INTRODUCTION

Mechanical forces have often been proposed as moldingfactors in morphogenesis. In motile organs, such as the devel-oping heart, possible morphogenetic mechanical stress is aconsequence of the organ’s function. In other cases morpho-genetic mechanical forces are more obscure and were onlydeduced on the basis of changes in the shape of the cellularand tissue components of the embryonic organs. In these casesthe cells appear to modify the surrounding extracellular matrixby mechanical traction causing a spatial realignment in thematrix, which in turn modifies the behavior of the cells; suchinteractions may provide positive feedback, which may beinvolved in complex shaping processes (Harris et al., 1981;Stopak and Harris, 1982).

Different types of molding mechanical effects have beendemonstrated to be responsible for events during limb mor-phogenesis. Extrinsic mechanical stress originating frommuscle contraction plays a significant role in joint cavitation(Mitrovic, 1982) and prevents degeneration of the long autopo-dial tendons (Kieny and Chevallier, 1979; Brand et al., 1985),but both joints and long tendons can be formed initially in theabsence of muscle activity (Mitrovic, 1982; Shellswell andWolpert, 1977; Kieny and Chevallier, 1979). Fine locallygenerated mechanical stress has also been proposed to beresponsible for limb morphogenetic events (Rooney et al.,1984). The mechano-chemical theory proposed by Oster et al.(1985) and Oster and Murray (1989) takes into account thegeneration of tension by condensing mesenchymal cells and itssubsequent transmission to the extracellular matrix as a

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In this work we have analyzed the presence of elastic com-ponents in the extracellular matrices of the developingchick leg bud. The distributions of elastin and fibrillin werestudied immunohistochemically in whole-mount prepara-tions using confocal laser microscopy. The association ofthese constituents of the elastic matrix with other compo-nents of the extracellular matrix was also studied, usingseveral additional antibodies. Our results reveal thetransient presence of an elastin-rich scaffold of extracellu-lar matrix fibrillar material in association with the estab-lishment of the cartilaginous skeleton of the leg bud. Thescaffold consisted of elastin-positive fibers extending fromthe ectodermal surface of the limb to the central cartilage-forming regions and between adjacent cartilages. Fibrillinimmunolabeling was negative in this fibrillar scaffold whileother components of the extracellular matrix including:tenascin, laminin and collagens type I, type III and type VI;appeared codistributed with elastin in some regions of thescaffold. Progressive changes in the spatial pattern of dis-tribution of the elastin-postive scaffold were detected in

explant cultures in which one expects a modification in themechanical stresses of the tissues related to growth.

A scaffold of elastin comparable to that found in vivo wasalso observed in high-density micromass cultures ofisolated limb mesodermal cells. In this case the elastic fibersare observed filling the spaces located between the carti-laginous nodules. The fibers become reoriented and attachto the ectodermal basal surface when an ectodermalfragment is located at the top of the growing micromass.Our results suggest that the formation of the cartilaginousskeleton of the limb involves the segregation of the undif-ferentiated limb mesenchyme into chondrogenic and elas-togenic cell lineages. Further, a role for the elastic fiberscaffold in coordinating the size and the spatial location ofthe cartilaginous skeletal elements within the limb bud isalso suggested from our observations.

Key words: limb development, chick embryo, chondrogenesis,extracellular matrix, elastin, fibrillin, collagen, morphogenesis

SUMMARY

Elastin exhibits a distinctive temporal and spatial pattern of distribution in the

developing chick limb in association with the establishment of the

cartilaginous skeleton

Juan M. Hurle1,*, Glen Corson2, Karla Daniels1, Rebecca S. Reiter1, Lynn Y. Sakai2 and Michael Solursh1,†

1Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242, USA2Shriner’s Hospital for Crippled Children, and Department of Biochemistry and Molecular Biology, Oregon Health SciencesUniversity, Portland, Oregon 97201, USA

*Author for correspondence at present address: Departamento de Anatomía y Biología Celular, Facultad de Medicina, Polígono de Cazona, Universidad de Cantabria,Santander, Spain†The authors dedicate this paper to the memory of Professor Michael Solursh, who died when this paper was in the course of publication

Journal of Cell Science 107, 2623-2634 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

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primary factor accounting for the morphogenesis of the limbskeleton. Experimental support for this hypothesis is scarce,but Stopak and Harris (1982) have clearly shown a moldingeffect of the growing cells on the surrounding extracellularmatrix under in vitro conditions. Further, these authors (Stopaket al., 1985) have also found that labeled collagen injected intothe limb bud is rearranged by traction of the limb mesenchy-mal cells. Whether this traction is also exerted in the matrixelaborated within the limb cannot be deduced from theseexperiments but the demonstration of extracellular matriceswith tensile properties (see Foster, 1982; Mecham and Heuser,1991; Pasquali-Ronchetti, et al., 1993; Rosenbloom et al.,1993) would be of great interest and would help to confirm therole of tension in normal development.

Elastic fibers are major components of the ECM in adult ver-tebrate organs and tissues subjected to mechanical tension,such as skin, lungs, blood vessels and ligaments (see Pasquali-Ronchetti et al., 1993). At the ultrastructural level elastic fibersconsist of amorphous tracts surrounded by small fibrils, 10-12nm in diameter, usually termed ‘elastin-associate microfibrils’(Cleary and Gibson, 1983). Elastin is the major component ofthe amorphous material and consists of a highly insolubleprotein with a characteristic rubber-like elasticity (Gray et al.,1973; Sage and Gray, 1979). Several glycoproteins have beenidentified in association with the microfibrillar component ofthe elastic fibers, including: fibrillin (Sakai et al., 1986),MAGP (microfibril-associated glycoprotein; Gibson et al.,1990), and emilin (elastic microfibril interface-located protein;Bressan et al., 1993). In general, little is known about thefunction of most of the elastin-associated glycoproteins, andall of them have been shown to have a wider distribution thanthat of elastin. The identification of alterations in elastin-asso-ciated glycoproteins in genetic diseases affecting the elasticproperties of the tissues, such as the Marfan syndrome (Dietzet al., 1991; Kainulainen et al., 1992; Ramirez et al., 1993),suggests an important role for at least some of these compo-nents in the elastic functions. The study of the distribution ofelastin and associated glycoproteins in new locations duringembryonic development may provide important insights into,and help to clarify the significance of, the molecular complex-ity of the elastic matrices. However, information on the distri-bution of the elastic matrix components in embryonic tissuesis scarce (Rosenquist et al., 1988; Foster et al., 1989; Gallagheret al., 1993; Holzenberger et al., 1993) and elastin is oftenthought to be a component produced in later stages of the fetalperiod (Clearly et al., 1967; Parks et al., 1988).

In this work we have analyzed the presence of elastic com-ponents in the extracellular matrices of the developing chickleg bud in vivo and in vitro. Elastin and fibrillin were detectedimmunohistochemically with monoclonal antibodies in whole-mount preparations using confocal laser microscopy. The asso-ciation of these proteins with other components of the extra-cellular matrix was also studied with a variety of monoclonalantibodies to components of the extracellular matrix. Ourresults reveal the transient presence of an elastin-rich scaffoldof extracellular matrix fibrillar material in association with theestablishment of the cartilaginous skeleton of the leg bud. Themost prominent components of this matrix scaffold are elastinfibers, extending from the ectodermal basal surface to thecentral chondrogenic region of the limb bud and betweenadjacent developing cartilages. A comparable scaffold is also

formed in micromass cultures of isolated limb mesenchymalcells between the developing chondrogenic nodules. Thescaffold underwent progressive changes in its spatial distribu-tion in explant cultures in which one expects a change in themechanical stresses of the tissues that is related to growth. Arole for the elastic fiber scaffold in coordinating the size andthe spatial location of the cartilaginous skeleton within the limbbud is suggested from our observations.

MATERIALS AND METHODS

MaterialsWhite Leghorn chick embryos ranging from stage 22 to 35(Hamburger and Hamilton, 1951) were used in the present study.

ImmunohistochemistryThe leg buds were dissected free and fixed at 4°C for up to 8 hoursin methanol plus 20% dimethyl sulfoxide (DMSO). The specimenswere then washed in Tris-buffered saline (TBS) and digested for 30(1-3 hours) minutes in 1600 units/ml of testicular hyaluronidase(Sigma). After a prolonged rinse in TBS they were immersed for 30minutes in 10% goat serum in 10 mM glycine and transferred to PBScontaining the primary antibody for 6 hours (undiluted hybridomasupernatant or purified antibodies diluted in PBS at a concentrationof 10

µg/ml). The specimens were washed again for 3-5 hours in TBS,incubated overnight in fluorescein-conjugated goat anti-mouse IgG(Cappel) diluted at 1:300, washed thoroughly in TBS and attachedwith Cell-Tack (Becton Dickinson Labware, Bedford, MA) toexcavated slides. After dehydration in methanol the cavity of the slidewas filled with a 2:1 (v/v) mixture of benzyl benzoate:benzyl alcoholand sealed with a coverglass attached to the slide by means of nail-polish. In some cases, after labeling with the second antibody thelimbs were carefully dissected under the microscope and attached tothe slides as described above. Controls omitting the first antibodywere also made.

Elastic components of the ECM were detected by monoclonal anti-bodies to elastin and to fibrillin (see below). Extracellular matrix com-ponents associated with the elastic matrix were studied with anti-bodies obtained from the Developmental Studies Hybridoma Bank,including monoclonal antibodies to: tenascin (M1-B4, from D. M.Fambrough), laminin (31, from D. M. Fambrough), collagen type II(II-II6b3, from T. F. Linsenmayer), collagen type III (3b2, from R.Mayne), and collagen type VI (39, from D. M. Fambrough). Mono-clonal antibodies to collagens type I (Linsenmayer et al., 1979) typeIV (Fitch et al., 1982) and type X (Schmid and Linsenmayer, 1985)were obtained from Dr T. F. Linsenmayer.

The specimens were examined in a Bio-Rad MRC-600 (Bio-RadLaboratories, Richmond, CA) scanning confocal microscopeequipped with a krypton-argon laser.

Antibodies to elastic matrix componentsThe monoclonal antibody for elastin (mAb 10B8) was produced byimmunizing Balb/c mice (Jackson Laboratories) with preparations ofchick tropoelastin from 17-day-old lathyric chick embryo aortae,extracted and purified according to established methods (Rich andFoster, 1982). The immunization schedule, fusion protocol andcloning of hybridomas were performed as described previously (Sakaiet al., 1982). Hybridomas were screened by immunofluorescenceusing sections of chick aorta. mAb 10B8 was selected and shown tobe specific for elastin by immunoblotting of purified chick tropoe-lastin and by immunoprecipitation of [3H]valine-chick tropoelastinfrom extracts of chick aorta organ cultures (see Fig. 1). We found thatthe epitope recognized by mAb 10B8 is conserved in bovine andhuman tissues, since it reacts with tissues from all three species. A

J. M. Hurle and others

2625Elastin in the developing chick limb

commercially available monoclonal antibody specific for bovineelastin (mAb BA-4) (Sigma), which crossreacts with human but notwith chicken elastin, was used as a control.

The specificity of the fibrillin monoclonal antibody (mAb 201) hasbeen described (Sakai et al., 1986). While the immunizing antigenwas human, mAb 201 recognizes chick fibrillin (Gallagher et al.,1993).

For these experiments, mAb 10B8 and mAb 201 were purified fromcell culture medium using a Protein G/Sepharose colum (Pharmacia).The antibody was eluted from the column with 0.1 M glycine/HCl(pH 2.5), neutralized, and dialyzed against phosphate buffered saline(PBS). The antibodies were typically stored at concentrations of 1-2mg/ml.

ImmunoassaysImmunoblotting of elastin obtained from cultures of human aorticsmooth muscle cells (Clonetics) was performed as described previ-ously (Sakai et al., 1986).

For immunoprecipitation, aortae from 17-day-old chicks weredissected and incubated overnight in Dulbecco’s modified Eagle’smedium (made without valine) with 50 µCi/ml [3H]valine(Amersham). The aortae were then extracted in 50 mM Tris-HCl, 0.5M NaCl, pH 7.5, in the presence of protease inhibitors (3 mM EDTA,1 mM phenylmethylsulfonyl fluoride). The extract was clarified bycentrifugation and the supernatant was applied to a 10B8 antibodyaffinity column, constructed by coupling antibody to CNBr-activatedSepharose (Pharmacia) according to the manufacturer’s instructions.The column was washed extensively with PBS containing 0.05%Tween-20. Bound protein was eluted with 0.1 M glycine/HCl (pH 2.5)and visualized by SDS-PAGE and fluorography.

Organ culturesOrgan cultures were made from explants from leg autopodia betweenstages 28 and 31. The whole autopodium and tissue fragments con-taining the following elements: (i) only an isolated digit; (ii) a digitplus the adjoining interdigital tissue; (iii) two digits with the inter-vening interdigital tissue; and (iv) only a tissue wedge obtained fromthe third interdigit; were transferred to 35 mm dishes and cultured for24-48 hours in Ham’s F-12 medium supplemented with 10% fetal calfserum and antibiotics. In some cases the explants were allowed toattach to the dish surface and in other cases they were maintainedfloating in the medium. At the desired stage, the explants were washedin saline and processed for immunohistochemistry as described above.

Micromass culturesHigh-density micromass cultures were prepared according to theAhrens procedure (Ahrens et al., 1977). Limb mesodermal cells weredisaggregated from whole limb buds at stage 22-23 after a shortdigestion with 0.1% trypsin and 0.1% collagenase in calcium- andmagnesium-free saline. The cells were then resuspended at a concen-tration of 2×104/µl in Ham’s F-12 medium (Gibco) supplementedwith 10% fetal calf serum and antibiotics. Drops (10 µl) of cell sus-pension were plated out in 35 mm tissue culture dishes. After 1 dayof culture a small fragment of ectoderm, isolated by trypsin-pancre-atin treatment from limb buds of stage 23, was located at the top ofsome cultures and allowed to grow for 3 days (Solursh et al., 1981).

Extracellular matrix distribution in the cultures was studiedimmunohistochemically in micromasses with or without ectoderm,following the procedure described above.

RESULTS

Monoclonal antibody to elastinMonoclonal antibody 10B8 reacted in ELISA with the chicktropoelastin used for immunization; this antibody also yielded

a lamellar immunofluorescent staining pattern in cross-sectionsof chick aorta (data not shown). mAb 10B8 immunoprecipi-tates chick tropoelastin (Fig. 1) and immunoblots humanelastin (data not shown). The commercially available mAbBA-4 immunoblotted the same band identified by mAb 10B8in preparations of human elastin. However, unlike mAb BA-4, mAb 10B8 binds to chicken elastin and can be used for tissuelocalization studies without requiring unmasking of tissuesection with trypsin.

The elastic matrix of the developing limb bud Two successive and distinct patterns of elastin distributionwere found in the developing limb bud. Initially, elastin wasobserved forming a complex scaffold within the undifferenti-ated limb mesenchyme. The scaffold was established in aproximo-distal sequence in the limb bud concomitantly withthe proximo-distal pattern of chondrification of the limbskeletal elements. During the next stages elastin was observedin the developing limb structures with expected elastic prop-erties such as joints and tendons.

The first sign of elastin scaffold formation was detected atstage 25-26. At this stage the limb bud appears elongated andshows a clear autopodial plate without distinguishable digitalelements. In whole mounts stained for cartilage the earliestanlage of the stylopodial (femur) and zeugopodial (tibia andfibula) cartilages are observed. A dorsal view of the limb underthe confocal microscope, immunostained for elastin, reveals a

Fig. 1. Monoclonalanti-elastin (mAb10B8) was used toimmunoaffinity purifytropoelastin extractedfrom aortae removedfrom 17-day-oldlathyric chick embryos.Organ cultures ofaortae weremetabolically labeledwith [3H]valine andextracted with 0.5 MNaCl, 0.05 M Tris-HCl,pH 7.5 (lanes 1 and 4).The soluble extract wasapplied to a mAb 10B8-Sepharose column;unbound proteinsflowed through thecolumn (lane 2). Afterwashing the column,bound protein waseluted (lanes 3 and 5).Proteins werevisualized after SDS-PAGE andfluorography. Samplesin lanes 1, 2, and 3were applied without 2-

mercaptoethanol; samples in lanes 4 and 5 contained 2-mercaptoethanol. The mobilities of prestained molecular massmarkers are indicated (kDa). Bound protein migrated, both with andwithout reduction of disulfide bonds, to a position (around 70 kDa)consistent with the electrophoretic characteristics of tropoelastin.

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precise distribution of elastin fibers in the anterior half of thedistal stylopodium and through the zeugopodium (Fig. 2A).The autopodial plate at this stage lacks elastin immunolabel-ing. Elastin fibers run perpendicularly to the longitudinal axis

of the limb from the anterior margin towards the central zoneof the bud, where they terminate. In transverse sections of thelimb the pattern of elastin fibers can be clearly appreciated(Fig. 2B). Fibers are always restricted to the anterior half of

J. M. Hurle and others

2627Elastin in the developing chick limb

the limb bud and appear to originate from the ectodermal basalsurface and run deeply towards the central cartilaginous axis.The fibers end in the contour of the cartilaginous elementswithout penetrating it.

An intense increase in elastic fibers was detected by stage27-28 but the basic scheme of a fibrillar system running fromthe ectoderm surface towards the central cartilaginous core ofthe limb is conserved. The fibrilar system observed in previousstages is no longer restricted to the anterior half of the limb butelastin fibers originate from most of the ectodermal surface andpenetrate deeply, reaching the surface of the developing carti-lages (Fig. 2C). In the next stages the pattern of elastin distri-bution in the zeugopod and stylopod becomes obscured by theformation of muscle bellies, which causes an extensive redis-tribution of the extracellular matrix components. In thefollowing stages elastin is detected at the zones of muscleinsertion and at the level of origin of the tendons (not shown).

From stage 28 the distribution of the elastic scaffold extendsto the developing autopodium where a sharp increase in elastinimmunoreactivity is observed in association with the formationof the metatarsal cartilages and digital rays. As can be seen inFig. 2D, numerous fibers are now detected radiating from thecondensing digital rays towards the ectodermal basal surfaceand towards the developing interdigital spaces. In the inter-digital spaces the fibers coming from each digit are ratherconfluent and tend to be directed towards the distal margin ofthe interdigit where they are lost in association with the distalsubridge mesenchyme. No anterior regionalization of theelastin fibers was detected in this region, since elastin fibersare associated with all the developing digital rays.

From stage 29 two distinct components of the elastin fiberscan be distinguish in the autopodium (Fig. 2E). One componentoriginates from the digital rays and radiates out towards theectoderm and interdigital space (Fig. 2E-F). The other

component occupies each interdigital space (Fig. 2E,G) andappears as a bundle extraordinarily rich in elastin fiberscondensed in the space limited by the distal end of themetatarsal cartilages and diverging in a fan-like fashion at thedistal portion of the interdigit where the fibers are lost eitherin the prechondrogenic tips of the digits or in the ectodermalbasal surface (Fig. 2H). From stage 33 the interdigital fibrillarcomponents become progressively lost in association withinterdigital tissue regression, and by stage 35 the elastin-positive material appears as a network of thick fibrillarbundles, which are concentrated at the distal margin of theregressing interdigit. The radiating elastin-containing fibersassociated with the digital rays become progressively substi-tuted in a proximo-distal fashion due to the rearrangement ofthe extracellular matrix associated with the development of thetendons. As can be seen in Fig. 3A, the developing tendons arepositive for elastin labeling and in the course of their trajec-tory along the surface of the developing phalanges they sendnumerous filamentous projection, which appear to anchor thetendons to the perichondrial surface, giving a feather-likeappearance to the tendons when observed at high magnifica-tion.

As we have mentioned above, in addition to the fibrillarscaffold associated with the developing skeleton, from stage29 elastin also appears associated with the developing musclesin the zones of insertion or tendon origin, and also in the devel-oping joints and in the long tendons of the autopodium, asdescribed above, but a detailed analysis of this elastin-positivematrix is beyond the scope of this study (unpublished data).

In contrast to elastin, fibrillin exhibited a more restrictedpattern of distribution (Fig. 3B,C). The early fibrillar patternof the limb bud at stage 25-26 and the elastin associated withthe digital rays and the interdigital tissue were all negative forfibrillin. The first appearance of fibrillin was detected in theautopodium at stage 28-29 in association with the formation ofthe earliest anlage of the developing tendons. At this stagefibrillin produced a fibrillar labeling pattern under the ectoder-mal surface of the developing ankle that was concentrated inthe proximal part of the developing digital rays. From stage 32fibrillin labeling was restricted to the perichondrium, joints andtendons. At the level of the joints fibrillin was codistributedwith elastin but elastin labeling was more intense. Fibrillinlabeling was also noted in the perichondrium (Fig. 3B), whichis also slightly positive for elastin. In the developing tendonsfibrillin was detected in the main tendinous blastemas (Fig. 3C)but not in the thin fibrillar component anchoring the tendonsto the developing phalanges.

Codistribution of other extracellular matrix componentswith elastin was observed from stage 28-29 during theformation of joints, tendons and perichondrium of the autopo-dial elements (Table 1). In addition, tenascin, laminin, andcollagens type I, type III and type VI, were also found takingpart in the formation of a complex extracellular matrix scaffoldassociated with the undifferentiated mesenchymal tissue.However, none of these matrix components paralleled thepattern of distribution of elastin. Fibrillar elements immuno-labeled for collagen type VI and tenascin were observed to bearranged in a preferential dorso-ventral pattern (Fig. 3D,E).These fibrils run through the undifferentiated mesenchyme ofthe autopodium from the ectodermal basement membrane ofopposing ectodermal surfaces (dorsal and ventral) in the

Fig. 2. Whole-mount chick limbs immunolabeled for elastin.(A-B) Stage 26 limb buds. (A) A dorsal view of the whole limbshowing elastin-positive fibrils distributed in its anterior half. Bar,200 µm. (B) A transverse section showing the restricted distributionof the labeled matrix in the anterior half of the limb bud. Note thatthe labeled matrix appears as fibrils running from the ectodermalsurface towards to central chondrogenic core of the limb. Bar, 100µm. (C) Transverse section through the distal stylopod of a limb budat stage 27 displaying a widespread distribution of the elastin-positive radial fibrillar matrix, which now originates from the entirecontour of the limb. C, cartilage. Bar, 100 µm. (D) Dorsal view ofthe autopode at stage 28 showing the presence of elastin labeledfibers radiating out from the chondrogenic digital rays (D). Note thepresence of fibers in the third interdigital space (I) running from thedigital rays towards the distal margin of the interdigit. Bar, 200 µm.(E) Dorsal view of the autopode at stage 29 showing elastin-positivefibers radiating from the digital ray and in the interdigital space. Bar,200 µm. (F) Longitudinal optical section through digit III (D) atstage 30 showing the fibrils associated with the digital rays. Bar, 100µm. (G) Micrograph of a stage 32 limb showing a detailed view ofthe fibrillar component located in the interdigital spaces. Note itsorigin in the proximal part of the interdigit (at the right). Bar, 200µm. (H) Transverse section of the autopod at stage 31 showing thetip of a digit (D) and the adjoining interdigital space. Note thearrangement of the fibers in the interdigital space with fibers endingat the margin of the adjoining digit and fibers ending in theectodermal surface. Bar, 100 µm.

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proximity of the developing digital rays. As can be seen in Fig.3D-E the number of these fibrils was much more reduced thanthe fibrillar component detected with elastin labeling. Further,both collagen type VI and tenascin were abundant componentsof the ectodermal basement membrane while elastin was absentfrom this structure. Laminin was mostly found in the ectoder-mal basement membrane but in the distal region of theautopodium laminin-positive tracts running up to 100 µm deepinto the subectodermal mesenchyme were also observed (Fig.3F). Collagen type I- and type III-positive fibrils were alsodetected in the interdigital spaces running in a proximo-distaldirection paralleling in some way the arrangement of the devel-oping tendons (Fig. 3G-I). At the distal part of the autopodium,collagen type I was also detected taking part in the fibrillar

system rich in collagen type VI and tenascin (Fig. 3H). Noneof these components of the extracellular matrix scaffold of thedeveloping autopodium was detected in association with theelastin fiber component located in the anterior half of the limbat earlier stages.

Elastin distribution in organ and cell cultures of thelimbExplant cultures of the whole autopodium for periods up to 48hours exhibit a pattern of elastin distribution similar to thatfound in vivo (Fig. 4A). Explants of autopodial fragments con-sisting of one or two digits also exhibited a normal pattern ofelastin distribution during the first 24 hours of culture. Inlonger culture periods modifications in the spatial distribution

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2629Elastin in the developing chick limb

of elastin were detected in relation to the characteristics of thecultured tissue fragments.

When the explants contain only a digit and adjacent inter-digital tissue, the fibrillar pattern radiating from the digitremains conserved but the former interdigital elastic systembecomes condensed at the margins of the explant (Fig. 4B).Explants containing two digits and the intervening interdigitresult in the formation of two digital rays, which tend to fuseat the distal end leaving an oval interdigital region betweenthem (Fig. 4C). In these cases, while the elastin fibrillarcomponent radiating out from the developing digits observedin vivo remained unaltered, the interdigital elastin componentlost its fan-like appearance of the in vivo condition (Fig. 4D).

An extreme modification of the interdigital elastic system isobtained in cultures of explants containing only a wedge ofinterdigital tissue. In these cases the interdigital mesenchymeforms a prominent cartilaginous element (Fig. 4E), whichexhibits a newly formed radiating complex of elastin fibrils(Fig. 4F) similar to that found in the digital rays. This elastin-positive fiber system exhibited a more precise radial arrange-ment when the explants were attached to the culture dish thanin explants floating free in the culture medium.

Elastin fibrils were also detected in high-density micromasscultures of dissociated limb mesoderm. As previouslydescribed (Ahrens et al., 1977) the limb mesoderm under these

culture conditions undergoes intense chondrogenesis, formingabundant cartilage nodules, detectable by Alcian Blue stainingafter 3 days of culture, surrounded by non-chondrogenic mes-enchyme. Elastin was very abundant between the chondrogenicnodules. The first identification of elastin fibers in thesecultures occurred at day 3, concomitant with the formation ofcartilage aggregates (Fig. 5A). At day 4 of culture the chon-drogenic aggregates were more prominent and elastin was veryabundant between the chondrogenic nodules (Fig. 5B). At thezones of confluence of several chondrogenic aggregates elastinfibers showed a well-defined radial pattern of distributionjoining the surface of adjacent cartilage nodules (Fig. 5C),resembling the pattern of elastin distribution observed in trans-verse sections of the interdigital spaces in vivo. Placement ofa small piece of ectoderm at the top of the micromass after 1day of culture causes a local inhibition of chondrogenesis(Solursh et al., 1981), and immunostaining for elastin showsan increased deposition of elastin in the subectodermal regionthat is devoid of cartilage (Fig. 5D). At high magnification andwith the advantage of the confocal microscope to study opticalsections of the tissue, it can be clearly observed that the elastinfibers in this region become aggregated in thick bundles offibrils that appear anchored by one of their ends to the ecto-dermal basal surface (Fig. 5E-F).

Fibrillin was also detected in the micromass cultures but itshowed a pattern of distribution very different from that ofelastin. The first identification of fibrillin was at day 2 ofculture in association with the earliest prechondrogenic aggre-gates (Fig. 5G). From day 3 of culture fibrillin showed anintense positivity on the outer surface of the chondrogenicnodules, which increased in the course of cartilage develop-ment (Fig. 5H). A diffuse labeling of the intercartilaginoustissue matrix was also observed. Location of a small piece ofectoderm on the top of the micromass caused the oppositeeffects on fibrillin distribution to those observed for elastin. Ascan be see in Fig. 5I, an intense halo of unlabelled matrixbecomes associated with the grafted ectoderm fragment by 2days of further culture.

Fig. 3. Whole-mount chick limbs immunolabeled for elastin (A),fibrillin (B,C), tenascin (D), collagen type VI (E), laminin (F),collagen type I (G,H), and collagen type III (I). (A) Detailed view ofa developing tendon at stage 35 after elastin immunolabeling. Notethe positive labeling of the main tendon blastema (T) and thepresence of fibrils anchoring the tendon to the surface of theadjoining phalange. Bar, 100 µm. (B,C) Two successive opticalsections of digits II and III at stage 35 after fibrillin immunolabelingBar, 200 µm. (B) The level of the digital cartilages showingpositivity in the perichondrium and developing joints. (C) Positivityof the developing tendons. Note that fibrillin does not label theanchoring fibers of the tendons observed A. (D) Distal tip of digit IIIand its adjacent interdigital tissue at stage 30 after collagen type VIimmunolabeling. The perichondrial surface of the developingcartilage and the ectodermal basement membrane (arrows) exhibitpositive labeling. Note the presence of positive fibrils crossing theinterdigital tissue. Bar, 100 µm. (E) Detailed view of the tip of digitIII (D) and its adjacent interdigital tissue at stage 29 immunostainedfor tenascin. Positive fibrils are observed running dorsoventrally inthe interdigital space. The surface of the developing cartilage ispositive for tenascin and displays tenuous fibrils running towards theinterdigit. Bar, 100 µm. (F) Detailed view of the distal part of theautopodium at stage 29 after laminin immunolabeling. Note that inaddition to the continuous labeling of the basement membranefibrillar tracts are also present running for a short trajectory into thesubectodermal mesenchyme. Bar, 100 µm. (G) Dorsal view of theautopodium at stage 32 showing the appearance of a developingtendon (T) after immunolabeling for collagen type I. Note theabundance of collagen fibers running parallel to the main tendinousblastema. Bar, 100 µm. (H) Distal tip of digit III and adjoininginterdigital tissue at stage 30 after immunolabeling for collagen typeI. Note the positivity of the ectodermal basement membrane and thepresence of fine tracts running longitudinally through the interdigit.Bar, 100 µm. (I) Distal part of the autopodium at stage 31 aftercollagen type III immunolabeling. The optical section corresponds tothe level immediately under the ectodermal surface of the interdigitand shows the presence of fibrils with a preferential longitudinalarrangement. D, digit III. Bar, 200 µm.

Table 1. Distribution of ECM molecules in relation toelastin during the development of the autopodium

Joints Tendons Perichondrium

Elastin ++ +++* +Fibrillin ++ ++ + +Collagen type I + +++ +Collagen type II† − − −Collagen type III +++ + +Collagen type IV‡ − − −Collagen type VI +++ +++ ++Collagen type X§ − − −Tenascin +++ +++ +++Laminin¶ +\− − +\−

*Elastin was the only element detected that formed fibrils anchoring thetendons to the developing phalanges (see Fig. 3A).

†Collagen type II was only detected in the ectodermal basement membraneand in the cartilage.

‡Collagen type IV was only detected in the wall of the developing bloodvessels of the limb.

§Collagen type X at the studied stages was restricted to the hypertrophiccartilage of the diaphisis of the skeletal pieces.

¶A faint labeling of the perichondrium and joints was detected with anti-laminin antibody.

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DISCUSSION

Elastin constitutes a unique extracellular matrix component

with rubber-like properties conferring tensile functions that arerequired to meet the demands of mechanical stress in vertebratetissue (Sage and Gray, 1979). Most studies of elastic matrices

J. M. Hurle and others

Fig. 4. Autopodial explants obtained at stage 29 and cultured for 48 hours. (A) Explant consisting of the whole autopodium immunolabeled forelastin to show the permanence of both the interdigital and the digital components of the elastin-positive fibrils in this culture condition. Bar,200 µm. (B) Explant consisting of a digit and the adjacent interdigital tissue showing the permanence of the elastin fibers radiating from thedigit, and the condensation and disarrangement of the interdigital fibrillar component in the margin of the explant (arrows). Bar, 200 µm.(C-D) Explants consisting of digits III and IV and its intervening interdigit. Bar 200 µm. (C) The appearance of the cartilaginous elements afterimmunostaining for collagen type II. (D) The pattern of elastin distribution showing the permanence of fibrils associated with the digits and thedisorganization of the fibrillar component of the interdigit (ID). (E,F) Explants consisting of the third interdigit. Bar, 100 µm. (E) Theappearance of an interdigital cartilage after immunostaining for collagen type II. (F) The formation of a new pattern of radiating fibrils afterimmunostaining for elastin reminiscent of the fibers associated with the developing digits.

2631Elastin in the developing chick limb

have been focussed on adult tissues and organs that arenormally subjected to periodic mechanical deformations suchas skin, pulsatile blood vessels, lung and some ligaments whereelastin comprises a very high percentage of their dry weight(Sage and Gray, 1979). On the basis of the data from thesestudies it has been largely assumed that elastin is a componentformed in late fetal periods of development and its function hasbeen mostly corelated with gross and continuous tissue defor-mation (Parks et al., 1988; Pasquali-Ronchetti et al., 1993).However, recent studies with the developing chick embryo,using in situ hybridization techniques, revealed that tropo-elastin mRNA is expressed early during development (Selminet al., 1991; Holzenberger et al., 1993), but the pattern ofelastic fiber distribution in relation to morphogenetic eventsremains to be clarified.

Our study reveals the involvement of elastin during the mor-phogenesis of the limb skeleton in vivo and in vitro. In thecourse of this study we have also observed that elastin inprecise patterns of fiber arrangement is present in many otherdeveloping embryonic tissues, including the outflow tract andatrioventricular cushion tissue of the heart, the early develop-ing lung, the notochord and the somites (unpublished data).Further, in contrast to previous studies on elastogenesis in vitroby bovine nuchal ligament fibroblasts (Mecham et al., 1984),we have also found that chondrogenic mesodermal limb cellcultures are very elastogenic. While nuchal ligament fibrob-lasts decrease elastogenesis markedly in vitro in the absence ofcontact with a previous elastic matrix, limb mesodermal cellsretain an intense elastogenic potential in vitro. All thesefindings point to new, previously unsuspected functions forelastic matrices during embryonic development.

In the limb bud prior to the establishment of muscle bellies,elastin exhibits a constant pattern of arrangement, forming afibrillar scaffold from the ectoderm to the chondrogenicskeletal pieces at the early stages of formation of the limbskeleton. There are several characteristics of this scaffold thatare particularly interesting as putative factors of importance inskeletal morphogenesis. The fibers of the scaffold are neverarranged randomly but exhibit a constant orientation repeatedat the same stage in all the embryos studied. Further, the patternof fiber arrangement follows closely the sequence of formationof the skeletal elements and remains unaltered in organ cultureconditions provided that the morphology of the growingexplant maintains the characteristic shape found in situ. Whenthe explant consists of a tissue fragment isolated from itsnormal neighboring elements, the elastin pattern undergoeschanges that parallel the altered pattern of growth and chon-drification of the explant. The appearance of the elastinscaffold both in vivo and in vitro is coordinated with the estab-lishment of distinct cartilage elements and it exhibits apermanent association with the ectoderm basal surface. Thislast feature is reinforced by the ability of the ectoderm to reori-entate and aggregate elastic fibers when located at the top ofmicromass cultures. Taking together all these features alongwith the well-known biomechanical properties of elasticmatrices (see Mecham and Heuser, 1991; Pasquali-Ronchettiet al., 1993; Rosenbloom et al., 1993), it can be proposed thatelastin scaffold formation may be linked to the occurrence ofmechanical stress associated with the anisotropic growth of thelimb with tissues of different physical characters. If thisassumption is true, the elastin scaffold provides molecular

evidence for the hypothesis involving mechanical tension inthe morphogenesis of the limb skeleton (Oster et al., 1985;Rooney et al., 1984). On the other hand, the precise arrange-ment of the elastin scaffold joining the different skeletal pieceswith the ectodermal limb cover might constitute an anchoringsystem accounting for the maintenance of the appropriatedistance between the different limb tissues and for the coordi-nation of growth.

A major question concerning the morphogenesis of the longbones of the limb is why, once the cartilaginous rudiments areformed, the early shape is maintained and growth is restrictedto their proximo-distal axis, while increase in thickness isalmost fully inhibited (Rooney et al., 1984). The perichon-drium has been proposed to be responsible for stopping lateraloutgrowth of the cartilage rudiments of the long bones (Rooneyet al., 1984), but the formation of the perichondrium, especiallyat the level of the epiphyseal regions, is a relatively late event.This study reveals the occurrence of a precise landmark ofelastic matrix that delimits, both in vivo and in vitro, the lateraledge of the developing cartilages from the surrounding, appar-ently undifferentiated mesenchyme. The sequential study ofmicromass cultures reveals that the formation of the cartilageaggregates is accompanied closely by the deposition of anelastic matrix in the adjoining nonchondrogenic tissue. Thiselastic matrix undergoes progressive differentiation showingprecise patterns of fibrillar arrangement, as observed in vivo,concomitantly with the growth and differentiation of thecartilage nodules. Further, and again resembling the eventsobserved in vivo, the location of an ectodermal fragment onthe top of the culture stimulates elastogenesis in precisefibrillar patterns. All these features suggest that the differen-tiation of cartilage aggregates in the developing limb budinvolves the segregation of the initially undifferentiated mes-enchyme into two distinct cell lineages: chondrogenic and elas-togenic. This concomitant segregation of the early undifferen-tiated mesenchyme may contribute to preventing furtherincorporation of cells into the chondrogenic foci. Ectodermappears to promote the elastogenic lineage and this mightaccount for the proven antichondrogenic effect of the ectodermin vitro (Solursh, 1984; Zanetti and Solursh, 1986) and in somein vivo conditions (Hurle and Gañan, 1986).

The restricted distribution of elastin to the anterior half ofthe limb bud between stages 25 and 26 is a striking feature,which might be explained by differences in the amount ofelastin associated with the formation of the anlage of the tibiain comparison to a more reduced amount associated with theformation of the fibula (see Archer et al., 1983; Hampe, 1960).However, since differences between mesodermal cells of theanterior and posterior segments of the limb have been found tobe of fundamental importance for limb patterning (Bryant andGardiner, 1992), in the absence of detailed information onpossible further functions of elastin (see Hornebeck et al.,1986) in developing systems, we cannot discount the possibil-ity of a relation between the anterior polarized distribution ofelastin and the previous distinctive commitment of the limbmesoderm.

In a previous paper we have described the occurrence of anextracellular matrix scaffold within the limb bud that isarranged in a dorsoventral pattern of fibrils attached to oppositeectodermal basal surfaces (dorsal and ventral) (Hurle et al.,1989). Tenascin, fibronectin and collagen type I were found to

2632

be the major constituents of this scaffold. In that study we iden-tified such a scaffold with a complex system of fibrils that wereobserved in paraffin sections stained with silver. In this studythe use of a wider set of antibodies, along with the advantageof using the confocal microscope to study the distribution of

immunolabeled matrix components in whole-mountspecimens, allowed us to clarify several aspects of this extra-cellular matrix scaffold. On one hand, the argyrophilic fibersystem clearly corresponds to the elastin scaffold describedhere in association with chondrogenesis. On the other hand,

J. M. Hurle and others

2633Elastin in the developing chick limb

this study reveals that the so-called ‘dorso-ventral fibrillarsystem’ constitutes a more complex scaffold containing addi-tional matrix components, including collagens type III and typeVI, and most likely laminin. Whether the components of thismatrix share common functions with the elastin scaffold cannotbe ascertained from this study. Elastogenesis by nuchalligament fibroblasts has been found to be critically dependenton the presence of other extracellular matrix components(Mecham et al., 1981, 1984). Further, assemblies of differentextracellular matrix components may be of fundamentalimportance in extracellular matrix function, including theestablishment of the patterns of adhesion to the cellular com-ponents. However, in this study none of the reported matrixcomponents exhibited a pattern of distribution paralleling theelastin scaffold, although they may be associated with partic-ular regions of the elastin scaffold.

The different patterns of distribution of elastin and fibrillinobserved in this study constitute an unexpected feature of theelastic matrix of the developing limb bud. Fibrillin is currentlyconsidered to be a constant component of the elastic matrix andhas been characterized as the fibrillar component associatedwith the amorphous tracts of elastin (Sakai et al., 1986). Earlystudies on elastogenesis showed that this fibrillar elasticcomponent precedes the appearance of the amorphousinsoluble elastin, suggesting a possible role as a precursor ofthe elastic matrices facilitating in some way the crosslinkingof tropoelastin to form insoluble elastin tracts (Ross andBornstein, 1969; Cleary et al., 1981). In this study fibrillin andelastin showed relatively similar patterns of distribution onlyin later stages of development in association with joints, peri-chondrium and tendon differentiation; but even in these casesdifferences in distribution were significant. Elastin, for

example, is associated with the fibrillar tracts anchoring thetendon blastemas to the developing phalanges, while fibrillinwas only present in the main tendon blastema. In early stagesof development fibrillin was not detected in the elastinscaffold. This different pattern of distribution was particularlyremarkable in micromass cultures where fibrillin and elastinshowed opposite patterns of distribution in the surface of thecartilage aggregates and in the extracellular matrix of the non-chondrogenic tissues, respectively. The location of an ectoder-mal fragment on top of the cultures emphasized this difference,causing a local increase in elastin fiber deposition and anintense inhibition of fibrillin distribution. While we cannotdiscard the possibility of the presence of masked fibrillin asso-ciated with the elastin scaffold of the limb or the occurrencein the chick of different types of fibrillins lacking the epitoperecognized by our antibody (see Ramirez et al., 1993), all theobservations mentioned above suggest that the differentpatterns of fibrillin and elastin distribution are characteristic ofthe elastic matrix of the developing limb.

Thanks are due to Karen Jensen for help and advice with theimmuhistochemical techniques, and to John Busse for photography.This work was funded in part by grants to J.M.H. (DGICYT, from theSpanish government), M.S. (NIH grant HD05505) and an award toL.Y.S. from the Snriner’s Hospital for Crippled Children.

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(Received 15 April 1994 - Accepted 24 May 1994)

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