PATTERNS & PHENOTYPES

Integrins in the Mouse Myotome: DevelopmentalChanges and Differences Between the Epaxialand Hypaxial LineageFernanda Bajanca,1,2 Marta Luz,1,2 Marilyn J. Duxson,3 and Solveig Thorsteinsdottir1,2*

Integrins are cellular adhesion receptors that mediate signaling and play key roles in the development of multicellularorganisms. However, their role in the cellular events leading to myotome formation is completely unknown. Here, wedescribe the expression patterns of the �1, �4, �5, �6, and �7 integrin subunits in the mouse myotome and correlatethem with the expression of several differentiation markers. Our results indicate that these integrin subunits may bedifferentially involved in the various phases of myogenic determination and differentiation. A detailed characterizationof the myogenic cell types expressing the �4 and �6 subunits showed a regionalization of the myotome anddermomyotome based on cell-adhesion properties. We conclude that �6�1 may be an early marker of epaxialmyogenic progenitor cells. In contrast, �4�1 is up-regulated in the intercalated myotome after myocytedifferentiation. Furthermore, �4�1 is expressed in the hypaxial dermomyotome and is maintained by early hypaxialmyogenic progenitor cells colonizing the myotome. Developmental Dynamics 231:402–415, 2004.© 2004 Wiley-Liss, Inc.

Key words: integrins; �4�1; �6�1; myotome; dermomyotome; epaxial; hypaxial; intercalated myotome; mouse; muscleprogenitor cells

Received 2 February 2004; Revised 27 April 2004; Accepted 27 April 2004

INTRODUCTION

The body wall muscles (e.g., ab-dominal, intercostal) and the deepback muscles originate from themyotomes, embryonic structures de-rived from the dermomyotome ofthe mesodermal somites (reviewedin Buckingham, 2001). The myotomecan be subdivided into three regionsdefined either morphologically or bythe expression of different markers(Sporle, 2001). These are the epaxialmyotome, dorsal to the notochord;the intercalated or adaxial myo-tome, which is a subdivision of the

epaxial domain and probably origi-nates from the early epaxial cells;and the hypaxial myotome, which islast to develop and lies ventral tothe notochord (Sporle, 2001).

Several models have been putforth to explain how cells leave thedermomyotome to invade the myo-tomal space and how the myotomegrows. In 1910, L.W. Williams elabo-rated the first model for epaxial myo-tomal colonization that suggestedthat the dorsomedial lip of the der-momyotome was the origin of myo-genic progenitor cells (MPCs). Re-

cent studies propose severaldifferent pathways of cell transloca-tion from the dermomyotome to themyotome, which suggest possibledifferences depending on the stageand species studied (Kaehn et al.,1988; Denetclaw et al., 1997; Kah-ane et al., 1998b; Venters et al.,1999; Eloy-Trinquet and Nicolas,2002). Although a consensus has notbeen reached between these differ-ent models (reviewed in Hollwayand Currie, 2003), they all includethe concept that the myotome isformed by several populations of

1Departamento de Biologia Animal, Centro de Biologia Ambiental, Faculdade de Ciencias, Universidade de Lisboa, Lisboa, Portugal2Instituto Gulbenkian de Ciencia, Oeiras, Portugal3Department of Anatomy and Structural Biology, University of Otago, Dunedin, New ZealandGrant sponsor: Fundacao para a Ciencia e a Tecnologia/FEDER; Grant numbers: PRAXIS XXI/P/PCNA/BIA/131/96; SFRH/BD/1359/2000;POCTI/BCI/47681/2002*Correspondence to: Solveig Thorsteinsdottir, Departamento de Biologia Animal, Faculdade de Ciencias, Universidade de Lisboa,1749-016 Lisboa, Portugal. E-mail: solveig@fc.ul.pt

DOI 10.1002/dvdy.20136Published online 5 August 2004 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 231:402–415, 2004

© 2004 Wiley-Liss, Inc.

MPCs, which arise at different timesand express specific factors.

A primary epaxial myotome isformed by MPCs expressing Myf-5 inthe mouse or Myf-5 and MyoD inavian embryos. These MPCs subse-quently differentiate and elongateto become desmin- and myosin-positive mononucleated myocytes(Kaehn et al., 1988; Ott et al., 1991;Williams and Ordahl, 1994; Denet-claw et al., 1997; Kahane et al.,1998b; Venters et al., 1999; Hirsingeret al., 2000). Several lines of evi-dence suggest that further growth ofthe myotome occurs by entry of asecond cell population that elon-gates and differentiates into mono-nucleated myocytes between, ormedial to, the primary myotomalcells, thus forming the secondarymyotome (Kahane et al., 1998a;Venters et al., 1999; Eloy-Trinquetand Nicolas, 2002). Although muchless is known about the develop-ment of the hypaxial myotome, it iscommonly accepted that it arises asa mirror image of the epaxial myo-tome (Dietrich et al., 1998; Cinna-mon et al., 1999; Sporle, 2001). Fi-nally, a third MPC population hasbeen described in the quail. In con-trast to the previous two, it inhabitsthe myotomal space in a proliferat-ing state for some time, expressingFREK (FGFR4) but no myogenic reg-ulatory factors (MRFs) before even-tually differentiating to give rise tothe great majority of myofiber nucleiof the trunk (Kahane et al., 2001).

While the morphogenic move-ments appear to be similar in theepaxial and hypaxial compartments(Dietrich et al., 1998; Cinnamon etal., 1999; Sporle, 2001), the extrinsicsignals leading to epaxial and hy-paxial myotome formation, as wellas the MRF activation pattern, differ(Cossu et al., 1996; Hirsinger et al.,2000; reviewed in Tajbakhsh andBuckingham, 2000). In particular, theexpression patterns of Myf-5 andMyoD and the analysis of Myf-5-nulland MyoD-null embryos have indi-cated that the epaxial lineage is pri-marily Myf-5–dependent, whileMyoD is also necessary for hypaxialmyotome development (Braun andArnold, 1996; Cossu et al., 1996; Ka-blar et al., 1997).

Integrins are heterodimeric trans-

membrane glycoproteins com-posed of � and � subunits. They bindto extracellular matrix (ECM) mole-cules promoting cytoskeletal reor-ganization and changes in the ac-tivity of intracellular signalingmolecules (Hynes, 1992; Tarone etal., 2000; van der Flier and Sonnen-berg, 2001; Bokel and Brown, 2002).Many developmental events, suchas cell migration, differentiation andsurvival, depend on integrin-medi-ated adhesion and/or signaling.Several integrin subunits (�1, �3, �4,�5, �6, �7, �9, �v, �1, �3) are ex-pressed during skeletal muscle de-velopment (reviewed in Gullberg etal., 1998). Of these, the roles of thefibronectin receptor �5�1 and thelaminin receptor �7�1 are the beststudied. �5�1 promotes proliferationand inhibits differentiation of myo-blasts in vitro (Blaschuk and Holland,1994; Boettiger et al., 1995; Sastry etal., 1996), whereas �7�1 is thought topromote the maturation and survivalof myotubes (Song et al., 1992; Va-chon et al., 1997).

Very few studies have addressedthe role of integrins in myotome de-velopment. Although the expressionof the �1 (Pow and Hendrickx, 1995),�1 (Duband et al., 1992), �4 (Steppet al., 1994), �6 (Bronner-Fraser et al.,1992; Thorsteinsdottir et al., 1995;Pow and Hendrickx, 1995), and �7(Velling et al, 1996) integrin subunitshave been described in the myo-tome, it is not clear whether their ex-pression patterns vary with the differ-entiation state of the myotomal cellsand/or among the different regionsof the myotome.

We recently mapped integrin ex-pression patterns in limb MPCs in themouse (Bajanca and Thorsteinsdot-tir, 2002). We showed that limb MPCsup- and down-regulate several inte-grin subunits during delaminationfrom the dermomyotome, migration,and early differentiation in the limbmuscle masses, suggesting a dy-namic regulation of cell–ECM inter-actions during early skeletal muscledevelopment in vivo. In the presentstudy, we aimed to identify the inte-grins expressed during the earliestphases of myotome development inthe mouse. Our results show that thelaminin receptor �6�1 is present onearly epaxial MPCs, and we suggest

it is a marker for the early Myf-5–pos-itive epaxial lineage. In contrast, thefibronectin receptors �4�1 and �5�1are only up-regulated in these cellsafter their differentiation. When hy-paxial myotome formation starts, thisdomain becomes enriched in �4�1,suggesting this integrin may be amarker for undifferentiated cells inthe hypaxial lineage. The other lami-nin receptors, �1�1 and �7�1, wereonly detected in later stage (fromE10.5) myotomes. Our data show im-portant differences in the cell adhe-sion properties between the epaxialand hypaxial domains of the mousemyotome, as well as between thedifferent stages of myotome devel-opment.

RESULTS

Early (Primary) MyotomeFormation

We analyzed the expression of �1,�4, �5, and �6 mRNA and �7 proteinin embryos at a stage where the pri-mary myotome is starting to form.Over the period from embryonicday (E) 8.5 to E9.0 only �6 was de-tected in the somites (Fig. 1B),whereas �4 and �5 were expressedin other tissues. �4 was found in thehead and branchial arches as de-scribed previously (Pinco et al.,2001), and �5 was widespread in allmesenchymal tissues (not shown).Neither �1 nor �7 were detected inthe embryo at this stage (notshown). The �1 expression patternhas not been described previouslyfor these stages in the mouse, butthe absence of �7 agrees with thedetailed study of Velling et al. (1996)who found �7 mRNA expressionstarting at E10.0/E10.5.

The �6 integrin subunit is presentvery early in development (Hierck etal., 1993; Thorsteinsdottir et al., 1995),and until E9.5, it is most strongly ex-pressed in the paraxial mesoderm,heart, headfolds/head, and pre-sumptive limb region (Fig. 1B,D). Inthe paraxial mesoderm, a faint sig-nal was detected in the rostralmostregion of the presomitic mesoderm(PSM), becoming slightly stronger inepithelial somites (not shown, seeFig. 2 for protein localization). Of in-terest, in E8.5 embryos �6 expression

INTEGRINS IN THE MOUSE MYOTOME 403

Fig. 1. mRNA expression of �1, �4, �5, and �6 integrins in embryonic day (E) 8.5–E10.5 embryos. A,B: Late E8.5. Myf-5 (A) and �6 (B) exhibitsimilar expression patterns in the early myotome (arrows), but �6 is also faintly expressed in epithelial somites. C–H: Early E9.0. MyotomalMyf-5 (C) and �6 (D) expression remains very similar, but �6 is also expressed in the overlying dermomyotome (arrows in H) and epithelialsomites. �-Cardiac actin expression reveals elongated myocytes at the forelimb level, starting in somite XIII (arrows in E), a level where�4 mRNA is first observed, in somites XI to XV (arrows in F, blue line in G). I–P: Late E9.5. Expression pattern of Myf-5 (I,M), �5 (J,N,P), �4 (K),and MLC1A (L,O). �5 is very strong in the presomitic mesoderm and first epithelial somite (yellow arrow in J, compare with Myf-5 in I). Notenarrow, striped myotomal pattern for �5 starting approximately in somite XII (arrows in J,N,P; longitudinal section in N, compare with M;forelimb level in P, compare with O). �4 mRNA is present more caudally (somite IV, black arrow in K) than MLC1A (somite XII, black arrowin L), and anteriorly, it is no longer restricted to elongated cells (compare with I and L). Q–W: E10.5. �5 expression remains in an elongated,thin stripe (arrows in Q,S); �4 mRNA (T) is now evident in caudal somites (black arrows), in interlimb level hypaxial myotomes (arrow-heads), in intercalated myotome, and, rostrally, in epaxial dermomyotomal lips (white arrows, see transverse section in R); �6 (U) isdown-regulated rostrally (arrows), but �1 (V, transverse section; W, longitudinal section) is strongly expressed in the whole myotome(arrows). Yellow arrows indicate the first clearly epithelial somite.

in the PSM or epithelial somites wasnever detected, and dermomyo-tomes are faintly labeled. The der-momyotome expression increases inintensity from E8.5 to E9.5 (Fig. 1B,D).From somite V onward, �6 expressionremains in the dermomyotome, isdown-regulated in the sclerotome(Fig. 1H), and is strongly present inthe nascent myotome (Fig. 1B,D,H).Comparing posterior, immaturesomites to progressively more rostraland mature somites shows that myo-tomal �6 expression is first detectedin the dorsomedial region of thesomite, then also in the mediorostralregion and later the expression do-main progressively expands ventro-laterally (Fig. 1B,D). Myf-5 is the ear-liest marker for MPCs in the mousemyotome and is also weakly ex-pressed in the presomitic mesodermand epithelial somites (Ott et al.,1991; Buckingham et al., 1992; Kieferand Hauschka, 2001). Its expressionpattern in the myotome is very simi-lar to that of �6 mRNA (compareFigs. 1A and B; C and D). The resultsobtained thus suggest that �6�1 isthe first and probably the only inte-grin present on the earliest myo-tomal MPCs.

Differentiation of EpaxialMyogenic Cells Into ElongatedMyocytes

After invasion of the myotomalspace, myogenic cells elongate ros-trocaudally in the mouse, giving riseto differentiated mononucleatedmyocytes (Venters et al., 1999).�-cardiac actin (Fig. 1E) is the earli-est marker for myocytes, appearingas early as E8.5 in the mouse (Buck-ingham et al., 1992), but myocytessoon also express MLC1A (Fig. 1L;Buckingham et al., 1992). The ex-pression of �6 mRNA remainedstrong along the whole extent of themyotome at this stage, always verysimilar to the Myf-5 pattern (com-pare Fig. 1D with 1C).

From early E9.5, mRNAs for the �4(Fig. 1F) and �5 (not shown) integrinsubunits were also detected in thecentral region of rostral myotomes(“central” meaning the middle on amedial to lateral axis). The �4 subunit(Fig. 1F) occurred in a striped pat-tern, similar to that of �-cardiac ac-

tin (Fig. 1E). Moreover, the dorsome-dial-most region of the myotomewas negative for �4 expression, anobservation confirmed in sections ofthese embryos (Fig. 1G), suggestingan up-regulation in elongated myo-cytes. This finding was in contrast tothe �6 mRNA expression pattern,which was widespread in the myo-tome at this stage (Fig. 1H). �1 wasstill not clearly detectable at E9.5(not shown). Thus, we conclude thatdifferentiating epaxial myocytes up-regulate the �4 and �5 integrin sub-units and that �6 expression is main-tained throughout the myotome.

Myotomal Growth

As the myotomes grow from lateE9.5 onward, integrin expression pat-terns become more dynamic. Ex-pression of �5 mRNA is now clearlypresent in all myotomes, forming astreak along the mediolateral axis(arrows in Fig. 1J,N,P,Q,S). This un-usual mRNA distribution pattern, re-stricted to a medial to lateral midlinein the myotome is similar to themRNA expression pattern describedfor slow myosins before myocyte in-nervation (Sacks et al., 2003). �4mRNA expression is now evident inmuch younger somites than beforeand expression is concentrated inthe hypaxial region of these somites(black arrows in Fig. 1K,T). Becauseexpression of the myocyte marker,MLC1A, is only detected more ros-trally (Fig. 1L), this finding indicatesthat �4 is expressed in less differenti-ated cells than observed at the pre-vious stage. Rostrally, �4 expression isalso very strong in the dorsomediallip of the dermomyotome (Fig. 1R,T).

Expression of �6 mRNA remainsstrong along the whole extent of thegrowing myotomes until approxi-mately E10.5, when it starts beingdown-regulated in rostral myotomes(arrows in Fig. 1U). At approximatelyE10.5, expression of the �1 integrinsubunit is clearly detected in themyotome (Fig. 1V,W), at a time that�7 is also up-regulated (not shown;Velling et al., 1996). This up-regula-tion of two new laminin receptorsand the down-regulation of theearly �6-containing laminin-receptorsuggest that the myotomes are en-tering a new myogenic phase.

�6�1 Protein Is Expressed byEpaxial Myf-5–Positive MPCsand Remains ExpressedThrough Their DifferentiationInto Myocytes

The similarity between the mRNA ex-pression patterns of �6 and Myf-5 inthe early epaxial myotome sug-gested that this integrin subunitcould be a feature of Myf-5–ex-pressing myogenic cells. However,�6 mRNA appeared to be morewidely expressed than Myf-5 in epi-thelial somites and dermomyo-tomes. By using immunohistochemis-try (IHC), we analyzed thedistribution of the �6 protein in theparaxial mesoderm, concentratingon early myotomal cells. �6 proteinwas found to follow the mRNA ex-pression pattern described abovefor epithelial somites, dermomyo-tome, and myotomal cells. It was notdetected on PSM cells but was con-centrated on the basal surface ofthe cells forming the cleft separatingthe first epithelial somite from thePSM (Fig. 2A), where its ligand, lami-nin, was also found (Fig. 2B). �6 isalso present on the surface of cells inepithelial somites (Fig. 2C). In 15-somite embryos, �6 is present in thewhole dermomyotome but immuno-reactivity is clearly stronger in themyotome (Fig. 2D), although at laterstages, the dermomyotomal �6 im-munoreactivity increases (Fig. 2E).Double IHC for �6 and Myf-5 showedthat all Myf-5–positive cells in thedorsomedial lip of the dermomyo-tome and in the myotome express�6 (Fig. 2D). This result confirms that�6 is present very early in myotomalmyogenesis.

Because the �6 mRNA patternsuggested that this integrin subunit isnot restricted to young MPCs, wedetermined whether it remainspresent as they differentiate intoelongated myocytes. Double IHC for�6 and the MRF myogenin, whichcomes up later than Myf-5 (Smith etal., 1993), or myosin, which is specificfor differentiating myocytes andmyofibers (Venters et al., 1999), con-firms that �6 is not lost immediatelyafter differentiation of myotomalcells (Fig. 2F–J). Cell dissociation ex-periments performed on interlimb

INTEGRINS IN THE MOUSE MYOTOME 405

level somites from E9.5 embryos con-firmed that elongated, differentiatedmyocytes express �6 (Fig. 2G–J).

In agreement with the in situ hy-bridization data, �6 immunoreactiv-ity starts to decrease at rostral levelsfrom E10.5 onward. Most of the myo-genin-expressing cells either nolonger express �6 or the level of im-munoreactivity is faint (compare Fig.2K with 2L).

�6 dimerizes with either the �1 orthe �4 integrin subunit (Sonnenberget al., 1990). To identify the particular�6-containing integrin present in theearly myotome, we exposed sec-tions of E9.5 embryos to antibodiesagainst �1 and �4 (Fig. 2M,N). �1 isstrongly present in myotomal cells,while �4 was absent, confirming thatE9.5 myotomal cells express the lami-nin receptor �6�1 on their surface.Immunoreactivity of laminin is seenlining the myotome medially at thisstage, and some punctate stainingis present within the forming myo-tome (Fig. 2O). Thus, laminin ispresent in close proximity to the �6-positive cells.

Taken together, our results showthat �6�1 is expressed very early inmyogenesis. It is strongly expressedon early epaxial MPCs, remaining inmyocytes while they differentiate,but is then gradually down-regu-lated.

MyoD-Positive Cells ExpressDifferent Laminin Receptors

MyoD starts being expressed in ros-tral myotomes at E10.5 in the mouse(Buckingham et al., 1992; Sassoon,1993). Because �6 expression de-creases at the same time, we hy-pothesized that �6 could be down-regulated in myogenic cells as theystart expressing MyoD. Double IHCfor �6 and MyoD at E11.5 shows thatthe majority of MyoD-positive cellsdo not express this integrin subunit(Fig. 3A,B). This absence of �6 wasrecorded on young MyoD-positivecells located either epaxially (Fig.3A) or hypaxially (arrow in Fig. 3B) aswell as along the dorsoventral extentof the mature myotome (Fig. 3B). Insome cases, both proteins werepresent in the same region (Fig. 3C).This region corresponds to a smallarea along the anterior–posterioraxis where MyoD has already beenup-regulated in the somites but �6has not yet been down-regulated.However, due to the density of cells,it was not possible to distinguishwhether the two proteins were coex-pressed in the same cells. It is possi-ble, thus, that a subpopulation ofMyoD-positive cells in the centralmyotome maintains �6 expression.Interestingly, the largest MyoD-posi-tive, �6-negative area is the ventro-

lateral, or hypaxial, region of themyotomes (see arrow in Fig. 3B). Thisobservation led us to investigatewhether the �6 protein was reducedor absent in the hypaxial side ofmyotomes. An analysis of �6 expres-sion at interlimb (trunk) levels in E11.5embryos reveals strong expression inthe intercalated myotome but lowor almost undetectable expressionlevels in the hypaxial region (Fig. 3D–F). Together, our results suggest that�6 is not expressed by the MyoD-positive epaxial MPCs and that itsexpression in the hypaxial myotomeis either transient or absent.

MyoD and myogenin are reportedto activate expression of the �7 inte-grin subunit in C2C12 skeletal myo-blasts (Ziober and Kramer, 1996). �7is not present in the mouse myotomewhen myogenin starts being ex-pressed (see above), but its up-reg-ulation coincides in time with the ap-pearance of MyoD (Velling et al.,1996). Thus, we used double IHC forMyoD and the �7 subunit to deter-mine whether these are expressedby the same cells in vivo. In trunk-level somites of E10.5 embryos, somemyotomal cells only express MyoD(Fig. 3G). However, in more rostralsomites, �7 was detected epaxiallyin elongated mononucleated myo-cytes with MyoD-positive nuclei (Fig.3H). In the mature myotomes of

Fig. 2. �6�1 is present in epithelial somites and then becomes strongly expressed in all cells of the early epaxial myotome. A,B:Longitudinal section of embryonic day (E)11.5 embryo tail bud showing �6 protein in the cleft separating the last formed somite and thepresomitic mesoderm (arrows in A) and laminin (B) assembled around tail bud and somites. C: Sagittal confocal section shows �6 proteinexpression in an epithelial somite of an E11.5 embryo. D: Transverse section of a 15-somite embryo shows that all the Myf-5-positive (red)cells in the myotome are �6-positive (green); note that, at this stage, the myotome shows a stronger immunoreactivity for �6 than thedermomyotome. E: Caudal section of an E11.5 embryo labeled for Pax-3 (red) and �6 (green) shows that �6 expression is strong in thedermomyotome (arrows) at this stage. F: Transverse confocal section of an E9.5 embryo shows that �6 protein (green) is present onmyogenin-positive (red) cells in the myotome. G–J: Dissociation of trunk somites of an E9.5 embryo confirms the presence of elongated,mononucleated cells that express both �6 (green) and myosin (red). Nuclei are visualized with 4�,6-diamidine-2-phenylidole-dihydro-chloride (DAPI) staining (blue). K,L: Sagittal sections of an E11.5 embryo showing trunk (K) and rostral (L) myotomes labeled withmyogenin (red) and �6 (green). Note the weaker, almost absent, immunoreactivity of �6 in rostral myotomes. M–O: Transverse sectionsof an E9.5 embryo show the presence of the �1 integrin subunit on myotomal cells (arrows in M), whereas the �4 subunit is not expressedin the myotome (arrows in N). At this stage, laminin lines the myotome medially (arrows in O) and some punctate staining is present withinthe forming myotome. bv, blood vessels; dm, dorsomedial; vl, ventrolateral. Scale bars � 100 �m in A–F, J (applies to G–J), K–O.Fig. 3. Expression of laminin receptors by MyoD-positive cells and in the hypaxial myotome. A–C: Sagittal sections of an embryonic day(E) 11.5 embryo show that most MyoD-expressing cells (red) are negative for �6 (green), both in young epaxial cells near the dorsomediallip (A) and in rostral mature myotomes (B). Note that the hypaxial domain is clearly �6-negative (arrow in B). Some MyoD-positive nuclei,however, are detected in trunk myotomes in areas containing �6-positive elongated myocytes (C). D–F: Sagittal section of an E11.5embryo at interlimb (trunk) level showing faint immunoreactivity for �6 (D, green in F) in the ventrolateral (hypaxial) portion of themyotome, whereas the whole myotome stains for myogenin (E, red in F). G–I: Immunohistochemistry for �7 (green) and MyoD (red) showsthat �7 is specific for MyoD-positive myocytes and myotubes. G: MyoD is clearly expressed in trunk-level myotomes of E11.0 embryos,before �7 starts being expressed on those cells. H: �7 first appears in elongated, MyoD-positive cells epaxially. Sagittal section of matureE11.5 myotomes shows extensive �7 immunoreactivity on elongated myocytes, which always stain for MyoD. I: Binucleated MyoD- and�7-positive cells (arrows) are also detected at this stage. dm, dorsomedial; drg, dorsal root ganglia; vl, ventrolateral. Scale bars � 100 �min A–C, F (applies to D–F), G–I.

406 BAJANCA ET AL.

Fig. 2.

Fig. 3.

INTEGRINS IN THE MOUSE MYOTOME 407

E11.5 embryos, �7 was widely ex-pressed in myocytes with MyoD-positive nuclei (Fig. 3I). We neverdetected MPCs expressing �7, sug-gesting that it is up-regulated in dif-ferentiated, elongated cells. In E11.5embryos, some binucleated cells ex-press both �7 and MyoD (arrows inFig. 3I). Our results suggest that �7�1,together with �1�1 (see above), arethe main laminin receptors presentin late myotome development,when �6�1 is being down-regulated.

�4 Labels the Hypaxial MPCsand Central ElongatedMyocytes

The in situ hybridization analysisabove, showed that the �4 integrinsubunit has a dynamic expressionpattern in the myotome, appearingfirst in elongated myocytes as thefirst myosin transcripts are detected,but by late E9.5, �4 appears morecaudally than the earliest myocytemarkers, suggesting an earlier ex-pression in MPCs. By using IHC, wefurther characterized the myogeniccells that express �4. In E10.0 em-bryos, the most posterior dermomyo-tomes showed intense reactivity for�4 (Fig. 4A). The first myogenic cellsexpressing �4 were present in a cen-tral position in young myotomes, in-tercalated with nonexpressing cells(arrows in Fig. 4B). In more rostralsomites of E10.0 embryos, �4 is alsopresent in the dermomyotomal lips,first appearing in the ventrolateraland then the dorsomedial lip (Fig.4C,D). As the myotomes grow and dif-ferentiate, �4 is present more widelybut it is always negative in the dorso-medial myotome (Fig. 4B–D,F). In aconfocal analysis of caudal myo-tomes of an E11.5 embryo, it is clearthat �4 is enriched in elongated myo-cytes (Fig. 4E). In addition, the ventro-lateral lip of the dermomyotomeshows immunoreactivity for �4, andsurprisingly, MPCs in the hypaxial myo-tome are also �4-positive (Fig. 4E).These observations suggest that the�4 integrin subunit has two distinctpatterns of expression in the myo-tome. First, it is up-regulated in elon-gated myocytes, and second, it is ex-pressed by hypaxial MPCs.

To characterize further these twodomains of �4 expression, we per-

formed double IHC for �4 and theMRFs Myf-5, MyoD, and myogenin.Double IHC for Myf-5 and �4 in cau-dal myotomes at E11.5 shows thatthese two proteins are expressed to-gether in most, but not all, cells in thecentral myotome, and only Myf-5 ispresent epaxially (Fig. 4F). At thissame stage, myogenin and �4 arecoexpressed in the central myo-tome (Fig. 4G), suggesting that alldifferentiated cells are �4-positive.With the markers used, we could notconfidently detect undifferentiatedcells in the myotome expressing �4at this stage. However, on the hy-paxial side, �4 immunoreactivity ex-tends further ventrally than the myo-genin-positive nuclei (data notshown). At E11.5, �4 is absent in asubset of MyoD-positive myogeniccells located dorsomedially, but it ispresent in the cells whose MyoD-positive nuclei are aligned centrallyin the myotome (Fig. 4H). However,due to the density of myocytes in thisarea of the myotome, it is not clearwhether �4 is present in all or just insome of these MyoD-positive cells.

The �4 expression domain in thedifferentiated areas of the myotomewas analyzed in detail. In E11.5 em-bryos, an extensive number of cellsin the myotome are already differ-entiated, fully elongated, and ex-press myosin (Fig. 5A). Myosin-posi-tive cells are also �4-positive;however, some cells that are myo-sin-negative express �4. These lattercells are detectable in the less ma-ture, caudal myotomes in a medialposition relative to the myosin-posi-tive myocytes and near the myo-tomal basal lamina, (arrows in Fig.5A). This pattern shows that �4 is up-regulated before full elongation ofthese cells.

Some elongated �4-expressingcells, show an enrichment of �4 im-munoreactivity on their tips (Fig.5B,C). This strong �4 immunoreactiv-ity is distal to the myosin labeling andreveals cell processes that pene-trate between the cells of the der-momyotome and come close to thetips of elongated myocytes from ad-jacent somites. This finding suggeststhat �4�1 could play a role in theelongation and/or attachment offully elongated myotomal cells. The�4 subunit must be dimerizing with �1

in the myotome, because its otherpossible partner, �7, is specific forhematopoietic cells (Postigo et al.,1993).

We analyzed the distribution pat-tern of the �4�1 ligands fibronectinand VCAM-1 in the myotome. Fi-bronectin is faintly expressed be-tween the myotome and the scle-rotome and is strongly expressedbetween adjacent somites (Fig. 5D),providing a potential extracellularsupport for the attachment of the�4-positive elongated cells. VCAM-1immunoreactivity is present in themesenchyme surrounding the somitesand in the sclerotome (Fig. 5E). There-fore, �4-positive myotomal cellspresent near the sclerotomal bordercould be attaching to VCAM-1-pos-itive cells in the sclerotome. Somecells already expressing myosin arelocated in a close proximity with theVCAM-1–positive sclerotomal cells(Fig. 5F). Taken together, these re-sults point to different roles for �4�1-mediated adhesion in elongated

Fig. 5. A–F: The intercalated myotomes ofadjacent segments in embryonic day (E)11.5 embryos make contact with eachother through �4-containing structures: lon-gitudinal sections (A–E) and sagittal section(F). A: Myosin-positive (red) myocytes ofthe intercalated myotome extend from ros-tral to caudal dermomyotome lips and ex-press �4 (green). Note that some �4-posi-tive cells do not express myosin (arrows),indicating that they are younger and lessdifferentiated. B,C: Higher amplificationshowing myosin (red in B), �4 (green inB,C), and 4�,6-diamidine-2-phenylidole-di-hydrochloride (DAPI; blue in C). Myosin-ex-pressing, �4-positive myocytes of the inter-calated myotome (B) extend their distal tipsbetween the cells of the dermomyotome(note oval dermomyotomal nuclei markedwith asterisks in B and C) and appear tocontact myocytes from the adjacent seg-ment (at arrows). D: Fibronectin (red) isstrongly expressed between segments andis colocalized with �4 (green) in those areas(arrows). E: VCAM-1 is present in the mesen-chymal cells surrounding the dermomyo-tome (arrowheads) and in the anterior por-tion of the sclerotome that is closest to themyotome (arrows) but is absent betweensegments (asterisks). F: Sagittal sectionthrough the most ventromedial part of themyotome, shows that the myosin-positive(red) cells of each myotome are in closeapproximation to VCAM-positive (green)cells in anterior sclerotome. dm, dorsome-dial; vl, ventrolateral. Scale bars � 100 �min A–F.

408 BAJANCA ET AL.

Fig. 4. �4 is dynamically expressed in the myotome and dermomyotome. A–D: Transverse sections of embryonic day (E) 10.0 embryoshowing different axial levels. B–D: Double immunohistochemistry (IHC) for �4 (green) and fibronectin (red). A: Caudally, dermomyotomestains strongly for �4, but myotome is negative. B: At the hindlimb level, some cells in the central myotome express �4 (arrows), but theseare intercalated with nonexpressing cells. C: At forelimb level, �4 is strongly expressed in the intercalated myotome (arrows) andhypaxially (arrowheads). D: Further rostrally, the epaxial lip (large arrow), intercalated myotome (arrows) and hypaxial region (arrow-head) are �4-positive. Note that fibronectin lines the myotome medially (B–D) and �4 is absent dorsomedially (asterisks, B–D). E: Confocaloptical section of E10.5 embryo shows strong �4 expression in intercalated myotome (between white arrows) and on cells in hypaxialmyotome (arrowheads) and dermomyotomal lip (yellow arrow), but �4 is absent between these two domains (asterisks). F–H: Double IHCfor �4 and differentiation markers. F,G: Transverse adjacent sections of an E11.5 embryo through a caudal somite shows that Myf5-positivecells near the epaxial lip are �4-negative (arrow in F) and �4 is expressed by myogenin-positive cells (G). H: Sagittal section of E11.5embryo shows absence of �4 in epaxial MyoD-positive cells (asterisks), but its presence on cells whose MyoD-positive nuclei are alignedalong the myotomal midline (arrows). dm, dorsomedial; vl; ventrolateral. Scale bars � 100 �m in A–H.

Fig. 5.

myocytes and in the early hypaxiallineage.

DISCUSSION

We have shown that the �1, �4, �5,�6, and �7 integrin subunits havevery specific expression patterns dur-ing the formation of the mouse myo-tome, suggesting myogenic cells altertheir integrin expression repertoire dur-ing their differentiation into myotomalmyocytes. We further show that clearregional differences exist in the celladhesion properties of myotomalcells along the medial to lateral axisin that the �6�1 integrin is expressedby all epaxial cells, while the �4�1integrin is expressed hypaxially aswell as in the intercalated myotome.These regional differences in �6�1and �4�1 expression are schemati-cally represented in Figure 6.

Laminin Receptor �6�1 inEpaxial Myotome Formation

�6�1 was the only integrin subunitdetected on early epaxial MPCs,and it is present on all cells that ex-press the MRF Myf-5. The �6 subunit isalso detected in the dermomyo-tome, but staining is much strongerin the Myf-5-positive myotomal cells,both at the mRNA and protein level,suggesting that �6�1 could play arole in events occurring as the MPCscolonize the myotome (see Fig. 6).

In vitro studies have shown that�6�1 is able to inhibit proliferationand stimulate differentiation of myo-blasts (Sastry et al., 1996, 1999) andlaminin promotes myoblast differen-tiation in vitro, inducing them to be-come postmitotic, fusion-capable,and myosin- and desmin-positive(von der Mark and Ocalan, 1989). Infact, it has been demonstrated re-cently that the ECM is necessary forskeletal muscle differentiation, inde-pendently of MRF expression, sug-gesting that, upon binding to theECM, integrins generate signals thatdrive skeletal muscle differentiation(Osses and Brandan, 2002).

A laminin-containing basementmembrane lines the myotome, bothin avian and mouse embryos (Leivoet al., 1980; Krotoski et al., 1986; thisstudy) and, early myotomal MPCsbecome postmitotic as they enterthe myotomal space, where they

soon differentiate into desmin- andmyosin-positive myocytes (Kahaneet al., 1998b; Venters et al., 1999).Thus, the increased �6 expressionobserved in early MPCs suggeststhat the �6�1 integrin could play arole in epaxial MPC differentiation.

It has been proposed recently that�6�1 may mediate the assembly oflaminin-1 into a matrix (Henry et al.,2001). This study showed that, al-though laminin-1 bound to mouseembryonic stem (ES) cells by meansof dystroglycan, an organized matrix

Fig. 6. Summary of �6�1- and �4�1-expressing cell dynamics during dermomyotomeand myotome development (embryonic day [E] 8.5–E10.5). Left panels shows dermomyo-tomal expression patterns in whole-mounts (without the underlying myotomal patterns),whereas the right panels only demonstrates representative myotomal patterns. The pres-ence of �6�1 is represented in green and of �4�1 in blue; nuclei expressing one or moremyogenic regulatory factors are red. A: At E8.5, �6�1 is faintly expressed in epithelialsomites and dermomyotomes (left). It becomes strongly expressed in early epaxial myo-genic progenitor cells (MPCs) and expression is maintained during myocyte differentiation(right). B: At E9.5, down-regulation of �6�1 starts hypaxially in limb level dermomyotomesand �4�1 is first detected in caudal dermomyotomes (left). Some epaxial myocytesup-regulate �4�1 at late E9.0, and soon after, all myocytes in the adaxial (intercalated)myotome express �4�1 (right). C: At E10.5, both �6�1 and �4�1 are present in caudaldermomyotomes. At interlimb levels, �4�1 is expressed in the hypaxial dermomyotomal lipand rostrally in the epaxial dermomyotomal lip. Dermomyotomal �6�1 expression hasdiminished rostrally (left). �4�1 is strongly expressed in epaxial myocytes, while �6�1expression is becoming reduced. Hypaxial myotome formation has started, and �4�1 isexpressed on hypaxial MPCs, whereas �6�1 is weak or absent (light green). For schematicsimplicity, �6�1- and �4�1-positive myocytes are shown as separate cells. DM, dermomyo-tome; ECM, extracellular matrix; NT, neural tube.

410 BAJANCA ET AL.

did not form in ES cells lacking �1.That �6�1 is the only laminin-bindingintegrin present in early MPCs, sug-gests that it could also play a role inthe organization of laminin as themyotome is being formed. Interest-ingly, myotome formation is delayedin Myf-5-null embryos, as myogeniccells fail to invade the myotomalspace and a laminin-containingbasement membrane does notform (Tajbakhsh et al., 1996). Theseresults indicate that the presenceof MPCs is essential for laminin as-sembly to occur. Nevertheless, trunkmuscles do eventually form in theseembryos (Kablar et al., 1997; Tajba-khsh et al., 1997; Kablar et al., 1998).That �6-null embryos have no re-ported defect in muscle develop-ment (George-Labouesse et al.,1996) does not necessarily meanthat �6�1 has no role in myotomeformation. Other laminin-receptorscould be up-regulated in its ab-sence (reviewed in Hynes, 1996). Al-ternatively, a delay in myotome for-mation can go undetected,because, as evidenced by the phe-notype of Myf-5-null embryos (Braunet al., 1994; Tajbakhsh et al., 1996),this delay does not necessarily leadto abnormal skeletal muscles lateron.

Specific Adhesion Properties ofthe Intercalated Myotome

The �4 subunit, in contrast with �6, isnot present during early epaxialmyotome formation, as evidencedby its timing of appearance and bythe consistent absence of �4-posi-tive cells in the dorsomedial myo-tome. However, �4 is up-regulated inthe fully extended cells in the centralmyotome (see Fig. 6). An extensivework by Sporle (2001) on the char-acterization of different regions ofthe somite, defines an intercalated(adaxial) region in the myotome. It ishypothesized that this region is com-posed of early epaxial myotomalcells, which arise dorsomedially anddue to further growth of the myo-tome become intercalated be-tween the more recently formeddorsal and the hypaxial myotomeregions (Sporle, 2001). Early expres-sion of Fzd9 and Cx40, markers fordifferentiated myocytes, is initially re-

stricted to the intercalated myo-tome and only later found in the dor-sal-most epaxial and hypaxialmyotome regions (Sporle, 2001).Thus, �4 seems to be specifically up-regulated in the intercalated (earlyepaxial, then adaxial) cells as theydifferentiate (see Fig. 6).

The up-regulation of �4 integrin indifferentiating myocytes suggests arole in the process of elongationand/or in the attachment of fullyelongated cells. In fact, the strong�4 immunoreactivity at the tips ofmyocytes and the formation ofplaques between somites is consis-tent with a role in cell attachment.The �4�1 receptor mediates cell–ECM interactions by binding to fi-bronectin (Wayner et al., 1989) andcell–cell interactions by interactingwith VCAM-1 on adjacent cells(Rosen et al., 1992). Our results showthat �4-positive myocytes may bindto the fibronectin matrix surroundingthe myotome, which is enriched atintersomitic borders. However, it isalso possible that these myocytes in-teract with VCAM-1-positive cells inthe mesenchyme around thesomite.

Although the exact function of �4in the intercalated myocytes re-mains unknown, our results demon-strate a developmental change inthe adhesive properties of early ep-axial MPCs. They first express a lami-nin receptor, �6�1, and then, as theydifferentiate into myocytes, theyalso up-regulate a fibronectin/VCAM-1 receptor, �4�1.

�7�1 and �1�1 Appear atLater Stages of MyotomeDevelopment

The �1 subunit mRNA was only de-tected in the myotome at E10.5. Thisfinding is in contrast to a previousstudy in the chick, where �1 wasfound present early in myotome de-velopment (Duband et al., 1992).This contradiction could be due to alesser sensitivity of the mouse probeor may reflect a real difference be-tween the two models. Some differ-ences exist in the expression ofgenes during myotome develop-ment in chick and mouse embryos.For example, the MRF MyoD ap-pears much earlier in the chick than

in the mouse. In the chick it is ex-pressed in the MPCs giving rise to theprimary myotome, while in themouse, the primary myotome differ-entiates through Myf-5 only (Ott etal., 1991; Williams and Ordahl, 1994;Venters et al., 1999).

In agreement with Velling et al.(1996), we show that �7�1 protein ispresent in the myotome from E10.5onward. MyoD has been shown toactivate this integrin in cell culturemodels (Ziober and Kramer, 1996;Bergstrom et al., 2002). Our in vivo re-sults show that �7 is up-regulated ep-axially in elongated, mononucleatedmyocytes after these start expressingMyoD. Moreover, we detected thepresence of �7 in binucleated, MyoD-positive myotubes.

The late appearance of �1�1 and�7�1 suggests that they could medi-ate the attachment of myotomalcells to laminin as �6�1 becomesdown-regulated.

Hypaxial Myotome Is Not aMirror Image of the EpaxialMyotome in Relation toIntegrin Expression Patterns

We show that the integrin subunit �4can be used as a marker for hypax-ial MPCs while �6 is only transientlyand faintly expressed here. It wassurprising to find that �4 was presentin hypaxial MPCs when in the epax-ial myotome it is up-regulated only inelongated myocytes, i.e., in a phaseof terminal differentiation. This obser-vation shows clear differences be-tween hypaxial and epaxial trunkmyogenesis in relation to cell adhe-sion properties (see Fig. 6).

The medial and lateral regions ofthe dermomyotome and myotomereceive distinctly different signals formyogenic induction. Shh and Wnt-1produced from axial structures actas medializing factors and lead toinitial specification of epaxial mus-cle. In contrast, the formation of hy-paxial muscles is induced by a com-bination of signals from ectoderm(Wnt-7a) and lateral plate meso-derm (BMP-4; reviewed in Currie andIngham, 1998). Additionally, differ-ent roles have also been assignedfor Myf-5 and MyoD in epaxial andhypaxial myogenesis (Kablar et al.,1997, 1998; Tajbakhsh et al., 1998; Ta-

INTEGRINS IN THE MOUSE MYOTOME 411

jbakhsh and Buckingham, 2000). Thisraises the interesting possibility thatdifferent regulators of myogenesiscould trigger different effectors,such as the integrins.

Although it is not clear what specificrole �4�1 plays in the hypaxial do-main, it has been shown to play animportant antiapoptotic role in mi-grating neural crest cells in vivo and invitro (Haack and Hynes, 2001), as wellas in other cell types (Koopman et al.,1994; Garcia-Gila et al., 2002). Hypax-ial MPCs are maintained in an undif-ferentiated state by BMP4-mediatedsignals secreted from the lateral platemesoderm (reviewed in Kablar andRudnicki, 2000). However, BMP-4 isalso a potent inducer of apoptosis(e.g., Schmidt et al., 1998), and it hasbeen suggested that the dermomyo-tome/myotome exhibits a gradient ofsensitivity to BMP-4–induced apopto-sis from highest epaxially and lowest inthe hypaxial domain (Schmidt et al.,1998). In the Pax-3 mutant Splotch, hy-paxial MPCs fail to develop properly inthat their delamination is impairedand they show increased apoptosis(Bober et al., 1994; Goulding et al.,1994). It is interesting, thus, to proposethat expression of �4�1 integrin maybe one of the mechanisms protectingearly hypaxial MPCs from apoptosis.

Differences in IntegrinExpression in theDermomyotome

The �6 and �4 integrin subunits areboth present in early dermomyo-tomes (see Fig. 6). However, whenmyotome formation starts, �4 ex-pression becomes restricted to thehypaxial bud of the dermomyotomewhile �6 expression becomes moreand more restricted to the dorsome-dial half from E9.5 onward. The ven-trolateral down-regulation of �6starts at limb levels, at a time whenthe limb MPCs delaminate and initi-ate their migration to the limb buds(Bajanca and Thorsteinsdottir, 2002).�6 expression then progressively de-creases in rostral and trunk dermo-myotomes, proceeding from ventro-lateral to dorsomedial, until itsexpression disappears completely.At later stages, the �4 subunit is alsodetected in the epaxial lip of thedermomyotome but remains absent

in the epaxial MPCs. The reason forthis exclusive presence in the dorso-medial lip is not clear but could berelated to later stages of epaxialmuscle development.

The expression pattern of the �4�1and �6�1 integrins raises the ques-tion of whether they could play arole in promoting the specificationand/or separation of the early epax-ial and hypaxial lineages. A study ofmouse LaacZ/LacZ chimeras dem-onstrated a clonal separation andregionalization of the medial andlateral lineages of MPCs (Eloy-Trin-quet and Nicolas, 2002). The authorssuggest that the acquisition of differ-ent adhesive properties betweenthe epaxial and hypaxial cells pre-paring to translocate to the myo-tome could prevent cell mixingamong the cells of the two clonaldomains or allow a separation of theepaxial and hypaxial cells throughcell sorting. Our results show that thelaminin-receptor �6�1 and the fi-bronectin/VCAM-1 receptor �4�1are potential candidates for a role inseparating epaxial and hypaxialMPCs during early myotome forma-tion in the mouse.

EXPERIMENTAL PROCEDURES

Embryo Collection

Pregnant females (Charles RiverCD-1; Harlan Interfauna Iberica, SA)were killed by cervical dislocationand embryos removed from theuterus in cold phosphate bufferedsaline (PBS). The day of the vaginalplug was designated E0.5. Precisestaging of embryos was obtained bycounting the number of somites aspreviously described by Houzelsteinet al. (1999). The most recentlyformed somite was identified assomite I (Ordahl, 1993).

In Situ Hybridization

The mRNA expression patterns of theintegrin subunits �1, �4, �5 (probeskindly provided by H. Gardner, J.T.Yang, and R.O. Hynes, respectively)and �6 (Thorsteinsdottir et al., 1995)were determined in whole-mountembryos from E9.0 to E10.5. To helpinterpret the patterns obtained, weused probes marking myogenic cellsin different phases of differentiation,

namely Myf-5, MLC1A, and �-car-diac actin (probes kindly providedby M. Buckingham). The transcrip-tion factor Myf-5 is the earliest MRFexpressed by myotomal cells, andthe cytoskeletal proteins MLC1A and�-cardiac actin are specific of differ-entiated, elongated myocytes(Smith et al., 1993; Buckingham etal., 1992; Kitzmann et al., 1998).

In situ hybridization (ISH) withdigoxigenin-labeled riboprobes wasperformed essentially as describedby Henrique et al. (1995). Embryosprocessed for whole-mount ISH werefixed in 4% paraformaldehyde (PFA)in PBS, dehydrated in methanol, andstored at �20°C. For ISH on sections,embryos were fixed in 4% PFA andfrozen for sectioning. Whole-mountswere hybridized at 55°C and cryo-stat sections at 65°C. Probe localiza-tion was detected with BM Purple(Roche). For a more complete anal-ysis of the distribution pattern of inte-grin-positive myogenic cells withinthe myotomes, some whole-mountswere embedded in hydroxyethylmethacrylate (Kulzer Histo-Technik8100) and sectioned.

Immunohistochemistry

Primary antibodies used were rat an-ti-�6 (GoH3; Serotec), rat anti-�7(CA5; a gift from A. Sutherland), rab-bit anti-laminin (Sigma), and rabbitanti-MyoD (a gift from A.J. Harris), alldiluted 1:100; rat anti-�4 (PS2; Sero-tec), rat anti–VCAM-1 (M/K-2; Sero-tec), both diluted 1:50; mouse anti-myosin (F-59; a gift from F.Stockdale) diluted 1:20; rabbit anti–Myf-5 (C-20; Santa Cruz Biotechnol-ogy) diluted 1:2,000 and mouse an-ti–Pax-3 (Venters et al., 2004; a giftfrom C. Marcelle), diluted 1:200.

Secondary antibodies used wereAlexa Fluor 350–conjugated goatanti-mouse immunoglobulin G (IgG),Alexa Fluor 568–conjugated goatanti-mouse IgG, Alexa Fluor 488–conjugated goat anti-rabbit IgG(Molecular Probes), all F(ab�)2 frag-ments and diluted 1:1,000; tetrarho-damine isothiocyanate (TRITC) -con-jugated goat anti-rabbit IgG,fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG, FITC-conjugated rabbit anti-rat (Sigma),all diluted 1:100 and TRITC-conju-

412 BAJANCA ET AL.

gated rabbit anti-rat IgG (Sigma), di-luted 1:160. Some sections were ex-posed to 5 �g/ml 4�,6-diamidine-2-phenylidole-dihydrochloride (DAPI)in 0.1% Triton X-100 in PBS for 2 min.

For IHC on frozen sections, E9.0–E11.5 embryos were fixed in 0.2%paraformaldehyde in 0.1 M phos-phate buffer (PB) with 0.12 mMCaCl2 and 4% sucrose overnight at4°C, washed in buffer only, and thenin PB with 15% sucrose, both over-night at 4°C. Embryos were then in-cubated in PB with 15% sucrose and7.5% gelatin for 1 hr at 30°C, imme-diately frozen in liquid nitrogen-chilled isopentane, and stored at�80°C until sectioned. Approxi-mately 10-�m-thick serial sectionswere collected on Super Frost slides,permeabilized in 0.2% Triton X-100 inPBS for 20 min and blocked for 30min with 10% normal goat serum inPBS containing 1% bovine serum al-bumin (except for anti-fibronectinantibody). Primary antibody incuba-tions were overnight at 4°C, fol-lowed by washing in PBS and sec-ondary antibody incubations werefor 1 hr at room temperature, fol-lowed by PBS washes. When sec-ondary antibodies were againstmouse IgGs, a final 30-min washingstep in 4� PBS was included to re-duce nonspecific background la-beling. Slides were mounted in 2.5%1,4-diazabicyclo-2,2,2-octane in 9:1glycerol:PBS or Vectashield (VectorLaboratories), and sealed after cov-er-slipping.

Whole-mount IHC was performedessentially as described in Ordahl etal. (2001). Embryos were fixed in 0.2%PFA in PBS and permeabilized with0.2% Triton X-100 for 20 min. Antibodystaining was performed as on sec-tions, except secondary antibody in-cubations were done overnight.

Dissociation of Somites

Groups of three to five somites cau-dal to forelimb level of E9.5 embryoswere dissected out and placed ontoSuperfrost Plus slides. They were thentreated with collagenase II (Sigma)at 37°C for 30 min to dissociate cellsand fixed in 0.2% PFA for 30 min(Venters et al., 1999). IHC was car-ried out as described for sections.

Imaging

Embryos processed for whole-mountISH were photographed by using anOlympus Camedia C-4040 digitalcamera coupled to a Wild M8 ste-reomicroscope. Sections processedfor ISH and IHC were photographedby using an Olympus DP50 digitalcamera coupled to an OlympusBX60 microscope equipped with No-marski optics and epifluorescence.Optical Z-series of whole-mount em-bryos were obtained in a Leica SP2confocal microscope. Images wereedited in Adobe Photoshop 7.0.

NOTE ADDED IN PROOFWhile this article was in its proofing

stage, a two-step mechanism formyotome formation in the chick wasdescribed (Gros et al. [2004] DevCell 6:875–882). Our results on inte-grin expression patterns in the mouseappear to be compatible with thismodel.

ACKNOWLEDGMENTSWe thank the following researchersfor their kind gift of cDNA plasmidsand antibodies: Drs. J.T. Yang (�4 in-tegrin cDNA), R.O. Hynes (�5 integrincDNA), M. Buckingham (Myf-5,�-cardiac actin, and MLC1A cD-NAs), H. Gardner (�1 integrin cDNA),A. Sutherland (rat anti-�7), A.J. Harris(rabbit anti-MyoD), F. Stockdale(mouse anti-myosin), C. Marcelle(mouse anti–Pax-3), and D. Henriqueand A. Manaia for helping with theISH technique. This study was fi-nanced by the project PRAXIS XXI/P/PCNA/BIA/131/96 (FCT/FEDER), thePhD grant SFRH/BD/1359/2000 (FCT)to F.B., and a student grant fromPRODEP to M.L. Also, F.B. and S.T. aremembers of the EU/FP6 Network ofExcellence, Cells into Organs.

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