INTRODUCTION

Tenascin is a multimeric glycoprotein found in the extracellu-lar matrix of embryos and tumors (Chiquet and Fambrough,1984; Chiquet-Ehrismann et al., 1986; Grumet et al., 1985).Tenascin is especially prominent in the developing centralnervous system, in the matrix lining the pathways of migratorycells, in mesenchyme at sites of mesenchymal-epithelial inter-actions and in developing connective tissues (for reviews seeErickson and Bourdon, 1989; Chiquet et al., 1991b; Chiquet-Ehrismann, 1993). Unlike fibronectin, which has a more ubiq-uitous distribution in embryonic mesenchyme, tenascin acts asan anti-adhesive substratum for cells in vitro. Cells will attachand migrate on tenascin-coated tissue culture plastic, but theyfail to spread and form strong adhesions (Spring et al., 1989;Halfter et al., 1989; Lotz et al., 1989). Moreover, when cellsare first allowed to spread on fibronectin or native basallaminae and tenascin is then added to the culture medium, thesecells will lose their strong attachments and become rounded(Chiquet-Ehrismann et al., 1988; Halfter et al., 1989; Lotz etal., 1989). If the cells used are from wing bud cultures, the cell

rounding apparently promotes chondrogenesis (Mackie et al.,1987). For these reasons, tenascin is believed to play a role inregulating cell-matrix interactions in a way that may promotecell motility and/or differentiation.

Several forms of tenascin have been found in the extracel-lular matrix that are the result of differential mRNA splicing.Three splice variants have been identified in the chicken (Joneset al., 1989; Spring et al., 1989) with apparent relativemolecular masses of 230

×103 (Tn230), 200×103 (Tn200) and190×103 (Tn190). These forms differ from one another in theirnumber of fibronectin type III repeats. Tn230 has elevenfibronectin type III repeats; two of these repeats are not foundin Tn200 and exons encoding three of these repeats are splicedfrom transcripts encoding Tn190. This diversity may reflectdifferent functional forms of tenascin, as splice variants havedifferent affinities for other matrix molecules (Chiquet-Ehrismann et al., 1991) and cell surface glycoproteins (Zischet al., 1992). Both immunohistochemistry with splice variant-specific antibodies (Matsuoka et al., 1990; Chiquet-Ehrismannet al., 1991; Kaplony et al., 1991) and in situ hybridization withsplice variant-specific probes (Prieto et al., 1990; Mackie and

637Development 120, 637-647 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

Previous studies have shown that several forms of the gly-coprotein tenascin are present in the embryonic extracel-lular matrix. These forms are the result of alternativesplicing, which generates tenascin variants with differentnumbers of fibronectin type III repeats. We have useddegenerate primers and PCR to isolate a novel tenascinexon from an avian genomic library. Genomic clonescontained a sequence encoding a fibronectin type III repeatthat corresponds to repeat ‘C’ from the variable domain ofhuman tenascin. To demonstrate that tenascin containingrepeat ‘C’ is actually synthesized by avian cells, a mono-specific antiserum was raised against a repeat ‘C’ fusionprotein. This antiserum recognized a novel high-molecular-weight variant on immunoblots of tenascin isolated fromchicken embryo fibroblast-conditioned medium, andstained tendons on frozen sections of chicken embryos. A

cDNA probe specific for mRNA encoding repeat ‘C’ wasused for in situ hybridization. This probe hybridized in asubset of the embryonic tissues labelled with a universaltenascin probe, including tendons, ligaments and mes-enchyme at sites of epithelial-mesenchymal interactions.Finally, we provide evidence that additional fibronectintype III repeats, one corresponding to a recently discoveredhuman repeat as well as one entirely novel sequence, alsoexists in chicken tenascin mRNA. These data indicate thattenascin is present in the embryonic matrix in a multitudeof forms and that these forms have distinctive distributionsthat may reflect more than one function for tenascin indevelopment.

Key words: cytotactin, splice variants, in situ hybridization,development, extracellular matrix

SUMMARY

Novel tenascin variants with a distinctive pattern of expression in the avian

embryo

R. P. Tucker1, J. Spring2, S. Baumgartner2, D. Martin2, C. Hagios2, P. M. Poss1 and R. Chiquet-Ehrismann2,*1Department of Neurobiology and Anatomy, Neuroscience Program, Bowman Gray School of Medicine, Wake Forest University,Winston-Salem, NC 27157-1010, USA2Friedrich-Miescher Institut, Postfach 2543, CH-4002 Basel, Switzerland

*Author for correspondence

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Tucker, 1992; Tucker, 1993) have revealed distinct tissue dis-tributions and cellular origins for the variants as well. Com-parison of avian tenascin with tenascins from other speciesshows that the spliced, variable domain of the molecule hasbeen conserved through evolution (Gulcher et al., 1989; Sagaet al., 1991; Weller et al., 1991).

It was noted earlier that the primary sequences offibronectin type III repeats from chicken and human tenascincorrespond with one another colinearly. That is, the first repeatfrom human tenascin is more similar to the first repeat inchicken than to other human fibronectin type III repeats, andso on. Within the variable domain, sequence analysis revealedthat one of the human fibronectin type III repeats, repeat ‘C’,did not have a counterpart in the chicken. For this reason, wehave undertaken a study of genomic sequence and have founda homologous avian exon. Monospecific antibodies raisedagainst avian repeat ‘C’ fusion proteins have identified thispreviously unknown splicing variant both in tenascin fromchicken embryo fibroblast-conditioned medium and in tissuesections. Finally, the tissue distribution of transcriptsencoding tenascin with repeat ‘C’ was examined by reversetranscriptase PCR and in situ hybridization. By investigatingthe fibronectin type III repeats surrounding repeat ‘C’-con-taining mRNA, two further novel fibronectin type III repeatswere discovered. Whereas one of these repeats represents anentirely new sequence, the other one is the homologue of therecently published human tenascin ‘AD1’ repeat (Sriramaraoand Bourdon, 1993). Thus our results provide further evidencethat the structure of tenascin is phylogenetically conserved,and that tenascin may have multiple functions in developmentthat are regulated via the expression of different forms of themolecule.

MATERIALS AND METHODS

Cloning and sequencing of tenascin variants andproduction of fusion proteinsGenomic clones of the tenascin gene were isolated from a library inEMBL3 (Clontech CL1004D) using a probe encompassing tenascinrepeats ‘A’ and ‘B’. One of the clones isolated ended in repeat ‘D’.This DNA was used as a template for the PCR reaction using degen-erate primers developed after the sequence of human tenascin repeat‘C’. The following oligonucleotides were used: 5′-GATGAATTC-GAYATHAAYCCNTAYGG-3′ and 5′-CTAGTCGACSWNAC-CATNACYTCRTA-3′ (corresponding to the regions marked in Fig.1). The resulting PCR product was subcloned and sequenced.Oligonucleotides corresponding to the obtained internal sequencewere used to sequence the entire chicken repeat ‘C’ plus the adjacentintrons of the original genomic clone. Oligonucleotides were then syn-thesized using the terminal sequences of this repeat to amplify theentire repeat for the subcloning into the bacterial expression vectorsand to screen a chicken embryo cDNA library (Clontech) to isolate acDNA clone containing repeat ‘C’. PCR products and cDNAs weresubcloned into the Bluescript vector (Stratagene). Both strands weresequenced using the Sequenase Kit (U.S. Biochemicals).

For the production of fusion proteins containing selectedfibronectin type III repeats of tenascin, the commercially availableplasmid pEX-1 (Genofit) was used. Since it was shown that after thedeletion of the C-terminal half of the β-galactosidase the stability andefficiency of the production of fusion proteins increased, the plasmidwas modified according to Wingender et al. (1989). The plasmid pEX-1 was cleaved at the unique

EcoRV and XbaI sites to delete part of

the β-galactosidase coding region and the two partially complemen-tary oligonucleotides 5′-GGTAATGGTGACCCGGGTAATTAAT-TAAT-3′ and 5′-CTAGATTAATTAATTACCCGGGTCACCAT-TACC-3′ were inserted at this site. With this insertion, the modifiedplasmid pEX-1 contains 3861 bp. After the truncated β-galactosidase-coding region, the aminoacids NGDP precede the restriction enzymerecognition site SmaI. Stop codons in all three reading frames followafter the SmaI site. Blunt end cloning into the SmaI site links the insertto the third position of the reading frame of the β-galactosidase part.Thus the fusion proteins produced contain the N-terminal part of theβ-galactosidase migrating at 54×103 Mr on SDS-PAGE plus the addedmolecular weight of the attached tenascin fragments. They werepurified from inclusion bodies according to the procedure ofWingender et al. (1989). Alternatively tenascin fragments wereexpressed as fusion proteins with glutathione-S-transferase using thepGEX series of vectors (Pharmacia). Since also these fusion proteinswere recovered in inclusion bodies, they were purified using the sameprocedure as for the β-galactosidase fusion proteins.

Antibodies, immunoblotting and immunohistochemistryThe monoclonal antibodies against tenascin and the location of theirepitopes have been described previously (cf. Chiquet-Ehrismann etal., 1991). The antiserum against the glutathione-S-transferase repeat‘C’ fusion protein has been raised in a rabbit using standard proce-dures. The antiserum was affinity purified using β-galactosidaserepeat ‘C’ fusion protein coupled to CNBr-activated Sepharose(Pharmacia). The monospecific antibodies were eluted using 0.1 Mglycine/HCl (pH 2.6) and the eluate was immediately neutralized.Their specificity was tested using an ELISA procedure as describedin Chiquet-Ehrismann et al. (1988).

The tenascin for the immunoblotting experiment was purified fromchicken embryo fibroblast conditioned medium according to Chiquetet al. (1991a). SDS-PAGE and immunoblotting was performed asdescribed earlier (Hofer et al., 1990), with the exception that a chemi-luminescence detection kit (ECL, Amersham) was used to develop thefilter. Immunofluorescence of cryosections was performed asdescribed by Mackie et al. (1987).

In situ localization of splice variantsTenascin variant transcripts were identified in frozen sections ofchicken embryos at a variety of developmental stages using in situhybridization. To identify the mRNA encoding repeat ‘C’, anupstream anti-sense primer corresponding to the first 22 nucleic acidsof repeat ‘C’ (5′-AGGCCATGCCCCCACTGGAAAA-3′) and adownstream sense primer corresponding to the final 21 nucleic acidsof repeat ‘C’ (5′-CTGTAACAGCCACAGTGCTTA-3′) were used togenerate a 272 bp reverse transcriptase PCR product (TNC). TNC wasidentified as a probe to the sequence encoded in the ‘C’ repeat by arestriction digest with HindIII. The in situ hybridization pattern withTNC was compared to the pattern obtained with two other probes:TN230 (Tucker, 1993), a 545 bp PCR product generated from primerscorresponding to the beginning of repeat ‘A’ and the end of the repeat‘B’; and cTn8, an 1899 bp chicken tenascin cDNA that correspondsto the portion of the tenascin mRNA encoding the heptad repeats, theEGF-like repeats and the first one and one-half fibronectin type IIIrepeats (Pearson et al., 1988). Thus, TN230 hybridizes to tenascintranscripts encoding variants with repeats ‘A’ and ‘B’, and cTn8hybridizes to all known variant mRNAs. All probes were labelled with35S-dCTP using random primers (Promega), and unincorporatednucleotides were removed with a Sephadex G-50 mini-spin column(Worthington). To control for spurious hybridization, a 955 bpTaqI/Sau3AI pUC19 restriction fragment was similarly purified andlabelled.

Chicken embryos (Hubbard Farms) at embryonic day 7 (E7), E10and E14 were fixed by immersion overnight in ice-cold 4%paraformaldehyde in phosphate-buffered saline (PBS), cryoprotectedovernight in 25% sucrose in PBS and embedded in OCT compound.

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639Novel tenascin variants

Serial 12-14 µm sections were cut in a cryostat and collected on poly-L-lysine-subbed slides. Details of the in situ hybridization protocolhave been published previously (Tucker and McKay, 1991). In brief,air-dried sections were prehybridized for 1 hour at room temperaturein 5× SSC and 5× Denhardt’s solution, with 100 µg/ml salmon spermDNA and 20 mM β-mercaptoethanol. Slides were then dehydrated inethanol, and 5×106 cts/minute of the appropriate probe in hybridiz-ation buffer (prehybridization buffer with 50% formamide, 20 mMTris, 0.1% lauryl sulfate and 1% dextran sulfate; all reagents fromSigma) was added and allowed to hybridize overnight at 42°C. Thenext day the slides were rinsed with 1× SSC at roomtemperature and at 42°C. Sections were exposed tohigh-resolution X-ray film (Hyperfilmβ, Amersham),then dipped in Kodak NTB2 nuclear track emulsion.

RT-PCR with chicken embryo fibroblastmRNAChicken embryo fibroblast cultures were preparedfrom skin of 11-day embryos and RNA was preparedafter the procedure of Chomczynski and Sacchi(1987). Poly(A) RNA was isolated using the mRNAkit from Pharmacia. For the RT-PCR (reverse tran-scriptase and Taq-polymerase, Boehringer-Mannheim) of chicken embryo fibroblast mRNA, thefollowing primers were used:

repeat ‘C’ forward (5′-TAGAATTCTAGCCAT-GCCCCCACTGGA-3′),

repeat ‘C’ reverse (5′-TAGAATTCTATGTAA-CAGCCACAGTGC-3′),

repeat ‘D’ reverse (5′-ATGTCGACGTCCAC-CTCGGGTTCTGC-3′),

repeat ‘5’ forward (5′-ATGTCGACCAACCAT-CAAGGGTTCGA-3′),

repeat ‘A’ forward (5′-ATGTCGACGGTGT-GATTCGAGGCTAC-3′),

repeat ‘6’ reverse (5′-TAGAATTCTATG-TTTTCAGAATTCCAG-3′),

repeat ‘B’ forward (5′-ATGAATTCATAAGT-GCTGCGGCTACA-3′),

repeat ‘AD1’ reverse (5′-ATGTCGACTGAGAT-GTGAGCGCTGCG-3′).

All products of the reactions shown in Fig. 9.II weresubcloned and sequenced.

RESULTS

Does a fibronectin type III repeat ‘C’exist in chicken tenascin?When the fibronectin type III repeats of chickenand human tenascin are aligned with each other,it is evident that the order of the fibronectin III

repeats is conserved. Thus, we assigned numbers to identify thehomologous constant repeats and letters to identify the corre-sponding repeats known to be subject to alternative splicing (cf.Vrucinic-Filippi and Chiquet-Ehrismann, 1993; Aukhil et al.,1993; Fig. 9). Repeat ‘A’ occurs in 4 versions in human tenascin,but only once in the chicken. Human repeat ‘C’ shares 35%identity with repeat ‘B’ of chicken tenascin, but this did not seemto be enough similarity to qualify as an authentic homologue,since all other homologous repeats have a counterpart in the

Fig. 1. DNA sequence of the chickentenascin repeat ‘C’ exon (capital letters)and flanking intron sequences (lowercase letters). The translated amino acidsequence is compared to the humanrepeat ‘C’ sequence (Siri et al., 1991)and the identical aminoacids are markedby a dash. Arrows above the DNAsequence mark the position of thedegenerate primers that were usedinitially to amplify a part of the chickenrepeat ‘C’ exon.

Fig. 2. (A) SDS-PAGE of the purified tenascin fusion proteins, glutathione-S-transferase repeat ‘C’ (GST-C), β-galactosidase repeat ‘C’ and β-Galactosidaserepeat ‘D’ (β-gal-D) and their recognition by the antiserum raised against GST-Cloaded on a β-gal-C affinity column (L), the flow through of this column (FT) and theeluate (E) containing monospecific antibodies against tenascin repeat ‘C’. Thenumbers are the absorbance values measured in an ELISA of wells coated with therespective fusion proteins. (B) Agarose gel analysis of RT-PCR products fromchicken embryo fibroblast mRNA using primers from the beginning of repeat ‘1’ tothe end of repeat ‘2’ (lane a), the beginning of repeat ‘C’ to the end of repeat ‘C’(lane b) and from the beginning of repeat ‘C’ to the beginning of repeat ‘D’ (lane c).The marker bands are from top to bottom 2322 bp, 2027 bp, 1353 bp, 1078 bp, 872bp, 603 bp, 310 bp, 281/271 bp and 234 bp (lane d). In each case the prominent bandis of the expected molecular weight, and the product of lane b has been subcloned andsequenced to confirm its identity. Lanes e-g show immunoblots of tenascin isolatedfrom chicken embryo fibroblast conditioned medium, reacted with a general anti-tenascin antiserum (lanes e,f), anti-tenascin repeat ‘C’ (lane g) and stained for protein(lane h). Anti-tenascin repeat ‘C’ recognizes a high-molecular-weight tenascin band,migrating above the three major tenascin variants seen after staining the tenascinprotein. The general tenascin antiserum recognizes the three major tenascin variants,but in lane e, where 10-fold more tenascin is loaded the minor repeat ‘C’-containingtenascin variant can also be detected. The arrowheads point to the repeat ‘C’-containing tenascin band. The protein molecular weight markers indicated to the rightof the gel correspond from top to bottom to 200, 97 and 67(×10−3) respectively.

Mr ×10−3

640 R. P. Tucker and others

Fig. 3. X-ray film overlay images of cross sections through E7 (A-H), E10 (I,K,M,O) and E14 (J,L,N,P) chicken embryos following incubationwith 35S-labelled cTN8 (8), cTN230 (230), cTNC (C) or a 955 bp pUC19 TaqI/SauAI restriction fragment (pUC). At E7 cTN8 hybridizes inareas of connective tissue morphogenesis, the dorsal aorta (a), the tips of lung (l) buds (arrows in A), and in kidney (k) mesenchyme (arrows inE). There is also a ‘spot’ of hybridization in the spinal cord (sc). cTN230 hybridizes in the same regions, but the signal is greatly reduced in areasof chondrogenesis. The signal with cTNC is similar to that seen with cTN230, except that no hybridization could be detected in the spinal cord.At E10 cTN8 (I) hybridizes in the spinal cord, in areas of chondrogenesis, at the base of developing feathers (small arrows) and in tendon (largearrow). The cTN230 (K) signal is similar, but as at E7 there is a very weak signal in areas of chondrogenesis. The only significant signal withcTNC (M) at E10 is in tendons (large arrow) and at the base of feathers (small arrows). At E14 both cTN8 (J) and cTN230 (L) hybridize inventral white matter, but the signal with cTNC (N) is indistinguishable from the control (P). f, feathers; g, gut; t, tendon; vb, vertebra; w, wing.

641Novel tenascin variants

chicken with over 50% amino acid identity. Therefore, we pos-tulated that in the chicken a previously unidentified ‘C’ repeatcould exist between the known repeats ‘B’ and ‘D’.

In our earlier studies (Spring et al., 1989), none of the cDNAclones analyzed revealed a potential repeat ‘C’. Therefore, wedecided to determine if the chicken tenascin gene contains anexon encoding repeat ‘C’. Genomic clones were isolated thathybridized with repeats ‘A’ and ‘B’. One of these clones endedwithin repeat ‘D’ and should, if repeat ‘C’ exists in chicken,

contain the putative exon. Using repeat ‘C’-specific degener-ate primers (derived from the human sequence correspondingto the two regions marked by arrows in Fig. 1), we were ableto amplify by PCR a product of the predicted length using thegenomic clone as a template. The sequence of the amplifiedfragment and of the corresponding exon in the genomic clone(including flanking intron sequences) confirmed that it wasindeed derived from an exon encoding a chicken repeat ‘C’ (Fig.2). This repeat has an identity of 80% with human repeat ‘C’.

Fig. 4. Cross sections through brachial-level chicken spinal cord at E7 (A-D) and E10 (E-G) stained with anti-tenascin (TN) or 35S-labelled cTN8(8), cTN230 (230) or cTNC (C). The sections treated for in situ hybridization are shown in dark field. Anti-tenascin staining first appears in theE7 spinal cord as a discrete portion of the ependymal layer just dorsal to the ventral floor plate (arrow in A). Both cTN8 and cTN230 hybridize inthis same region (arrows in B and C). The signal with cTNC (D), however, is indistinguishable from controls (not shown). At E10 there is anintense hybridization signal with both cTN8 (E) and cTN230(F) in the spinal cord ependymal layer in cells found in the ventral horn and ventralwhite matter. In contrast, the cTNC signal (G) is identical to controls (not shown). Thus, ‘C’ repeat-containing mRNA can not be detected in thedeveloping spinal cord by in situ hybridization, indicating that cTN230 is the most abundant high-molecular-weight form of tenascin.

642 R. P. Tucker and others

Fig. 5. Cross sections through E7 (A-F) and E10 (G-I) chicken embryos incubated with 35S-labelled cTN8 (8), cTN230 (230), cTNC (C) orstained with anti-tenascin (TN). At E7, all three tenascin cDNA probes (A-C,) hybridize in the connective tissue linking adjacent vertebrallaminae (vl). There is no signal in the dorsal root ganglion (drg). Similarly, cTN8 (D), cTN230 (E) and cTNC (F) all hybridize in theendoderm-derived epithelium at the growing tips of the lung bronchioles (b, arrows). (G) At E10, the only tissues where cTNC hybridizes aretendons (t) and in the mesenchyme at the base of feather buds (fb, arrow). Anti-tenascin also stains the base of feather buds (H, arrow) andtendons (I, t) in nearby sections. Thus, repeat ‘C’-containing mRNA is relatively abundant in some embryonic connective tissues, as well as atsites of epithelial-mesenchymal interactions.

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Do chicken fibroblasts express tenascin containingthe ‘C’ repeat?An antiserum was raised against a recombinant glutathione-S-transferase repeat ‘C’ fusion protein (GST-C, Fig. 2A). Inorder to purify the antibodies from this serum specific for the‘C’ repeat, a second fusion protein between β-galactosidaseand repeat ‘C’ was prepared (β-gal-C). β-gal-C was coupled toCnBr-activated Sepharose 4B and the antiserum was appliedto the column. Bound antibodies were eluted by low pH andthe eluate was immediately neutralized. The specificity of theantiserum and the purified monospecific antibodies were thentested by ELISA (Fig. 2A). The antiserum loaded onto thecolumn (L) reacted both with GST-C and β-gal-C, but not witha different β-galactosidase fusion protein (β-gal-D). The flowthrough (FT) still reacted with the GST fusion protein, but notwith β-gal-C. The eluate (E), as expected, reacts with bothtypes of repeat ‘C’ fusion proteins.

To determine whether chicken embryo fibroblasts producemRNA and tenascin protein containing repeat ‘C’, mRNA wasisolated from chicken embryo fibroblast cultures and used forRT-PCR. Primers to amplify the constant repeats ‘1’ and ‘2’were used in a control reaction (Fig. 2B, lane a). Primers ofsequences from either end of repeat ‘C’ (Fig. 2B, lane b) or asense primer from the beginning of repeat ‘C’ and an anti-senseprimer from the beginning of repeat ‘D’ (Fig. 2B, lane c) wereused to test for the presence of repeat ‘C’. In all cases, we were

able to amplify a PCR product of the appropriate size. Theproducts also contained the predicted internal restriction sites(not shown). Thus, repeat ‘C’ is present in fibroblast tenascinmRNA adjacent to repeat ‘D’. Furthermore, tenascin isolatedfrom the conditioned medium of chicken embryo fibroblastswas analyzed by immunoblotting using the ‘C’ repeat mono-specific antibody. The ‘C’ repeat antibody recognized exclu-sively a high-molecular-weight tenascin variant that migratedjust above the larger of the three major tenascin bands recog-nized by an antibody that recognizes all tenascin forms (Fig.2B, lanes e-h). Thus, both the mRNA encoding the ‘C’ repeat,and a tenascin variant containing the ‘C’ repeat, are made inprimary cultures of chicken embryo fibroblasts. However, therepeat ‘C’-containing tenascin is only a minor fraction of thetenascin produced in these cells, since it is not visible in thelane containing the purified tenascin stained by CoomassieBlue and it could only be detected with the general anti-tenascin antiserum in a lane loaded with a ten-fold highertenascin concentration than was used to reveal the three majorvariants (Fig. 2B).

Tissue distribution of tenascin containing repeat ‘C’The distribution of tenascin mRNA encoding repeat ‘C’ wasdetermined by in situ hybridization in sections of chickenembryos at several stages of development. The distribution ofrepeat ‘C’ transcripts was compared with the distribution of

Fig. 6. Indirect immunofluorescence of sequential sections of a chicken wing at E10 stained with anti-TnM1 (A,B; recognizing all tenascinvariants), anti-Tn32 (C; recognizing repeat ‘D’), anti-Tn26 (D; reacting with repeats ‘A’ or ‘B’), and anti-tenascin repeat ‘C’ (E). All antibodiesstain the flight tendon (arrow in A), which is the only structure specifically stained by the repeat ‘C’ antiserum. The area of the sections aroundthe tendon marked in A is shown in B-E. The connective tissue area marked by a star is brightly stained only by the anti-TnM1, reflecting thepresence of the smallest tenascin variant exclusively. Bars, 250 µm (A-C), 100 µm (D-I).

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mRNAs containing the variable ‘AB’ repeats as well as the dis-tribution of total tenascin transcript.

Fig. 3A-H shows low magnification overviews (X-ray filmoverlays) of cross sections through two axial levels of E7chicken embryos hybridized with tenascin probes and a pUCcontrol probe. cTn8, which hybridizes to the mRNAs of allknown tenascin forms (Spring et al., 1989), hybridizes in theperichondrium, in endothelial cells of the aorta, in the connec-tive tissue associated with forming vertebrae, as well as in lungbuds and kidney mesenchyme (Fig. 3A). Some of these regionsare shown at higher magnification in Figs 4 and 5, which showdark-field images of silver grains following emulsion autora-diography. The small, punctate signal seen near the center ofthe E7 spinal cord with cTn8 at low magnification is clearlypresent in a restricted portion of the ependymal layer justdorsal to the ventral floor plate (Fig. 4B; Tucker and McKay,1991). This ‘spot’ corresponds to the strongest concentrationof tenascin itself as seen with anti-tenascin staining of a nearbysection (Fig. 4A). The strong cTn8 hybridization signal seenassociated with the vertebrae at low magnification is actuallypresent in connective tissue linking adjacent vertebral laminae,i.e. the developing ligamentum flavum (Fig. 5A). In lung, thecTn8 signal is present within the epithelial cells at the very tipof the bronchiole buds, as described previously (Koch et al.,1991). The signals seen with TN230, which recognizes mRNAcontaining sequences encoding variable repeats ‘A’ and ‘B’,are a subset of the regions where cTn8 hybridizes: kidney mes-enchyme, bronchiole tips, ligamentum flavum, aorta endothe-lium and spinal cord ependyma are all ‘AB’ positive (Figs3B,F, 5B,E, 4C). In turn, the hybridization pattern with TNCis a subset of the TN230 pattern. The TNC hybridization signalis strong in the ligamentum flavum and at the tips of bronchi-oles, and is also seen (albeit more weakly) in the kidney mes-enchyme and aorta endothelium (Figs 3C,D, 5C,F). The TNCsignal in the E7 spinal cord, in contrast to cTn8 and TN230, isindistinguishable from controls (Fig. 4D).

At later stages in development, the differences in the hybrid-ization signals seen with the tenascin splice variant probes areeven more distinct than at E7. Low magnification overviewsof E10 chicken sections, for example, show a strong signal inperichondrium, tendons, the base of feather buds and in spinalcord with cTn8 (Fig. 3I). TN230 hybridizes in spinal cord,tendons and at the base of feather buds (Fig. 3K), whereas theTNC hybridization signal is limited to tendons and the base offeather buds (Fig. 3M). Higher magnifications emphasize theabsence of a significant hybridization signal with TNC in thespinal cord (Fig. 4G), in contrast to the robust signal in glia

seen with cTn8 and TN230 (Fig. 4E,F). At E14, TNC still failsto hybridize within the spinal cord white matter, in contrast tothe other probes (Fig. 3J,L,N,P). The strongest signal seen withTNC is found in tendons and at the base of feather buds (Fig.5G), in the same regions where anti-tenascin stains the extra-cellular matrix (Fig. 5H,I). Thus, tenascin mRNA encodingrepeat ‘C’ is concentrated in a subset of the cells that synthe-size tenascin with repeats ‘A’ and ‘B’. Repeat ‘C’ transcriptsare not detectable by in situ hybridization in perichondrium orin the spinal cord, though other tenascin mRNAs are found inthese regions.

The restricted distribution of repeat ‘C’-containing mRNAand the prominent expression in embryonic tendon wasconfirmed by immunostaining. We stained sections of chicken

R. P. Tucker and others

Fig. 7. DNA sequence of thefibronectin type III repeats ‘AD2’ and‘AD1’ and the translated amino acidsequence of the chicken ‘AD2’(chAD2) and the chicken ‘AD1’(chAD1) repeat. The comparison ofthe amino acid sequence of thechicken ‘AD1’ repeat with thecorresponding human ‘AD1’ repeat(huAD1; Sriramarao and Bourdon,1993) reveals an amino acid identityof 59% (dashes in the human sequencemark identical amino acids).

Fig. 8. RT/PCR products obtained from mRNA from chickenembryo fibroblast cultures. All bands containing tenascin sequencesare marked by white symbols. Molecular weight marker is shown inlane a and the band sizes are marked in bp. Reactions using forwardprimers of either repeats ‘5’, ‘A’, or ‘B’ in combination with areverse primer of repeat ‘AD1’ are shown in lanes b-d, respectively.The products of the reactions using primers of repeats ‘5’ to ‘D’ areshown in lane e, ‘A’ to ‘D’ in lane f, ‘B’ to ‘6’ in lane g, and ‘5’ to‘C’ in lane h. The products of lanes b-d were subcloned andsequenced and the band marked with a star contained the novelrepeat ‘AD2’. Reaction III resulted in up to five bands (marked bywhite dots) spaced by about 270 bp, the size of single fibronectintype III repeats. To determine the combination of repeats in each ofthese bands, aliquots of the reaction were digested with restrictionenzymes that cut within one of each repeat exclusively. Theinterpretation of the data is schematically shown in figure 9.

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embryos with the ‘C’ repeat monospecific antibody. The onlyprominent staining was found in the flight tendon of a chickenwing in a 10-day embryo (Fig. 6). Fig. 6A shows an overviewover the section stained with anti-TnM1, which stains alltenascin variants and therefore shows the most widespreaddistribution. This staining was compared to the stainingpattern obtained by other monoclonal tenascin antibodies andthe repeat ‘C’ antibody as shown Fig. 6B-E. Anti-Tn32 doesnot recognize the smallest tenascin variant without extrarepeats and shows a subset of the anti-TnM1 staining pattern,reflecting the presence of tenascin variants containing extrarepeats ‘A’ and ‘B’ (Fig. 6C). Even more restricted is thestaining using anti-Tn26, which recognizes a large tenascinvariant containing extra repeat ‘D’ (Fig. 6D). The ‘C’ repeatantibody consistently stained the tendon exclusively (Fig. 6E).There was no staining of tendons with control antiserum (notshown). Whereas the perichondrium was equally stained bythe three monoclonal anti-tenascin antibodies (only shown forM1, Fig. 6A), another connective tissue structure (marked bya star) was brightly stained by anti-TnM1, moderately stainedby anti-Tn32 and not stained at all by anti-Tn26 or repeat ‘C’antibody.

Are there more tenascin variants?The next set of experiments was undertaken toinvestigate the nature of the tenascin variantscontaining repeat ‘C’. First we screened achicken embryo cDNA library with a probespecific for repeat ‘C’. One of the clones isolatedcontained repeat ‘D’, preceded by repeat ‘C’,which surprisingly was not joined to a knownrepeat, but was preceded by a novel fibronectintype III sequence (Fig. 7). This new repeatrevealed an identity of 59% to a recently dis-covered additional repeat in human tenascin andtermed ‘AD1’ by Srimarao and Bourdon (1993).We thus decided to adopt this nomenclature andrefer to this repeat as the chicken ‘AD1’ (cf Fig.7; chAD1). We then performed three sets of RT-PCR reactions using chicken embryo fibroblastmRNA. In the first set using different combina-tions of primers from the published chickentenascin repeats, bands corresponding to the pre-viously described three tenascin variants could beamplified exclusively (Figs 8e-g, 9.I). To oursurprise, when we performed RT-PCR with aprimer from repeat ‘AD1’ together with primersfrom repeats ‘5’, ‘A’ and ‘B’, we obtained, in thelatter case, a product of about 270 bp larger sizethan expected (Figs 8d, Fig. 9.II). The cloningand sequencing of this product revealed a furtherentirely novel fibronectin type III sequence,which we decided to term ‘AD2’ (Fig. 8;chAD2). A third set of RT-PCR reactions usingprimers of repeat ‘5’ together with repeat ‘C’primers resulted in a number of bands spaced byabout 270 bp (marked by white dots in Fig. 8h),which by restriction digests could be identified asthe splicing variants shown in Fig. 9.III. Thus,chicken tenascin mRNA can contain up to sixfibronectin type III repeats subject to alternative

splicing as shown in the model of Fig. 9, and repeat ‘C’ occursin multiple tenascin splice variants.

DISCUSSION

The importance of tenascin for development has recently beenquestioned by Saga et al. (1992), since they discovered that micelacking tenascin developed apparently normally. This result ledErickson (1993a) to propose that tenascin could be a superflu-ous protein and that the sites of prominent expression of tenascinare not necessarily the sites where tenascin has a function.Tenascin is the product of a member of a multigene family con-sisting of three or four members (Erickson, 1993b;Chiquet-Ehrismann et al., 1993). Clearly the knocking out ofgenes belonging to multigene families often has no or only minoreffects on the deficient mice and frequently the defects are asurprise and unpredictable. However, since in most of thesecases the loss of a gene product is compensated for by othergenes, the normal function of the deleted gene can not be foundby this approach. Certainly redundant proteins are not superflu-ous, and if a protein has any function (which presumably everyprotein has cf. Brookfield, 1992), this function has to be

Fig. 9. Model of human tenascin in comparison to chicken tenascin. The structuralfeatures shown are the central domain at the N terminus (sector), followed by heptadrepeats (waved line), EGF-like repeats (diamonds), fibronectin type III repeats(rectangles) and the C-terminal globe homologous to the globular domain of γ- and β-fibrinogens. The homologous constant repeats are numbered, whereas the homologousrepeats subject to alternative splicing are letter coded. Numbers above the chickenfibronectin type III repeats designate the amino acid identity with the homologousrepeat in human tenascin. The question mark above chicken repeat ‘AD2’ designates anovel repeat of chicken tenascin, which as yet has not been described in tenascin ofany other species. The location of the epitopes of the antibodies used in the presentstudy are indicated above the chicken tenascin model. Below the chicken tenascinmodel the fragments obtained in three sets of RT-PCR reactions (I-III) seen in Fig. 8are shown. Reaction set I shows that with the use of primers of previously knowntenascin repeats only the major tenascin variants are amplified in detectable amounts.In reaction set II using a reverse primer within repeat ‘AD1’ and forward primers fromrepeats 5 or ‘A’, the expected products were obtained, whereas using a forward primerwithin repeat ‘B’, the only product that contained tenascin sequences harboured thenovel repeat ‘AD2’. In a reaction using primers from repeat 5 together with ‘C’ repeatprimers (III), a number of bands spaced by the size of fibronectin type III repeats wereobtained reflecting the occurrence of repeat ‘C’ in several different splicing variants.

646

performed where the protein is present. We therefore believe thatit is still important to investigate the structure, function andexpression pattern of tenascin in normal embryos as well as intissue culture models to find possible functions of tenascin.

Evidence from both biochemical and morphological studiesindicate that different splice variants of tenascin may havedifferent functions. For example, low-molecular-weighttenascin has a higher affinity for both fibronectin (Chiquet-Ehrismann et al., 1991) and contactin/F11 (Zisch et al., 1992),than tenascin containing the variable domain. Moreover, whenbovine endothelial cells are exposed to a fusion protein corre-sponding to the variable domain of human tenascin, they losetheir focal adhesions (Murphy-Ullrich et al., 1991). In theembryo, individual motile cells like osteoblasts and glial cellsmake high-molecular-weight tenascin, whereas non-motilechondroblasts are a primary source of low-molecular-weighttenascin (Mackie and Tucker, 1992; Tucker, 1993). A possiblerole for high-molecular-weight tenascin in promoting invasivebehavior is also provided by studies of tumors and in vitrotransformation. For example, Borsi et al. (1992) have recentlyfound that high-molecular-weight tenascin is preferentiallyexpressed by invasive human breast carcinomas, whereas low-molecular-weight tenascin is expressed in normal breast tissueand by benign breast lesions. Moreover, when chicken embryofibroblasts are transformed by Rous sarcoma virus middle T(Matsuoka et al., 1990), or when 3T3 cells are treated withbFGF (Tucker et al., 1993), there is a selective induction oftenascin containing the variable fibronectin type III repeats.Taken together, these data indicate that the key to understand-ing tenascin function may lie in our understanding the differ-ences between alternatively spliced variants of the molecule.

Comparison of the amino acid sequences of the fibronectintype III repeats of human (Gulcher et al., 1989) and chicken(Spring et al., 1989; Jones et al., 1989) tenascin indicated thatthe ‘C’ repeat from the variable domain of human tenascin hadyet to be identified in the chicken. Through a combination ofgenomic cloning with probes flanking the putative ‘C’ repeatand PCR amplification with degenerate oligonucleotides, wehave found a ‘C’ repeat in chicken tenascin. Here we provideevidence that this previously unknown fibronectin type IIIrepeat is actually present in avian tenascin: (1) monospecificantibodies to an avian repeat ‘C’ fusion protein recognize aband corresponding to a high-molecular-weight splicing variantof tenascin from medium conditioned by chicken embryofibroblasts, (2) these antibodies stain a subset of the embryonicextracellular matrix stained by antibodies that recognize alltenascin forms, (3) appropriately sized PCR products withrestriction sites characteristic for repeat ‘C’ can be amplifiedfrom chicken embryo fibroblast mRNA using primers designedfrom avian ‘C’ repeat sequence, and (4) a DNA probe specificfor mRNA encoding repeat ‘C’ hybridizes within a subset ofthe embryonic tissues where other tenascin probes hybridize.Previously, we have shown that tenascin containing repeats ‘A’and ‘B’ is made by migrating cells (osteoblasts and glia cells),at the sites of epithelial-mesenchymal interactions (featherbuds, kidney and at the tips of growing lung bronchioles), andis found in embryonic tendons (Tucker, 1993). We now knowthat the high-molecular-weight tenascin found in these diverseregions is not all the same type. Within the CNS, most of thehigh-molecular-weight tenascin apparently lacks the ‘C’ repeat.This is evident from the absence of a significant hybridization

signal in developing spinal cord with TNC. In contrast, thestrong signal with TNC in tendon, lung and kidney and at thebase of feather buds indicates that tenascin with repeat ‘C’ is amajor component of high-molecular-weight tenascin concen-trated at sites of epithelial-mesenchymal interactions as well asin embryonic tendon. These different sites of expressionimplicate repeat ‘C’ in functions that may be associated withinductive events, cell proliferation, or the binding of non-neuronal matrix components.

With the aim of determining in which type(s) of splicingvariants the fibronectin type III repeat ‘C’ occurs, we discov-ered two novel repeats preceding repeat ‘C’. The homologueof one of them has also been identified recently in humantenascin and has been termed repeat ‘AD1’ for alternativedomain 1 by Sriramarao and Bourdon (1993). We thus decidedto adopt this nomenclature for the chicken tenascin repeats andtermed our novel repeats ‘AD1’ preceded by ‘AD2’. In the caseof the human ‘AD1’ repeat, it was suggested that its expressionmay be tumor associated (Sriramarao and Bourdon, 1993). Itwill be the subject of future studies to investigate theexpression pattern of these novel repeats in the developingchicken. Furthermore, it remains to be seen whethermammalian tenascin also contains a homologue of the chicken‘AD2’ repeat. Repeat ‘C’ does not always occur together with‘AD1’, but appears to be present in a multitude of splicingvariants in mRNA isolated from chicken embryo fibroblastcultures. These mRNAs are, however, a minority of thetenascin mRNA in these cultures, since they can only bedetected using primers specific for either of the three novelrepeats. In future studies, it will be most interesting to find outwhether the novel repeats confer new activities to tenascin.

The sequences reported in this article can be obtained from theEMBL data base under the accession number X73833.

This work was supported in part by a grant from the NationalScience Foundation (BNS-9021124) to R. P. T.

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(Accepted 9 December 1993)

Note added in proofWe have recently sequenced the entire genomic region of about9 kb of the chicken tenascin gene encompassing the exonencoding repeat “A” to the exon encoding repeat “C”. Weconfirmed the presence of the exons of all extra repeatsdescribed in the present manuscript, but did not find any otherunknown exons encoding novel repeats. Therefore, we canconclude that the tenascin model presented in this paper mostlikely includes a complete set of all possible fibronectin typeIII repeats of chicken tenascin.