem 91120, Israel.

*Author for correspondence (e-mail: kalcheim@nn-shum.cc.huji.ac.il)

Accepted 18 January 2006

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perspective of a clearer cellular picture underlying morphogenesis.In the present study, we investigate the precise expression and roleof N-cadherin in each of the described waves of somitic myogenesis.

N-cadherin belongs to a family of Ca2+-dependent cell adhesionmolecules (Hatta et al., 1988; Hatta et al., 1987; Hatta and Takeichi,1986; Volk and Geiger, 1984; Volk and Geiger, 1986a; Volk andGeiger, 1986b) and is crucial for a diversity of steps duringembryonic development (Gumbiner, 2000; Nelson and Nusse,2004). It is characterized by five extracellular cadherin-bindingdomains that are separated by Ca2+-binding pockets, atransmembrane domain and an intracellular �-catenin-bindingdomain (Tepass et al., 2000). Among numerous other systems, N-cadherin is also dynamically expressed in the paraxial mesoderm(Duband et al., 1987; Duband et al., 1988). In vitro studiesimplicated N-cadherin in the regulation of the specification anddifferentiation of muscle progenitors (George-Weinstein et al., 1997;Goichberg and Geiger, 1998; Holt et al., 1994; Knudsen et al., 1990),but knowledge of its role(s) in vivo is still very limited. A pivotal rolefor N-cadherin in somite morphogenesis was demonstrated(Horikawa et al., 1999; Linask et al., 1998; Radice et al., 1997).However, this early requirement during somite formation, and thepremature death of embryos carrying a mutation in N-cadherin,precluded phenotypic analysis at muscle-forming stages, thus,genetic evidence awaits the analysis of conditional knockouts. Inavians, adenovirus-mediated missexpression of N-cadherin mutantsrevealed a role in myofiber arrangement, yet this technique allowedanalysis of only an early subset of myofibers (Horikawa andTakeichi, 2001). In zebrafish, an interplay between N- and M-cadherins was reported to control the migration of slow muscle cellsfrom their paranotochordal region of origin to the most lateralsurface of the myotome (Cortes et al., 2003).

In a previous study, we determined that the dual generation ofdermal and mitotic muscle progenitors from the central DM isassociated with a sharp change in the plane of cell division from theyoung epithelium, in which symmetrical divisions occur parallel tothe mediolateral plane of the DM, to the dissociating DM, in whichcell divisions become mostly perpendicular with one daughter cellfacing the dermis and the other positioned adjacent to the myotome(Ben-Yair and Kalcheim, 2005). Here, we report that during thegrowth phase of the DM sheet, when the orientation of mitoticspindles is parallel to the mediolateral extent of the epithelium, N-cadherin is inherited by both daughter cells. Prior to DM dissociationinto dermis and muscle progenitors, when mitoses becomeperpendicularly oriented, N-cadherin remains associated with onlythe apical cell located in apposition to the myotome, and is absentfrom the basally oriented cell, thus generating molecular asymmetrybetween basal and apical progeny. In line with this differentialsegregation, local gene misexpression confirms that N-cadherin-mediated adhesion is sufficient for promoting myotomecolonization, whereas interfering with the above processprematurely drives the cells towards the dermal domain with noPAX-positive progenitors colonizing the myotome. Hence, N-cadherin-mediated adhesion is both necessary and sufficient for thedifferential allocation of DM progenitors into the myotome. Notably,cells overexpressing N-cadherin that translocate into the myotomedifferentiate into myofibers, rather than remaining as mitoticallyactive PAX+ progenitors, as has been reported for their normalcounterparts, suggesting that N-cadherin is also involved in musclespecification. Likewise, interference with the activity of N-cadherin-mediated cell adhesion in the bordering lips (DML, rostral andcaudal) caused the rapid dissociation of the DM epithelium.Consequently, no cells entered the myotome and no fibers

differentiated. Notably, in the rostral and caudal lips of the DM, earlyelectroporation prevented the onset of Myod transcription andmyofiber generation, further implying that N-cadherin-mediatedadhesion is required for proper myogenic specification. In contrastto DM lip-derived fibers, differentiation of the pioneer fibers was notaffected. This finding provides the first molecular evidence that thepioneer population is unique and distinct from the later myofibersderiving from the DM and, in particular, from the DML.

MATERIALS AND METHODSEmbryosFertile quail (Coturnix coturnix Japonica) eggs from commercial sourceswere used.

Expression vectors and electroporationFour different expression vectors were employed: pCAGGS-AFP, whichserved as control (Momose et al., 1999); full-length chicken N-cadherin; adominant-negative version of N-cadherin lacking part of the extracellulardomain (cN390�); and N-cadherin lacking its intracellular �-catenin-binding domain (CBR–) (Fujimori and Takeichi, 1993; Nakagawa andTakeichi, 1998). The latter three were subcloned into the pCAGGS vectorand fused in frame to a GFP-encoding sequence.

Electroporations were performed under a dissecting microscope. DNA (4�g/�l) was microinjected into the center of flank-level epithelial ordissociating somites at specific stages as depicted in the figure legends. Fivedifferent types of electroporation were performed: to the medial epithelialsomite, to the DM sheet, to the DML and to the rostral and caudal lips (Fig.1). To this end, tungsten electrodes were mounted on two separatemicromanipulators and placed to reach each somitic domain as described inFig. 1. The direction of the electric field is indicated by the black arrow. Afour parameter PulseAgile square wave electroporator (PA-4000, Cyto PulseSciences) was used to deliver three groups of sequential pulses as follows:3�30 V, 20 mseconds each; 1�38 V, 5 mseconds; 3�30 V, 20 msecondseach.

The number of embryos showing a given phenotype out of the totalnumber of transfected embryos for each treatment is presented in thecorresponding figure legends.

Tissue processing, immunocytochemistry and in situ hybridizationEmbryos were fixed with 4% formaldehyde in PBS, embedded in paraffinwax and sectioned at 8 �m. Immunostaining for desmin was as described(Kahane et al., 2001). Rabbit anti-GFP (Molecular Probes) was used at adilution of 1:500, alone, in combination with desmin immunolabeling, orwith in situ hybridization for Myod, Alx4 or Pax7 (Cinnamon et al., 2001).Monoclonal anti-N-cadherin antibodies were from Zymed and were used ata 1:100 dilution in PBS containing 5% fetal calf serum and 0.1% Triton X-100. PAX7 immunolabeling, staining of centrosomes with �-tubulinantibodies and Hoechst nuclear staining were as described by Ben-Yair andKalcheim (Ben-Yair and Kalcheim, 2005). Whole-mount embryopreparations and sections were photographed using a DP70 (Olympus)cooled CCD digital camera mounted on a BX51 microscope (Olympus).

RESULTSExpression of N-cadherin at different stages ofmyotome developmentEpithelial somites and the pioneer wave of myotomeformationThe progenitors of the first wave arise along the medial surface ofepithelial somites. In embryos aged 22-25ss, these progenitorsexpress Myod and Myf5 and reveal a low level of DNA synthesisrelative to the rest of the somite (Kahane et al., 1998a; Kahane et al.,1998b). N-cadherin is homogeneously distributed throughout theepithelium, including the medial domain, with predominant stainingin the apical surfaces and weaker expression in the somitocoele (Fig.2A). Upon somite dissociation, pioneer myoblasts bend underneaththe forming DM, dissociate into mesenchymal cells and engage in

RESEARCH ARTICLE Development 133 (6)

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caudorostral migration (Kahane et al., 1998b; Kahane et al., 2002)(see also Fig. 2F and Fig. 8C). Slightly before this process begins,pioneer myoblasts that express desmin downregulate N-cadherin toa basal level (arrowheads in Fig. 2B-G), similar to the behavior ofdissociating sclerotomal cells (Fig. 2B,C) (Duband et al., 1987). Theyoung DM retains strong apical N-cadherin expression (Fig. 2B-I).Following polarized migration, the first myotomal fibers aregenerated in rostrocaudal and mediolateral directions (Kahane et al.,1998a; Kahane et al., 1998b; Kahane et al., 2002), a stage at whichthey regain N-cadherin protein on their surface (Fig. 2H,I). Hence,pioneer myoblasts are positive for N-cadherin at the epithelial stage,lose the protein during dissociation and migration, and re-express itduring differentiation into myofibers.

Dermomyotome and the second and third waves ofmyotome colonizationThe second wave of myogenesis generates fibers and emanates fromall four lips of the DM. Rostral and caudal lips that express Myod ina scattered pattern directly generate fibers into the myotome (Kahaneet al., 2001; Kahane et al., 1998a) (see also Fig. 7), and express N-cadherin primarily in the apical aspect of the epithelial cell, asrevealed by N-cadherin staining of cells transfected with a GFP-encoding DNA (Fig. 3D-G).

The DML and VLL are also N-cadherin positive (Fig. 3H-M). Wehave previously shown that these lips contribute indirectly tomyofiber generation, by releasing intermediate progenitors thatlocalize underneath these lips in a region we termed the sub-lipdomain (SLD). Similar to the DML and VLL, the SLD is also

1103RESEARCH ARTICLEN-cadherin and regulation of somite myogenesis

Fig. 1. Electroporation strategies used to transfect distinctsomite domains. (A-D) Electroporation of pioneers (A),dermomyotome sheet (B), dorsomedial lip (C), and rostral and caudallips (D, only rostral is depicted, the location of the positive electrode ischanged to the opposite edge for caudal transfections). The shapesand placement of the electrodes are drawn in white. Arrows indicatethe direction of the current; red blocks represent the targeteddomains.

Fig. 2. Expression of N-cadherin protein during development of the pioneer myoblasts. (A,E,G,I) N-cadherin; (B-D,F,H) overlay of desminand N-cadherin. (A) A newly-formed somite in a 22ss embryo showing homogeneous expression of N-cadherin, including the medial region thatgenerates pioneer myoblasts (P). Note enhanced immunoreactivity in the apical adherens junctions. (B) Somite 26 in a 28ss embryo showing theinitial bending of desmin-positive pioneers (green) under the forming DM. Desmin-positive pioneers still in the epithelium (arrowhead), and thebending cells, no longer express N-cadherin. (C-E) Somite 23 in two 28ss embryos. Arrowheads indicate mesenchymal pioneers in the dissociatedsomite that are desmin+/N-cadherin– during migration (D,E). N-cadherin is enriched at the apical pole of DM cells (E, arrow). (F,G) Frontal section asin C-E, revealing desmin+/N-cadherin– pioneers (delimited by a thin white line) underneath the DM. Pioneers are predominantly located in the rostraldomain of the segment. (H,I) Somite 22 in a 30ss embryo. Upon differentiation into myofibers, pioneers reexpress N-cadherin (arrowheads). DM,dermomyotome; EC, ectoderm; NT, neural tube; SC, somitocoele; Scl, sclerotome. Scale bar: 40 �m in A-C,H-I; 20 �m in D-G.

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negative for desmin but, unlike the DML and VLL, it is positive forMyod, Myf5 and FREK (Cinnamon et al., 2001), and also for N-cadherin, even though these cells are no longer epithelial (Fig. 3H-J).

Finally, the DM sheet is composed of cells that generate bothPAX-positive mitotic muscle progenitors and dermis (Ben-Yair andKalcheim, 2005). This part of the epithelium also reveals N-cadherinimmunoreactivity enriched in the apical pole of the cells(arrowheads in Fig. 3A-C). N-cadherin protein is, however,downregulated in the emerging dermis (Fig. 3K-M), but is stablymaintained in desmin-expressing myofibers that, contrary to theDM, are decorated in a homogeneous pericellular pattern (Figs 3, 4).

Differential segregation of N-cadherin to the progeny ofDM sheet cells during mitosisFollowing dissociation, many individual DM sheet progenitorsgenerate both mitotic myotomal progenitors as well as dermis. Thisis associated with a shift in the orientation of cell divisions which,in the young DM epithelium, are parallel to the mediolateral axis ofthe DM and prior to DM dissociation progressively change tobecome perpendicularly oriented (Ben-Yair and Kalcheim, 2005).To begin approaching the relationship between mitotic orientations,dissociation of the DM epithelium and fate segregation, wedetermined the localization of N-cadherin protein during mitosis(Fig. 4). As in other types of epithelia, the mitotic phase of the cellcycle in the DM occurs at the apical pole where N-cadherinexpression is predominant (Fig. 4A). In all parallel cell divisions,daughter cells remain side by side, associated with the apical surfaceof the epithelium, and N-cadherin is symmetrically inherited by both

cells (Fig. 4B). In perpendicular mitoses, one daughter cell pointstowards the dermis and the other towards the myotome. In all suchcases, N-cadherin remains associated only with the apical celllocated in apposition to the myotome and is lost from the basallypositioned cell (Fig. 4C,D), thus generating a molecular asymmetrybetween the basal and apical progeny. Notably, N-cadherin inoblique mitoses was similarly restricted to the apical progeny,similar to what was observed in the perpendicular cell divisions (Fig.4E), further validating by molecular means the classificationpreviously employed to distinguish between parallel andperpendicular mitoses (Ben-Yair and Kalcheim, 2005; Cayouette etal., 2001).

Effects of N-cadherin-mediated adhesion on celltranslocation and fate segregation of DM sheetprogenitorsThe observation that myotomal cells are N-cadherin positive whereasdermal progenitors downregulate the protein (Figs 3, 4), together withthe finding that N-cadherin becomes differentially segregated to theapical daughter cells during mitosis (Fig. 4), suggest that, of thesingle cells previously shown to generate both muscle and dermalprogenitors, the N-cadherin-positive DM daughter cell translocatesinto the N-cadherin-expressing myotome as a result of homophilicinteractions, whereas the cell lacking N-cadherin generates dermis.To examine this possibility, the nascent DM sheet was focallyelectroporated (see Fig. 1B), with control GFP, wtN-cadherin-GFPor N-cadherin bearing a deletion in the extracellular domain(cN390�-GFP) (Fig. 5). This mutation was documented to interfere

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Fig. 3. Expression of N-cadherin during the second wave of myotome development. (A,H,K) Desmin (green); (B,E,F,I,L) N-cadherin (red);(C,J,M) overlay of desmin and N-cadherin, nuclear Hoechst is blue. (D-G) Electroporation of GFP/DNA into a rostral DM lip (green in D and G) co-stained for N-cadherin (E-G). (A-G) Flank somites of E3 embryos. (A-C) The apical surface of the DM and desmin+ fibers express N-cadherin(arrowhead). (D-G) Semi-frontal section showing that the GFP+ epithelial cells in the rostral lip co-express N-cadherin. Note in F and G that, like inthe DM sheet, N-cadherin is restricted to the apical aspect of the cells (arrows). (H-M) Transverse sections through flank somites at E4. (H-J) Highmagnification of the DML region showing the myotome and DML positive for N-cadherin. The latter displays strong apical N-cadherin staining(arrowhead). Cells within the SLD (delineated between arrows) express N-cadherin but are desmin negative; note that their cell and nuclear shapesare rounder than the elongated epithelial cells of the DML. (K-M) Dermal cells (D) downregulate N-cadherin expression to a basal level. Scale bar:80 �m in A-C,K-M; 40 �m in D-E; 20 �m in F-I.

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Fig. 4. Differential allocation of N-cadherin to the apical daughters of perpendicularly dividing DM progenitors. (A) Transverse sectionshowing the DM and underlying myotome (M) co-stained with nuclear Hoechst (blue) and N-cadherin (green). Cell divisions occur at the apicalportion of the DM (arrowhead). (B-E) High magnifications of dividing cells stained with Hoechst (blue), N-cadherin (green) and �-tubulin to labelcentrosomes (red). (B) A cell dividing parallel to the mediolateral axis of the DM. Both daughter cells retain N-cadherin staining at their apicalaspects. (C,D) Perpendicularly dividing DM cells in which only the apical progeny remains N-cadherin positive. (E) Oblique mitosis showing a similarsegregation of N-cadherin to the apical daughter cell. In contrast to the DM, myotomal cells express homogeneous N-cadherin on their surface.Panels in second and third rows show, separately, Hoechst staining of nuclei associated with �-tubulin and N-cadherin with �-tubulin. n, at least 60mitoses were scored with equal N-cadherin distribution in progeny of all parallel mitoses, and apically localized N-cadherin in all perpendiculardivisions. Scale bar: 33 �m in A; 10 �m in B-E.

Fig. 5. Effects of N-cadherin-mediated adhesion on cell translocation and fate segregation of DM sheet progenitors. (A,E) Control GFP-treated somites (n=31/31 embryos). (B,G-I) cN390�-GFP–treated somites (n=27/30 embryos). (C,F) Full-length N-cadherin-treated somites(n=27/30). (D) N-cadherin (CBR–)-treated somites (n=12/12). Desmin is red, GFP is green and Hoechst staining is blue in A-D. The blue products inG-I are in situ hybridizations for Alx4 (G,H) and Pax7 (I). Electroporations were performed in somite 22 of 28ss embryos (A-H). (A-D) Transversesections two days after electroporation, showing (A) the distribution of control GFP-treated cells among both the dermis and myotome,(B) cN390�-GFP–treated cells in the dermal domain only, and (C,D) wild-type N-cadherin or N-cadherin (CBR–)-treated cells in myotome only.(E,F) Whole mounts showing that wild-type N-cadherin-treated DM cells generate fibers (F), whereas GFP only-treated cells remain mesenchymal(E). (G,H) DM cells that received cN390�-GFP relocate into the nascent dermis while maintaining expression of Alx4 (arrows). (I) Prematuredissociation of DM cells induced by focal electroporation of cN390�-GFP 10 hours after electroporating an epithelial somite. Dissociating cellsdownregulated Pax7 mRNA (arrowheads) when compared with similar cells still resident in the epithelium (arrows) or with untransfected cells. Scalebar: in A, 40 �m; in B, 30 �m; in C,D, 50 �m; in G,H, 60 �m; in I, 15 �m.

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with activity of endogenous cadherin by competing for interactionswith cytoskeletal components, resulting in the abrogation ofcadherin-mediated cell adhesion (Fujimori and Takeichi, 1993). Thetransfection method used did not reach the bordering lips and wasconfined to the center of the DM sheet. Up to 18 hours aftertransfection, the GFP-labeled DM was still epithelial in control-treated segments, but in cells expressing cN390�-GFP the beginningof de-epithelialization could be detected as early as 5 hours followingelectroporation; this was also revealed by a gradual loss of basementmembrane-associated laminin immunoreactivity, which is observedin the basal surface of the DM (see Fig. S1 in the supplementarymaterial, data not shown). By 30 hours, the DM sheet had alreadydissociated in control segments, and GFP-labeled cells localized inboth dermis and myotome (Fig. 5A) (Ben-Yair and Kalcheim, 2005).In clear contrast, the cells expressing cN390�-GFP failed to colonizethe myotome and localized instead to the prospective dermal domain.There, they sorted out from the unlabeled progenitors to becomeaggregated subectodermally (Fig. 5B). These data suggest that N-cadherin-mediated adhesion is necessary for myotome colonization.To examine whether it is also sufficient, wtN-cadherin-GFP wassimilarly delivered. In contrast to control GFP and to cN390�-GFP,all cells overexpressing N-cadherin translocated into the myotomewith no contribution to dermis (Fig. 5C). This effect was mimickedby overexpression of a mutant of N-cadherin in which theintracellular �-catenin-binding domain (CBR–) was deleted (Fig.5D). Altogether, these results suggest that the extracellular domainof N-cadherin that mediates homophilic cell adhesion is bothnecessary and sufficient ‘in vivo’ to drive cell translocation into themyotome.

Next, we analyzed the fates of cells overexpressing either wtN-Cadherin-GFP or cN390�-GFP. As previously documented, controlGFP-treated cells remained as mesenchymal Pax7-positive muscleprogenitors in the myotome (Fig. 5E) (Ben-Yair and Kalcheim,2005). By contrast, N-cadherin (wt or CBR–)-treated cells enteredthe myotome as Pax-positive progenitors, gradually downregulatedPax by 24-30 hours (data not shown) and then differentiated intofibers by 48 hours after transfection (Fig. 5F). Thus, N-cadherin isnot only necessary and sufficient for cell translocation into themyotome, it also triggers myogenic specification followed bydifferentiation in cells that otherwise remain as proliferativemyotomal progenitors.

We then examined the phenotype of cells that receivedcN390�-GFP and localized subectodermally to where dermisdevelops (Fig. 5B). Similar to control GFP-treated cells, thesecells remained mitotically active (see Fig. S2 in the supplementarymaterial). Like normal dermis progenitors, they maintainedexpression of Alx4, a marker of the central DM region that is laterexpressed in dermis (Cheng et al., 2004) (Fig. 5G,H), and theyrapidly downregulated Pax7 mRNA expression (Fig. 5I), whilestill exhibiting a low level of Pax7 protein; the protein hadcompletely disappeared by 20 hours after transfection (see Fig. S2in the supplementary material, data not shown). Thus, cN390�-GFP-treated cells display features of early dermal progenitors(maintenance of Alx4 and downregulation of Pax genes). Becauseof a relatively rapid loss of the transfected signal, we were unableto trace them after E5, when more mature markers of dermisdevelopment, such as Dermo1, or differentiation features ofmature dermis become apparent.

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Fig. 6. cN390�� causes DML dissociationand lack of myotome colonization.(A-F) Control GFP; (G-L) cN390�-GFP.Electroporations were directed to the DML ofearly dissociating somites 22-23 of 28ssembryos. GFP is green; desmin, red.(A-D) Five hours after transfection, labeledDML cells retain their epithelial structure asrevealed in dorsal views (A,B) and transversesections (C,D, arrowheads, n=15/17).(E) Thirty hours after electroporation, GFP-positive myofibers derived from the DMLoccupy the medial-most aspect of themyotome (arrowheads). The DML itselfretains its epithelial structure (n=12/14).(F) Dermal stage showing a larger number oflabeled DML-derived fibers in the desmin-positive myotome with a still epithelial DML.(G-J) Five hours after transfection, DML cellsthat received cN390�-GFP have begundissociating, as revealed in dorsal views(G,H, arrows) and transverse sections(I,J, arrowheads, n=8/10). (K) By 30 hours,cN390�-GFP-treated cells have lost theirepithelial morphology and have locatedsubectodermally (arrows). None of themgenerated myofibers (n=8/11). (L) Dermalstage to further emphasize the dissociation ofDML cells and the lack of myotomalcolonization. Scale bars: 20 �m in C,D,I,J;10 �m in E,F,K,L.

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Effects of N-cadherin-mediated adhesion on theontogeny of the myotomeThe dynamic expression of N-cadherin to pioneer muscleprogenitors and to DM lips, as well as its effects on cells originatingin the DM sheet, prompted us to further examine its involvement inthe development of the first two myogenic waves that generatemyofibers.

Effect of N-cadherin-mediated adhesion on the DMLIn previous studies, we subdivided the contribution of the medialsomite into early pioneer myoblasts and later DML-derived fibers.Here, we wished to determine whether these two populations canbe further distinguished by a differential sensitivity to N-cadherin(see Figs 6, 8). To examine its effect on DML progenitors, thenascent DML was electroporated with control GFP or withcN390�-GFP (Fig. 1C). In control-treated segments, the DMLremained both epithelial and labeled for the entire duration of theexperiments (Fig. 6A-F), further suggesting that this domain actsas a continuous source for myofibers (Ordahl et al., 2001); this isdifferent from pioneers, which are a limited cell subset leaving noresidual labeled cells after myogenesis (see Fig. 8). Approximately20 hours after electroporation, no DML-derived fibers were yetapparent (data not shown); this was again different from pioneers,many of which by this time had already differentiated intomyofibers (see Fig. 8D). Thirty hours following electroporation,the first labeled fibers became apparent in the desmin-positivemyotome, being restricted to its medial region (Fig. 6E). In strikingcontrast, shortly after electroporation of the mutant cadherin, somelabeled cells had already adopted a round morphology and becomelocalized subectodermally (Fig. 6G-J). This phenotype becamestronger by 30 hours, when, in addition, the labeled cellscompletely failed to enter the myotome (Fig. 6K). This effect doesnot reflect a delay in myotome colonization because a similarpicture was observed 42 hours after transfection, at stagesfollowing DM dissociation and dermis formation (Fig. 6L,compare with 6F).

Effect of N-cadherin-mediated adhesion on the rostral andcaudal DM lipsWhen dissociating somites were electroporated with control GFP attheir rostral and caudal edges (Fig. 1D), expression of the transgeneappeared shortly afterwards in the rostral and caudal lips of the DM,respectively (not shown). A day later, fibers emanating from allalong the corresponding lips elongated toward the opposite direction(Fig. 7A,C, see also Fig. S2A in the supplementary material), andPax7/GFP-immunoreactive cells were also apparent in the myotome(Fig. S2 in the supplementary material, arrows in A and B). Bycontrast, overexpression of cN390�-GFP at this stage caused thedissociation of the lip cells into round progenitors apparent in theintersomitic region, and no myofibers or Pax7-positive progenitorscolonized the myotome (Fig. 7B,D, and Fig. S2D-F in thesupplementary material). By contrast, overexpression of wtN-cadherin robustly generated myofibers (Fig. 7E).

To begin to understand the mechanism underlying the failure ofmyofiber differentiation observed in cN390�-GFP-treated lips,control and experimental embryos were in situ hybridized to detectMyod. In previous studies, we have shown that Myod mRNAappears in the extreme DM lips in a scattered fashion followingestablishment of the pioneer myotome, about a day after epithelialsomites had formed (Kahane et al., 2001; Kahane et al., 1998a). Bythis time, control GFP-positive epithelial cells in both lips co-expressed Myod (Fig. 7F,H,J). However, cN390�-GFP-treated cells

were Myod negative (Fig. 7G,I,K). These results suggest that thecN390�-induced early dissociation of the somite epitheliumprevented the onset of Myod transcription and the subsequentformation of myotomal fibers. Together with our finding that forcedexpression of wtN-cadherin or N-cadherin CBR– stimulate myofiberdifferentiation in otherwise mitotic progenitors, these data supportthe notion that, in vivo, N-cadherin triggers a cascade of eventsleading to muscle specification and differentiation.

Loss or gain of N-Cadherin function does not affect thegeneration of early pioneer myofibersThe origin, migration and pattern of differentiation of pioneermyoblasts was previously documented using lineage tracing withthe lipophilic dye DiI, pulse-chase experiments with tritiatedthymidine, and expression of Myod, Myf5 and desmin (Kahane etal., 2002). To interfere with N-cadherin activity in pioneers, we firstdetermined conditions to specifically transfect DNA into this cellpopulation. A GFP-encoding DNA was electroporated into themedial somite as shown in Fig. 1A; this method differentiatesbetween the pioneer population and the prospective DML and DMsheet (Fig. 1B,C). The position of fluorescent cells observed 5 hoursafter transfection (Fig. 8A,B, see also 8E) confirms the specificityof labeling. Consistent with previous results, by 16 hours aftertransfection, labeled cells have mesenchymalized and arepredominantly localized to the rostral half of the segment, revealinga general triangular pattern (Fig. 8C) that, as directly demonstratedelsewhere, reflects caudorostral cell movement (Kahane et al.,1998b; Kahane et al., 2002). In addition, the first fibers to arise arelocated adjacent to the medial edge and are anchored to the rostrallip of the segment (Fig. 8C, arrow), further substantiating themedial-to-lateral and rostral-to-caudal directions of fiber generation(Kaehn et al., 1988) [see also figure 1B,C in Kahane et al. (Kahaneet al., 2002)]. By 24 hours, full-length myofibers are already presentthat span a significant fraction of the mediolateral extent of thesomite (Fig. 8D).

Next, we examined the function of N-cadherin on the formationof the pioneer fibers by electroporating the medial epithelial somitewith cN390�-GFP (Fig. 8E,F). Electroporation of 23-25ss embryosrevealed the formation of a normal myotome (Fig. 8G), similar tocontrol GFP-transfected somites. Similar electroporationsperformed on younger embryos aged 15-16ss also resulted in theformation of normal myofibers. which were already apparent by 20hours (Fig. 8H,I). To further test whether preventing the normaldownregulation of N-cadherin that takes place upon cell dissociation(see Fig. 2B-E) has any effect on the development of the pioneerfibers, full-length N-cadherin-GFP was similarly delivered. N-cadherin overexpression did not prevent the dissociation of medialepithelial progenitors (data not shown). Furthermore, no effect onthe differentiation of pioneer fibers was detected, with the earliestfull-length fibers being generated medially and the lateral somite stillcontaining mesenchymal cells 20 hours after transfection (Fig.8J,K). Thus, N-cadherin-mediated adhesion is not necessary for thedevelopment of the first wave of myotomal fibers. Moreover, forcedexpression of N-cadherin does not impair either their dissociation ortheir ability to generate fibers.

DISCUSSIONWe addressed the effects of N-cadherin on the development of thethree waves that constitute the avian myotome. Although the firstwave of pioneer myoblasts is not affected, specification anddifferentiation of the second wave emanating from the DM lips isseverely harmed by a lack of N-cadherin-mediated intercellular

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interactions (see Table S1, in the supplementary material). Hence,the present results show for the first time a differential role for N-cadherin in myogenic specification and fiber differentiation.

Likewise, the DM sheet, which generates a large proportion ofmitotic myotomal progenitors (third wave), as well as dermis,dissociates in the absence of cadherin-mediated adhesion, and cells

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Fig. 7. Electroporation ofcontrol GFP, cN390��-GFP orwtN-cadherin to rostral andcaudal lips of the DM.(A-D) Electroporations ofcontrol GFP (A,C) and cN390�-GFP (B,D) to caudal (A,B) androstral (C,D) DM lips to somites22-23 of 28ss embryos.(E) Similar transfection of wtN-cadherin-GFP to a caudal lip.Embryos were re-incubated for24 hours. In control-treatedsegments, the labeled lip cellsgenerated fibers thatelongated toward the oppositedirection (arrows in A, whole-mount view, and in C, frontalsection); a similar phenotype isobtained upon wtN-cadherinoverexpression (whole-mountview, E, n=7/7). In cN390�-GFP-treated lips (B,D), labeledcells lost their epithelial shape,remained in the inter-somiticregion and did not generatefibers. Arrowheads indicate theinter-somitic spaces.(F-K) Similar electroporations asin A-D. Whole mounts (F,G)and frontal sections (H-K) werein situ hybridized with a Myodprobe and stained for GFP.Each panel shows separatelyand in combination Myodtranscripts and GFP expression.Both caudal and rostral lip cellstreated with control GFP, co-express Myod (arrowheads in F,arrows in H,J) and generatedmyofibers (n=8/9). cN390�-GFP-expressing cells (G,I,K)dissociated from theepithelium, do not expressMyod (arrowheads in G, arrowsin I,K) and did not generatemyofibers (n=10/11). DM,dermomyotome; EC, ectoderm;IS, intersomite; R and C, rostraland caudal; Myo, myotome;Scl, sclerotome. Scale bars: inK, 10 �m for C,D; 20 �m forH-K.

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populate the dermal domain but fail to colonize the muscle.Conversely, full-length N-cadherin or N-cadherin lacking its �-catenin-binding domain (CBR–) trigger translocation of DMprogenitors into the myotome at the expense of dermis, followed bymuscle differentiation. This process is associated with asymmetriclocalization of N-cadherin to the apical daughter cells during theapical-basal type cell divisions in the mature DM, suggesting thatcadherin is involved in coupling asymmetric cell divisions with cellfate. Altogether, N-cadherin-mediated adhesion is both necessaryand sufficient for myotome colonization by both second and thirdwave myoblasts, thus demonstrating substantial, yet distinct,spatiotemporal effects of N-cadherin on somitic myogenesis invivo.

A possible involvement of N-cadherin inasymmetric cell division and fate segregationAn important observation is that N-cadherin differentially segregatesto the apical, but not to the basal daughter cells during mitosis in themature DM. This occurs at a time when a significant proportion ofmitotic figures become perpendicularly oriented with respect to themediolateral axis of the epithelium. In addition, loss of cadherin-mediated adhesion drives cells to the dermal domain where theyexhibit features of early dermal cells. Conversely, overexpression ofwtN-cadherin or of N-cadherin (CBR–) triggers DM cells totranslocate into muscle. Taken together, these lines of evidencesuggest that during normal development of the DM sheet, apicaldaughter cells maintaining N-cadherin translocate into the N-

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Fig. 8. N-cadherin is not required for the development of the earliest myotome composed of pioneer fibers. (A-D) Electroporation ofcontrol GFP to the medial region of newly-formed epithelial somites in 23-25ss embryos. (A) Five hours after transfection, the pioneer cells(arrowhead) express GFP, whereas the dorsal somite (delineated between arrows) is devoid of labeled cells (n=20/24). (B) Dorsal view of a somite 5hours after transfection. Note the homogeneous rostral-to-caudal distribution of labeled cells. (C) Sixteen hours later, pioneers have dissociated, andare localized preferentially in the rostral half of the segment, forming a triangular shape (arrowhead); in addition, a few partial-length fibers thatelongate rostrocaudally (arrow) and localize medially close to the neural tube (NT) are apparent (n=10/10). (D) Twenty-four hours after transfection,full-length myofibers formed (n=13/17). (E-G) Electroporation of cN390�-GFP to the medial region of newly-formed epithelial somites in 23-25ssembryos. (E,F) Transverse (E) and dorsal (F) views 5 hours after transfection. Labeled cells are localized to the pioneer region (arrowhead) but not tothe prospective DM and DML (between arrows, n=14/17). (G) Twenty-four hours after electroporation, full-length myofibers formed (n=15/16).(H,I) Electroporation of cN390�-GFP to the medial region of newly-formed epithelial somites in young 15ss embryos. Twenty-four hours latermyofibers had formed normally and already span a significant mediolateral extent of the segment (n=9/10 embryos). (J,K) Electroporation of wtN-cadherin-GFP to the medial region of newly-formed epithelial somites in 23-25ss embryos. As early as twenty hours post-transfection, continuousexpression of the protein is compatible with the normal formation of myofibers. In addition, some labeled mesenchymal cells are still apparent inthe lateral domain of the somite (arrowheads, n=8/8). H and J depict GFP+ cells on a phase contrast background, I and K are GFP only. Note inD,G,H and K that in controls and all experimental treatments, formation of the pioneer fibers occurs as a discrete process leaving no residual GFPlabeling of the DML region. The labeled cells observed at 5 hours in the medial domain of panels B and F correspond to the labeled cells in thetransverse sections in A and E, respectively (i.e. ventrally located with respect to the future DML). Scale bars: 20 �m for A,C,E.

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cadherin-positive myotome via homophilic interactions, whereasbasal daughter cells that lose N-cadherin become dermis. Thissupports a role for N-cadherin as a cell surface determinant requiredfor the fate segregation of DM progenitors, a function accomplishedby directing cell translocation into myogenic versus dermogenicprimordia. These results also suggest that the shift in the orientationof cell divisions in the DM (Ben-Yair and Kalcheim, 2005) reflectsa change to an asymmetric mode of cell division, and is therefore offunctional significance for fate segregation, as shown during thedevelopment of several invertebrate and vertebrate systems(Betschinger and Knoblich, 2004).

Differential sensitivity of pioneers and DML-derived fibers to N-cadherin further emphasizesthe uniqueness of these two medial somiticpopulationsPrevious studies postulated that the contribution of the medial somiteto the formation of the myotome occurs via a single mechanismwhereby stem cells constituting the DML directly translocate intothe myotome and differentiate without migration (Denetclaw et al.,2001; Gros et al., 2004). We found instead that the medialcontribution is subdivided into two components: first, the pioneerwave; and, later, fibers from the DML. This distinction is based onseveral findings. Pioneer myoblasts arise in the medial wall of thestill epithelial somite, defining a region different from the futureDML. This region expresses Myod and Myf5 and is characterized bya significantly lower rate of cell proliferation, including the presenceof post-mitotic cells (Kahane et al., 1998b), suggesting that this is alimited myogenic subpopulation. Hence, it differs from the laterDML, which in avians lacks expression of Myod or Myf5, andreveals a continuously high rate of cell proliferation (Ben-Yair et al.,2003). This is consistent with the suggestion that DML cells behaveas a continuous source of myoblasts (Ordahl et al., 2001; Venters andOrdahl, 2002). The distinction between fibers derived from pioneersrelative to DML was further confirmed in this study, in which GFP-DNA was differentially electroporated into the medial versus thedorsomedial regions of the somite. In the first case, followingdissociation of the epithelial pioneers and fiber formation, noresidual GFP-labeled cells remained in the epithelium (indicative ofa finite cell subset), whereas a similar transfection to the DMLalways revealed residual labeling of the lip (suggesting a stem-likemechanism). Moreover, pioneer myoblasts generated a myotomewith a significant amount of full-length fibers present already by 20hours after GFP transfection, whereas DML-derived fibers were stillabsent after 24 hours.

Another unique feature of pioneer myoblasts is that they undergoa polarized migration towards the rostral edge of the DM, fromwhich initial myofiber generation proceeds in a rostral to caudaldirection (Kahane et al., 2002). This particular migratory behaviorwas further confirmed in this study by tracing the cells after focalGFP electroporation (see Fig. 8). The results of our lineage analysesare consistent with the observed expression patterns of Myod, Myf5,desmin (Kahane et al., 2002; Kalcheim et al., 1999), and theflamingo homologue c-fmi (Formstone and Mason, 2005) in thedeveloping myotome.

This mode of initial myotome formation clearly differs from thecontribution of the DML that generates fibers through anintermediate SLD (Cinnamon et al., 2001) (see also Denetclaw etal., 2001; Gros et al., 2004). The medial cells involved in generationof the second myogenic wave (the DML itself and the SLD) and theresulting fibers, express N-cadherin throughout the entire process.Consistent with this continuous expression, cN390�-GFP caused

the rapid dissociation of the DML, as well as that of the DM sheet,with cells that sorted out to become localized subectodermally, andconsequent failure of myotome colonization. These data suggestthat, in addition to keeping the epithelial integrity of the somite, N-cadherin-mediated homophilic attraction between DM progenitorsand the underlying pre-existing myofibers is necessary forprogenitor translocation into the myotomal domain.

By contrast, we report that N-cadherin-mediated adhesion is notnecessary for establishment of the first wave of pioneer fibers. Thismight be related to the early downregulation of the protein that isapparent immediately before the dissociation and rostralwardmigration of the pioneers. It is, however, unlikely to depend upon thestate of specification of these progenitors, which already expressMyod and Myf5 at epithelial stages, as a similar electroporation ofcN390�-GFP to epithelial somites of younger embryos (aged 15ss),prior to significant medial expression of the myogenic genes and totheir withdrawal from the cell cycle (N. Kahane and C.K.,unpublished), also had no effect on myofiber development.

Overexpression of wtN-cadherin at this stage was also withouteffect, strongly suggesting that N-cadherin is neither necessary norsufficient for the earliest progenitors to migrate and generate amyotome. This further highlights the molecular differences betweenpioneers and second wave progenitors, and suggests thatmechanisms other than, or additional to, N-cadherin operate toregulate the formation of this cell population. Unlike the avianpioneers, differential cell adhesion mediated by N- and M-cadherinsin the zebrafish myotome drives the migration of adaxial cells(Cortes et al., 2003). Adaxial cells, like the avian pioneers, originatein a medial position in the somite, where they express Myod andMyf5, and migrate laterally to localize superficially in the myotome(Devoto et al., 1996). Furthermore, on their way, zebrafish adaxialcells also exhibit an intermediate phase of rostralward migration(Cortes et al., 2003). Nevertheless, whereas zebrafish adaxialscontinuously express N- and M-cadherins from their site of originand throughout migration (Cortes et al., 2003), avian pioneers loseN-cadherin expression prior to or concomitant with the onset ofdelamination from the medial somite, migrating as cadherin-negative progenitors. Hence, the molecular mechanisms underlyingthe migration of the avian pioneers seem to differ from those of thezebrafish adaxial cells.

Our findings are consistent with results in which the infection ofthe chick segmental plate with adenoviruses expressing either full-length N-cadherin or cN390� had no effect on early myofibergeneration (Horikawa and Takeichi, 2001). Collectively, a wealth ofindependent evidence, including lineage tracing with DiI and GFP(this study), patterns of marker expression, distinct proliferativebehavior, and also functional data on the differential sensitivity toN-cadherin-mediated adhesion (this study), fully validates the notionthat pioneers and DML cells are distinct myogenic populations.

N-cadherin mediated adhesion and its significanceto myogenic specification and differentiationDuring myogenesis, the onset of the second wave of musclecolonization generally follows the formation of the pioneermyotome (Kahane et al., 1998a; Kahane et al., 1998b). Theproduction of myofibers from the extreme lips is associated with thelocal upregulation of Myod, with transcripts appearing scattered tosubpopulations of lip cells (Kahane et al., 2001; Kahane et al.,1998b). The factors inducing the transcription of Myod in these lipshad remained unknown. Here, we show that the disruption of N-cadherin-mediated adhesion in the nascent lips prevents thesynthesis of Myod and, consequently, the generation of myofibers.

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Hence, N-cadherin acts by mediating specific adhesive interactionsamong neighboring lip cells, and/or between lips cells and the pre-existing pioneer myofibers that attach to the lips. These interactionsin turn promote signaling events that trigger myogenic specification.

This notion is further supported by the finding that, in the DMsheet, overexpression of N-cadherin not only drives cell entry intothe myotome at the expense of dermis, but also causes myofiberformation rather than maintenance of mitotic myotomal progenitors,as is observed under normal conditions. The reason for thisphenotypic shift is unclear. One possibility is that in normaldevelopment, Pax3/Pax7-positive mitotic cells that enter themyotome as N-cadherin-expressing cells subsequently lose theprotein in order to remain in a progenitor state. An additionalpossibility is that the relatively high amount of protein in N-cadherin-transfected cells is not compatible with maintenance of amitotic state, whereas physiological amounts are. As this phenotypewas mimicked by N-cadherin (CBR–), we favor the interpretationthat N-cadherin-mediated adhesion rather than N-cadherin-mediatedsignaling is responsible both for cell translocation anddifferentiation. This is further strengthened by the observations thatN-cadherin delivered to the central DM sheet, which is normallydevoid of Myod, was not sufficient to upregulate Myod in theepithelium (Y.C., unpublished), and that the N-cadherin-overexpressing cells entered the myotome as Pax3/Pax7-positiveprogenitors, which generated fibers only 24 hours after transfectioninto the DM sheet. Therefore, N-cadherin is likely to indirectly affectmuscle-specific gene transcription and myogenesis by mediatinghomophilic cell interactions that enable cell translocation into anappropriate environment. Our results are consistent with the notionproposed on the basis of in vitro paradigms, that local cell-cellinteractions are required for myogenesis (Cossu et al., 1995; Gurdonet al., 1993), and that N-cadherin is capable of mediating suchinteractions by inducing first the assembly of adherens-typejunctions and then the expression of muscle-specific markers(Goichberg and Geiger, 1998; Holt et al., 1994). Also, in vitro,activation of N-cadherin was found to stimulate Rho GTPaseactivity, which in turn activates the serum response factor thatenhances muscle-specific transcription and promotes myogenesis(reviewed by Krauss et al., 2005). This molecular cascade coupleschanges in cell adhesion and morphology with patterns of geneexpression and cell differentiation.

We thank all members of our group for discussions and, in particular, Irit Tamirfor subcloning full-length N-cadherin-GFP, cN390�-GFP and N-cadherin(CBR–)-GFP. We also thank J. Yisraeli for critical reading of the manuscript. Weare indebted to M. Takeichi for the N-cadherin DNAs. This work was supportedby grants from the Israel Science Foundation (ISF), the EEU 6th Frameworkprogram Network of Excellence MYORES, the March of Dimes, ICRF and DFG(SFB 488) to C.K.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/133/6/1101/DC1

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RESEARCH ARTICLE Development 133 (6)