Correlation of GDF5 and Connexin 43mRNA Expression During Embryonic

DevelopmentCYNTHIA M. COLEMAN,1,2 GRACE A. LOREDO,2 CECILIA W. LO,3,4 AND

ROCKY S. TUAN1,2*1Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, andMusculoskeletal and Skin Diseases, National Institutes of Health, Department of

Health and Human Services, Bethesda, Maryland2Department of Orthopaedic Surgery, Thomas Jefferson University,

Philadelphia, Pennsylvania3Biology Department, University of Pennsylvania, Philadelphia, Pennsylvania

4Laboratory of Developmental Biology, National Heart, Lung and Blood Institute,National Institutes of Health, Department of Health and Human Services,

Bethesda, Maryland

ABSTRACTGrowth/differentiation factor 5 (GDF5) regulates connexin expression and enhances

embryonic chondrogenesis in a gap junction-dependent manner, suggesting that GDF5 actionon developmental skeletogenesis is coordinated with gap junction activities. The resultsshown here demonstrate concordance between the mRNA expression profiles of GDF5 andthe gap junction gene, Cx43, in the mouse embryonic limb, spine, and heart, consistent withcoordinated functions for these gene products during developmental organogenesis. Anat RecPart A 275A:1117–1121, 2003. © 2003 Wiley-Liss, Inc.

Key words: growth/differentiation factor 5; connexin 43; chondrogenesis;heart development; spine development; tendon development

Growth/differentiation factor 5 (GDF5) is a key regula-tor of limb skeletal development (Storm et al., 1994; Stormand Kingsley, 1996). GDF5 mRNA expression has beenlocalized to the condensing mesenchyme of the limb and infuture joint spaces, directly correlating with the position,number, and timing of developing joints (Chang et al.,1994; Storm and Kingsley, 1996, 1999; Francis-West et al.,1999). Natural mutations in GDF5 result in the brachypod(bp) phenotype in mice (which consists of a normal axialskeleton and skull, with severely malformed limbs, jointfusions, absent skeletal elements, and bone spurs) andsupporting a role for GDF5 in proper skeletal developmentand joint formation (Gruneberg and Lee, 1973; Storm etal., 1994; Storm and Kingsley, 1996; Clark et al., 2001;Mikic et al., 2001, 2002; Shum et al., 2003). In the firstpublished description of the anatomy of the bp mouse,Gruneberg and Lee (1973) hypothesized that limb malfor-mations resulted from decreased cellular ability to un-dergo condensation. In vitro, bp limb mesenchymal cellsflatten and are unable to adhere to the tissue cultureplate, which suggests that these cells are unable to adhereto and/or communicate with one another (Elmer and Sell-eck, 1975; Owens and Solursh, 1982). In further support of

this hypothesis, we recently demonstrated that GDF5 reg-ulates connexin gene expression, and that the require-ment of gap junction communication in GDF5 stimulatescellular condensation during limb mesenchymal chondro-genesis (Chatterjee et al., 2003; Coleman and Tuan,2003a).

Grant sponsor: NIH; Grant numbers: RO1 ES07005; RO1CA71602; ZO1 AR041131.

Grace A. Loredo’s present address is Sacramento VA MedicalCenter, VA Northern California Health Care System, Mather,California.

*Correspondence to: Rocky S. Tuan, Ph.D., Chief, CartilageBiology and Orthopaedics Branch, National Institute of Arthritis,and Musculoskeletal and Skin Diseases, National Institutes ofHealth, Building 50, Room 1503, MSC 8022, Bethesda, MD20892. Fax: (301) 402-2724. E-mail: Tuanr@mail.nih.gov

Received 9 May 2003; Accepted 6 June 2003DOI 10.1002/ar.a.10125

THE ANATOMICAL RECORD PART A 275A:1117–1121 (2003)

© 2003 WILEY-LISS, INC.

Gap junctions are transmembrane channels made up ofsubunits known as connexins (Cx). Specifically, duringlimb patterning, gap junction-mediated interactions arecritical factors in cellular synchronization and communi-cation (Allen et al., 1990; Coelho and Kosher, 1991a, b;Laird et al., 1992; Dealy et al., 1994; Green et al., 1994;Meyer et al., 1997). It has been demonstrated that gapjunctions play a role during precartilage mesenchymalcondensation by compacting cells, presumably to aid incellular communication and organization (Zimmermannet al., 1982). Connexin 43 (Cx43) has been implicated inearly cartilage development. It has been hypothesizedthat Cx43-containing gap junctions aid in the organizationand compartmentalization of tissues, and permit thetransmission of instructive cues during patterning anddifferentiation (Ruangvoravat and Lo, 1992). Cx43 mRNAexpression is observed in the condensing limb mesen-chyme, but decreases as cells differentiate into chondro-cytes, eventually localizing to the perichondrium. Thissuggests that Cx43-containing gap junctions play a role incompartmentalizing the differentiating chondrogenic corefrom adjacent nonskeletogenic tissues (Dealy et al., 1994;Green et al., 1994; Meyer et al., 1997; Lecanda et al., 1998;Donahue, 2000; Lecanda et al., 2000; Levin 2002).

To further assess the possible functional linkage be-tween GDF5 and Cx43 during developmental chondrogen-esis, we profiled GDF5 and Cx43 mRNA expression duringorganogenesis in mice to ascertain whether there is asimilar spatiotemporal profile during the development ofembryonic structures. CD1 mice (Charles River Laborato-ries, North Franklin, CT) were mated and staged. At 12.5,13.5, 14.5, and 15.5 days postcoitus (dpc), the femaleswere killed. Embryos were collected, fixed overnight in 4%paraformaldehyde, and processed for paraffin embeddingand in situ hybridization according to the method of Waw-ersik and Epstein (2000). The protocol was approved bythe institutional animal care and use committees ofThomas Jefferson University and the National Institutesof Health. For analysis of gene expression, [35S]-labeledantisense and sense probes were generated using cDNAsencoding the 3�UTR of mouse Cx43 (Ruangvoravat andLo, 1992) or a fragment encoding the first 445 base pairsof the mouse GDF5 cDNA (kindly provided by GeneticsInstitute, Cambridge, MA). All transcription reactionswere carried out in the presence of [35S]-UTP (NEN LifeScience Products, Boston, MA). Sections were viewed us-ing dark- and bright-field optics on a Jenaval microscope(Jena, Germany). Images were acquired utilizing a Kon-tron Progress 3012 digital camera (Munich, Germany).

Our results revealed similar expression patterns in thedeveloping limb, tendon, spine, and heart, consistent withthe hypothesis that GDF5 and Cx43-containing gap junc-tions act in conjunction with each other during organogen-esis. In addition, we demonstrated for the first time GDF5mRNA expression in the heart and the cartilage primor-dium of the neural arch.

RESULTSCx43 and GDF5 Expression in the DevelopingLimb

GDF5 mRNA expression was localized to the condens-ing digit and long bones of the hindlimb at 12.5 dpc (datanot shown). Cx43 transcripts were previously documented

in the condensing mesenchyme of the limb at a similarstage (Meyer et al., 1997). GDF5 and Cx43 transcriptswere both localized to the perichondral regions of theforelimb of a 13.5 dpc embryo, with GDF5 localizationpersisting to 14.5 dpc (Fig. 1A and C, and data not shown).GDF5 transcripts were also localized to the presumptiveelbow joint (Fig. 1A) and hip joint surrounding the femoralhead from 12.5 to 14.5 dpc (data not shown).

The expression of GDF5 also mirrored that of Cx43during tendon development. GDF5 mRNA was observedin the tendon of the hip in a 14.5 dpc embryo (Fig. 1E).Cx43 mRNA transcripts were detected in the dermis andsurrounding the tendons in each toe of a 15.5 dpc mouse(Fig. 1G).

Cx43 and GDF5 Expression in the DevelopingSpine

Cx43 and GDF5 transcript expression patterns weresimilar during vertebral development. GDF5 mRNA sig-nal was observed in the cartilage primordium of the neu-ral arch from 12.5 dpc to 14.5 dpc (Fig. 1I and data notshown). Cx43 was observed in the spine of a 13.5 dpcembryo, specifically associated with the internal and ex-ternal lining of the neural tube, as well as surrounding thecondensing vertebrae (Fig. 1K). GDF5 and Cx43 tran-scripts are thus expressed in closely adjacent structures(the cartilage primordium of the neural arch and the ver-tebrae, respectively).

GDF5 and Cx43 Expression in the Heart at 13.5dpc

A transverse section of a 13.5 dpc embryo revealedGDF5 expression in the heart (Fig. 1M). This pattern wasobserved only at 13.5 dpc of development. GDF5 tran-scripts were also localized in the expected pattern in thedeveloping limb, including the shoulder and elbow joints,and the perichondrium of the radius/ulna and digits. Cx43transcripts (Fig. 1O) were detected in the heart ventriclein a spatiotemporal expression pattern similar to that ofGDF5.

DISCUSSIONIn this study, we profiled GDF5 and Cx43 mRNA ex-

pression during mouse organogenesis. The results showeda large degree of similarity in their spatiotemporal expres-sion profiles during the development of embryonic struc-tures, including the developing limb, tendon, spine, andheart, consistent with the hypothesis that GDF5 andCx43-containing gap junctions act in conjunction with oneanother during organogenesis.

In the developing appendicular skeleton, expression ofboth GDF5 mRNA and Cx43 transcripts localizes to thecondensing mesenchyme and the perichondrium, and inthe interdigital mesenchyme (Meyer et al., 1997). Thissimilarity in expression profiles supports our recent find-ings that GDF5 action depends on functional gap junctioncommunication (specifically Cx43-containing junctions)during the condensation phase of endochondral ossifica-tion (Coleman and Tuan, 2003a). In vivo, gap junction-mediated communication at this stage may initiate and/orpropagate the synchronization of cells in the condensingmesenchyme to initiate cellular differentiation, as well assegregate the maturing chondrocytes of the long bones

1118 COLEMAN ET AL.

from the adjacent mesenchyme by creating a cell commu-nication barrier along the perichondral regions. However,GDF5 is expressed in the developing joint, where Cx43 isabsent. It has been hypothesized that GDF5 influenceschondrocyte maturation during joint development, possi-bly through a different pathway from that involved inprecartilage condensation (Francis-West et al., 1999;Coleman and Tuan, 2003b).

Cx43 and GDF5 share a similar spatiotemporal profileof mRNA expression during tendon development in the

limb. Supporting a role for GDF5 in tendon formation,ectopic GDF5 protein was previously demonstrated tostimulate neotendon growth (Wolfman et al., 1997). Addi-tionally, studies in the bp mouse have also shown that inthe absence of active GDF5, tendon placement is alteredand the overall strength of the tendons is reduced(Gruneberg and Lee, 1973; Mikic et al., 2001). Therefore,we propose that GDF5 influences tendon developmentthrough a mechanism similar to that observed in chondro-genesis, including Cx43-containing gap junctions, to orga-

Fig. 1. Developmental expression of GDF5 and Cx43 mRNA inmouse embryonic limb, tendon, spine, and heart. Sections of mouseembryos at 12.5 (I and J), 13.5 (C, D, and K–P), 14.5 (A, B, E, and F), and15.5 (G and H) dpc were probed with GDF5 and Cx43 antisense probesas indicated. Dark-field images and corresponding Mallory-stainedclosely adjacent sections are presented. In the developing limb (A–H),GDF5 transcripts localized to the elbow joint (A). Cx43 transcripts wereobserved in the perichondrium of the long bones (C). GDF5 transcriptslocalized to the tendons of the hip (E), just as Cx43 transcripts hybridizedin the dermis and tendons of the foot (G). In the developing spine, GDF5transcripts localized to the developing cartilage primordium of the neural

arch (I). Cx43 transcripts were seen in the lining of the neural tube andsurrounding the condensing vertebrae (K). In the developing heart, GDF5transcripts localized to both the ventricle and the atrium of the heart (M).Additionally, as reported previously (Chang et al., 1994; Storm et al.,1994; Storm and Kingsley, 1996; Francis-West et al., 1999), GDF5 tran-scripts were detected in the shoulder and elbow joints and the perichon-dral regions of the digit. Cx43 mRNA was detected in the ventricle of theheart (O). Abbreviations: a, atrium; c, cartilage primordium of the neuralarch; d, digit; de, dermis; e, elbow; f, femur; lu, lungs; n, neural tube; r/u,radius/ulna; te, tendon; v, vertebrae. Scale bar is 500 �m in all panelsexcept E–H, where the scale bar is 5 mm.

1119CORRELATION OF GDF5 AND CX43 EXPRESSION

nize cellular populations and regulate their placementand maturation during development.

GDF5 and Cx43 are also coexpressed in immediatelyadjacent tissues during vertebral development. Cx43 ex-pression is specifically localized to the lining of the neuraltube and surrounding the condensing, differentiating ver-tebrae. GDF5 is expressed in regularly spaced intervalsadjacent to the condensing vertebrae, localizing to thecartilage primordium of the neural arch. Although bothCx43 and GDF5 are expressed in the same general regionat similar times, their expression is actually localized totwo distinct structures. GDF5 in the cartilage primordiumof the neural arch may function by activating cellularresponses, including the expression of Cx43, to maintainthe segmented vertebral structure. A similar mechanismof action during joint development has been proposed forGDF5 (Hartmann and Tabin, 2001; Coleman and Tuan,2003b). Additionally, GDF5 may induce the expression ofconnexins other than Cx43 in the cartilage primordium ofthe neural arch, thereby stimulating a similar mechanismof cellular communication through gap junctions leadingto the differentiation of the neural arch. The expressionprofiles of other connexin genes in this region have notbeen explored in this study.

GDF5 mRNA expression is observed in the developingheart, specifically at 13.5 dpc. Cx43 expression duringcardiac development begins at 9.5 dpc and continuesthroughout organogenesis (Ruangvoravat and Lo, 1992);therefore, it is most likely regulated by a factor(s) otherthan GDF5. However, it is very interesting that GDF5,which is known for its chondro-stimulatory activity, isexpressed during cardiac development. During endochon-dral ossification in the limb, GDF5 enhances cellular ad-hesion, proliferation, and maturation. It is possible thatduring cardiac development GDF5 plays a similar role ininfluencing cell adhesion and proliferation. Alternatively,GDF5 may regulate cellular synchronization by influenc-ing gap junction-mediated cellular communication or cel-lular organization by regulating the expression of otherconnexins. This interesting finding warrants further ex-ploration in the future.

In conclusion, we have demonstrated similar spatiotem-poral developmental mRNA expression patterns of Cx43and GDF5 in the developing limb, tendon, spine, andheart. These findings are consistent with the hypothesisthat GDF5 influences cellular communication and the re-sultant synchronization and organization of tissues dur-ing differentiation and organogenesis, and does sothrough Cx43-containing gap junctions.

LITERATURE CITEDAllen F, Tickle C, Warner A. 1990. The role of gap junctions in

patterning of the chick limb bud. Development 108:623–634.Chang SC, Hoang B, Thomas JT, Vukicevic S, Luyten FP, Ryba NJ,

Kozak CA, Reddi AH, Moos Jr M. 1994. Cartilage-derived morpho-genetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones duringhuman embryonic development. J Biol Chem 269:28227–28234.

Chatterjee B, Meyer RA, Loredo GA, Coleman CM, Tuan RS, Lo CW.2003. BMP regulation of the mouse connexin-43 promoter in osteo-blastic cells and embryos. Cell Adhes Commun 10:37–50.

Clark RT, Johnson TL, Schalet BJ, Davis L, Gaschen V, Hunziker EB,Oldberg A, Mikic B. 2001. GDF-5 deficiency in mice leads to disrup-tion of tail tendon form and function. Connect Tissue Res 42:175–186.

Coelho CN, Kosher RA. 1991a. Gap junctional communication duringlimb cartilage differentiation. Dev Biol 144:47–53.

Coelho CN, Kosher RA. 1991b. A gradient of gap junctional commu-nication along the anterior-posterior axis of the developing chicklimb bud. Dev Biol 148:529–535.

Coleman CM, Tuan RS. 2003a. Functional role of growth/differentia-tion factor 5 in chondrogenesis of limb mesenchymal cells. MechDev 120:823–836.

Coleman CM, Tuan RS. 2003b. Growth/differentiation factor 5 en-hances chondrocyte maturation. Dev Dyn 228:208–216.

Dealy CN, Beyer EC, Kosher RA. 1994. Expression patterns of mRNAsfor the gap junction proteins connexin43 and connexin42 suggest theirinvolvement in chick limb morphogenesis and specification of thearterial vasculature. Dev Dyn 199:156–167.

Donahue HJ. 2000. Gap junctions and biophysical regulation of bonecell differentiation. Bone 26:417–422.

Elmer WA, Selleck DK. 1975. In vitro chondrogenesis of limb meso-derm from normal and brachypod mouse embryos. J Embryol ExpMorphol 33:371–386.

Francis-West PH, Abdelfattah A, Chen P, Allen C, Parish J, Ladher R,Allen S, MacPherson S, Luyten FP, Archer CW. 1999. Mechanismsof GDF-5 action during skeletal development. Development 126:1305–1315.

Green CR, Bowles L, Crawley A, Tickle C. 1994. Expression of theconnexin43 gap junctional protein in tissues at the tip of the chicklimb bud is related to the epithelial-mesenchymal interactions thatmediate morphogenesis. Dev Biol 161:12–21.

Gruneberg H, Lee AJ. 1973. The anatomy and development of brachy-podism in the mouse. J Embryol Exp Morphol 30:119–141.

Hartmann C, Tabin CJ. 2001. Wnt-14 plays a pivotal role in inducingsynovial joint formation in the developing appendicular skeleton.Cell 104:341–351.

Laird DW, Yancey SB, Bugga L, Revel JP. 1992. Connexin expressionand gap junction communication compartments in the developingmouse limb. Dev Dyn 195:153–161.

Lecanda F, Towler DA, Ziambaras K, Cheng SL, Koval M, SteinbergTH, Civitelli R. 1998. Gap junctional communication modulatesgene expression in osteoblastic cells. Mol Biol Cell 9:2249–2258.

Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, CivitelliR. 2000. Connexin43 deficiency causes delayed ossification, cranio-facial abnormalities, and osteoblast dysfunction. J Cell Biol 151:931–944.

Levin M. 2002. Isolation and community: a review of the role ofgap-junctional communication in embryonic patterning. J MembrBiol 185:177–192.

Meyer RA, Cohen MF, Recalde S, Zakany J, Bell SM, Scott Jr WJ, LoCW. 1997. Developmental regulation and asymmetric expression ofthe gene encoding Cx43 gap junctions in the mouse limb bud. DevGenet 21:290–300.

Mikic B, Schalet BJ, Clark RT, Gaschen V, Hunziker EB. 2001.GDF-5 deficiency in mice alters the ultrastructure, mechanicalproperties and composition of the Achilles tendon. J Orthop Res19:365–371.

Mikic B, Battaglia TC, Taylor EA, Clark RT. 2002. The effect ofgrowth/differentiation factor-5 deficiency on femoral compositionand mechanical behavior in mice. Bone 30:733–737.

Owens EM, Solursh M. 1982. Cell–cell interaction by mouse limb cellsduring in vitro chondrogenesis: analysis of the brachypod mutation.Dev Biol 91:376–388.

Ruangvoravat CP, Lo CW. 1992. Connexin 43 expression in the mouseembryo: localization of transcripts within developmentally signifi-cant domains. Dev Dyn 194:261–281.

Shum L, Coleman CM, Hatakeyama Y, Tuan RS. 2003. Morphogen-esis and dysmorphogenesis of the appendicular skeleton. Birth De-fects Res-C: Embryo Today (in press).

Storm EE, Huynh TV, Copeland NG, Jenkins NA, Kingsley DM, LeeSJ. 1994. Limb alterations in brachypodism mice due to mutationsin a new member of the TGF beta-superfamily. Nature 368:639–643.

1120 COLEMAN ET AL.

Storm EE, Kingsley DM. 1996. Joint patterning defects caused bysingle and double mutations in members of the bone morphogeneticprotein (BMP) family. Development 122:3969–3979.

Storm EE, Kingsley DM. 1999. GDF5 coordinates bone and jointformation during digit development. Dev Biol 209:11–27.

Wawersik S, Epstein JA. 2000. Gene expression analysis by in situhybridization. In: Tuan RS, Lo CW, editors. Developmental biologyprotocols. Vol. III. Totowa: Humana Press, Inc. p 87–96.

Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N,Dube JL, DiBlasio-Smith E, Nove J, Song JJ, Wozney JM, Rosen V.1997. Ectopic induction of tendon and ligament in rats by growthand differentiation factors 5, 6, and 7, members of the TGF-betagene family. J Clin Invest 100:321–330.

Zimmermann B, Scharlach E, Kaatz R. 1982. Cell contact and surfacecoat alterations of limb-bud mesenchymal cells during differentia-tion. J Embryol Exp Morphol 72:1–18.

1121CORRELATION OF GDF5 AND CX43 EXPRESSION