DEVELOPMENTAL

BIOLOGY

148,219-i?32

(1991)

Characterization of a Newt Tenascin cDNA and Localization of Tenascin mRNA during Newt Limb Regeneration by in Situ Hybridization HIROAKI Departments

ONDA,* MATTHEW of *Molecular

Genetics

L. POULIN,* and tInterna1

ROY A. TASSAVA,* Medicine,

Accepted

August

The Ohio

AND ING-MING

State University,

CHIU*+’

Columh~s,

Ohio &%?10

1, 1991

We previously showed that tenascin, a large, extracellular matrix glycoprotcin, exhibits a temporally and spatially restricted distribution during urodele limb regeneration. To further investigate the role of tenascin in regeneration, we cloned a newt tenascin cDNA, NvTN.1, that has 70% homology to the chicken tenascin sequence. A deduced amino acid sequence of NvTN.1 showed a modular structure unique to tenascin characterized by epidermal growth factor-like and fibronectin type III repeats. To determine the cellular origin of tenascin protein during limb regeneration, we localized tenascin transcripts by in situ hybridization using a riboprobe synthesized from NvTN.1. Transcripts could not be detected in normal limb tissues but first became detectable in the wound epithelium at 2 days and in the distal mesoderm at 5 days after amputation. These wound epithelial cells are probably the source of tenascin protein found within and immediately underneath the wound epithelium. At preblastema stages, hybridization was seen in cells associated with most of the distal mesodermal tissues but not in dermis. At blastema stages, essentially every mesenchymal cell contained tenascin transcripts. Thus, regardless of origin, blastemal mesenchymal cells may share a common regulatory mechanism that results in tenascin gene transcription. Finally. during redifferentiation stages of regeneration, tenascin gene transcription was associated with both differentiation and growth. The results show that initiation of tenascin gene expression is an early event in regeneration and continued tenascin gene transcription is associated with some of the important processes of regeneration, namely wound epithelial-mesenchymal interactions, dedifferentiation, initiac 1991 Academic press. IN. tion of cell cycling, blastema outgrowth, and cellular differentiation. INTRODUCTION Tenascin is an extracellular matrix’ (ECM) glycoprotein abundantly present in many tissues of developing embryos such as small intestine (Probstmeier et al., 1990), brain and other neural derivatives (Crossin et al., 1986, Prieto et al., 1990), tooth mesenchyme (Vainio et cd, 1989), myotendinous junctions, cartilage, and smooth muscle (Chiquet and Fambrough, 1984), condensing mesenchyme of cartilage and bone (Mackie et al., 1987), limb bud (Onda et ah, 1990), kidney (Aufderheide et al., 1987), and mammary glands and hair follicles (Chiquet-Ehrismann et al., 1986). The distribution of tenascin becomes very restricted in normal adult tissues (Lightner et al., 1989, Daniloff et al., 1989, Ferguson et al., 1990), but reexpression occurs during malignant tumor formation (Inaguma et al, 1988), wound healing (Mackie et ah, 1988), and peripheral nerve regeneration (Daniloff et al., 1989); thus, clearly the tenascin gene is developmentally regulated. Correlation of tenascin synthesis with these developmentally orchestrated events

suggests a role for this large, hcxameric glycoprotein in morphogenetic processes such as cell-to-cell interactions, cell adhesion, cell migration, and cell division (Chiquet-Ehrismann et al., 1986). Using a monoclonal antibody (mAb MTl) specific to urodele tenascin, we have identified this matrix protein in the blastema of the regenerating newt and axolotl limb (Onda et ah, 1990). Tenascin appears at 5 days after amputation in the area of dedifferentiation and increases in the distal stump during preblastema stages. Some tenascin appears to be present within cells of the wound epithelium but mesodermal reactivity is primarily extracellular. During blastema stages, an abundance of tenascin is seen in the blastema matrix and in a thick layer underlying the wound epithelium. Tenascin persists to late digit stages of regeneration (Onda et al, 1990). Based on this temporal and spatial appearance, we postulate a possible involvement of tenascin in mesodermal cell dedifferentiation, migration, proliferation, and/or epithelial-mesenchymal interactions (see Thornton, 1968; Singer and Salpeter, 1961). A role for tenascin in newt tail regeneration has also been implied (Arsanto et al., 1990).

i To whom correspondence should be addressed at Department of Internal Medicine, The Ohio State University, 480 W. 9th Ave., Columbus, OH 43210. *Abbreviations used: ECM, extracellular matrix; mAb, monoclonal antibody; EGFL, epidermal growth factor-like; PN, fibronectin.

It is possible that tenascin is synthesized by a subset of the blastema cell population. However, the cellular origin of tenascin could not be determined using mAb MTl. Our goal in the present study was to examine the 219

0012-1606&l Copyright All rights

$3.00

Q 1991 by Academic Press, Inc. of reproduction in any form reserved.

220

DEVELOPMENTALBIOLOGY V0~~~~148,1991 GGGGACTGCGGCCAGGAGA~CTGCCAAGT;GAATGTAGT~AGTTTGGAA~ATGTGTGAA~GGACAATGT~TGTGCGATG~AGGTTTCAC;GGTGMGAC;GCAGTGAGC~ACGCTGCCC~ GDCGPEICQVECSEFGKCVNGQCVCDEGFTGEDCSEPRCP

120

MCMTTGCMCMTCGTGGGCGTTGTGT~GAGGATG~~GCGTCTGCG~CGAAGGATT~ACAGGAGAT~ACTGCAGCG~ACTGATCTG~CCCAATGAC~GCTTCGACC~AGGCCGTTG; NNCNNRGRCVEDECVCDEGFTGDDCSELICPNDCFDRGRC

240

ATCAACGGAGTCTGCTTCTGTGACGAGGG~TTCACAGGGGAGGACTGTG~GGAACTGACATGCCCCAAC~ACTGCAATA~TCGTGGTCGCTGTGTGAAT~GCCTCTGTG~CTGTGATGA~ INGVCFCDEGFTGEDCGELTCPNNCNNRGRCVNGLCVCDD

360

GGATTCCAAGGAGATGACTGCAGTGAATTGCGATGTCCCAATGATTGCAATGACCGTGGCCGATGTGTCAATGGAAAATGTGTGTGCAAAGAAGGCTTCATGGGTGMGACTGTGCTGAC GFPGDDCSELRCPNDCNDRGRCVNGKCVCKEGFMGEDCAD

480

TTGAGATGTCCCAATGACTCCAACAACCG~GGTAGATGT~TCAACGGGC~ATGCGTGTG~GACGAAGGC~TCATGGGCG~AGACTGTAG~GATTTAAGG~GCCCTGGTG~CTGTAACAA~ LRCPNDCNNRGRCVNGPCVCDEGFMGEDCSDLRCPGDCNN

600

CGTGGTCGATGCGTCAATGGCCAGTGCGTGTGTGATG~GGCTTCCGAGGTGAAGACTGTGGTGAGCTCAGATGCCCAGATGACTGC~CAACCGTGGTGTTTGTGTTA~TGGTCAGTGC RGRCVNGPCVCDEGFRGEDCGELRCPDDCNNRGVCVNGQC

720

ATCTGCGAT~MGGCTTCA;GGGAGMAA~TGTGGTGAA~TGCGGTGTC~AAACGACTG~AAGAACCGC~GCCGGTGTG;CAACGGACA~TGCATTTGT~ATGATGGCTiCAAAGGTGA; ICDEGFMGENCGELRCPNDCKNRGRCVNGQCICDDGFKGE

840

GACTGCAGTGAGCTCCGGTGCCCTGATGA~TGTAATGATCGTGGCCGCT~TATCAATGGACAATGTGTGTGTGCAGAAG~CTTCACTGGAGAGAACTGT~ATTCCTTGG~CTGCTTGAA~ DCSELRCPDDCNDRGRCINGCICVCAEGFTGENCDSLACLN

960

AACTGCAACGACCGGGGCC;TTGTGTCAA;GGCCAATGCCTCTGTGAAGAAGGCTTCTTCGGCGAAGAC;GCTCTGAAG;TTCCCCTCCAAAAGACCTGACAGTGACAGACGTGACAACA NCNDRGLCVNGQCVCEEGFLGEDCSEVSPPKDLTVTDVTT

1080

CAGTCTGTAAACCTGGAGTGGGCGAACGAAAGTCACAGAGTACCTTATCACCTACATCCCCACCAGCCCCGGTGGCCTTGAACTGGACTTCAGGGTGCCAGGAGACCAGACCACT PSVNLEUANEMKVTEYLITYIPTSPGGLELDFRVPGDQTT

1200

GCCACCATCCMGMCTTGAGCCTGGTGTGGAGTATTTTGTTCGGGTCT~TGCCATTCTAAGG~TCAGAGGAGTATTCCAGTCAGTGCTCGAGTCGCAACTCATCTGC~~CMCTGA~ ATIQELEPGVEYFVRVFAlLRNClRSIPVSARVATHLPTTD

1320

GACTTGAGGTTCAAATCAGTCAAAGAGAC;TCAGTGGAGGTGGAGTGGGACCCTCTGGA~ATCTCCTTT~ACACTTGGG~TCTCATTAT;CGTAACACG~AAGAAGAG~TGGAGAGAT~ DLRFKSVKETSVEVEUDPLDISFDTUDLIIRNTKEENGEI

1440

AGCACAAGC~TGCAMGGC~TGTGACTTC~TATGTGCAA~CAGGCTTAG~ACCAGGCGA~ACCTACAAC;TCTCCATCC~TGTTGTGAA~AACAGCACC~GAGGACCTG~ACTTGCCAA; STSLPRPVTSYVPTGLAPGETYNFSIHVVKNSTRGPGLAK

1560

GTGACCACCACACGGCTGGATGCCCCAAGTCAGGTTGAGGTGCGCGATGTCACCGACTC~ATGGCTCTGGTCACCTGGT~CAGGCCGCT~GCACAAATT~ATGGCGTTA~CCTATCCTA~ VTTTRLDAPSQVEVRDVTDSMALVTUFRPLAQIDGVILSY

1680

GGGACAGGAAGTCAGCCGCCCACTGTCGTCCAACTCTCT~AAGATGAAA~CCAGTACTC~CTGGGA~T~TGATCCCAG~TACCGAGTA~GAGGTGACA~TGCTCTCAC~GCGTGGCTT~ GTGSQPPTVVELSEDESQYSLGNLIPDTEYEVTLLSRRGL

1800

ATGACAAGTGACCCAGTGACTGAGACCTTTACTACAGATTTGGATGCTCCGAAGAATCTGAGACGGGTGTCCCAGACTGACACTACCATCACCTTGGAG;GGAAGAACACTCAAGCCAA; HTSDPVTETFTTDLDAPKNLRRVSQTDTTITLEiJKNSClAN

1920

GTTGATCTTTACAGGATCACC VDLYRIT

1941

FIG. 1. Nucleotide NvTN.1

was determined

sequence of NvTN.1 and deduced amino acid sequence. The DNA sequence of the 1.9 kb EcoRI fragment of cDNA clone by dideoxy sequencing and the amino acid sequence was translated from the DNA sequence. Accession No. M76615.

distribution of cells transcribing the tenascin gene during limb regeneration. Here we report the isolation and characterization of a cDNA encoding newt tenascin and the spatial and temporal distribution of tenascin transcripts during regeneration by in situ hybridization using a riboprobe made from this cDNA. We show that both wound epithelial and distal mesenchymal cells transcribe the tenascin gene, a result suggesting a dual source of tenascin protein during newt limb regeneration. MATERIALS

AND

METHODS

Animals Adult newts, Notophthalmus viridescens, were collected in southern Ohio, maintained in aged tap water at room temperature, and fed raw beef twice weekly. Limbs were amputated through the midradius/ulna and protruding bones were trimmed to the level of soft tis-

sues with a razor blade. Newts were then returned to water and allowed to regenerate to the desired stage. Regenerates were collected at 1,2,3,&g, 11, and 14 days after amputation (preblastema stages), and at earlybud, mid-bud, late-bud, palette, and digit stages (staged according to Iten and Bryant, 1973). From two to six limbs/regenerates were sampled at each stage. Operations were performed while animals were anesthetized with MS-222 (ethyl m-aminobenzoate methanesulfonate; Sigma, St. Louis, MO). Isolation

of Newt Tenascin cDNAs

A 1.9-kb EcoRI fragment from a chicken tenascin cDNA clone, cTN8 (Pearson et ah, 1988), was used to screen a newt mid-bud blastema cDNA library in Xgtll (Ragsdale et al., 1989). Duplicate filters from 10 plates containing approximately 1 X lo6 plaques were screened with a 32P-labeled cTN8 probe under low stringency

Tenascin

ONDAETAL.

A

ECOR I I

sac I

XhOl

-

mRNA

221

in Regenerates

ECOR I

I NvTN.1

anmense

*sense GALTNl Mlkb FN Type

SEVSPPKDLTVTDVTTQSVNLEWANEMKVTE

III repeats

GDC 3 GPEI_WVE~SEFGKfJ'NGC!~a)EGFTGED~ 34 SEPR~PNN~NNRGR&VEDE~&BEGFTGDD& 65 SELl~PND~FDRGR~lNGV~Fa)EGFTGED~ 96 GELT&PNN~NNRGR~NGL&'&DDGFC!GDDC 127 S.ELR~PND~NDRGR~NGKCV_a(ECFMGED~ 158 ADLR~PNDCNNRGRtJfNGQ~~EGFMGED~ 189 SDLR~PGDCNNRGRlJ'NGQCJ'~EGFRGED~ 220 GELR&PDDtJdNRGV&VNGQCI&DEGFMGENC 251 GELRlJPND~NRGR&'.'NGQ~llJDDGFKGED~ 282 SELR~DD~NDRGREINGQ&.V&4EGFTGEN~ 313 DSLACLNN_UrDRGLlJ/NGQ~cV_nECFLGED~ 344 YLITYIPTSPGGLELDFRVPGDQTTAT~QE~PGVEYFVRVFAILRNQRSIPVSARVA~

HLPTTDDLRFKSVKETSVEVEWDPLDISFDTUDLIIRNTKEENGEISTSLQRPVTSYVQTG~PGETYNFSIHWKNSTR TRLDAPSQVEVRDVTDSHALVTWFRPLAQIDGV DLDAPKNLRRVSQTHTTITLEWKNSQANVDLYRIT

ILSYGTGSQPPTVVE

434 GPGLAKVTT

LSEDESQYSLGNLIPDTEYEVTLLSRf&lTSDPVTETFTT

523 612

647

FIG. 2. (A) Alignment of NvTN.1 with a chicken tenascin cDNA sequence (GALTNl; Spring et al., 1989) coding for the largest subunit of the chicken tenascin monomer. Restriction sites for XhoI and Sac1 used for construction of riboprobes and arrows indicating the orientation of the antisense and the sense probes are shown. The block between the white vertical lines is the region corresponding to the chicken cDNA probe, cTN8, used to screen the newt cDNA library. A structural model of a chicken tenascin monomer based on Spring et al. (1989) is shown below. The various structural characteristics of tenascin are represented by different symbols. The most prominent features of the molecule, the 13; EGFL repeats and the 11 contiguous FN type III repeats are indicated by open diamonds and open squares, respectively. The lines connecting cTN8 and the structural model indicate the corresponding coding region of cTN8. (B) The modular structure of a portion of newt tenascin revealed by the deduced amino acid sequence. By aligning conserved cysteine residues (underlined), 11 contiguous repeats of EGFL homology are apparent. Following immediately are three and one half FN type III repeats. The type III repeats are arranged so that the invariable amino acid residues tryptophan, leucine, and threonine are aligned. The tripeptide RGL in the third type III repeat is indicated by (0).

(43% formamide, 5X SSC, 5~ Denhardt’s solution, 1% SDS, 200 pg/ml salmon sperm DNA in 50 mMphosphate buffer, pH 6.5, at 37°C). Initially, 12 positive plaques were identified. After subsequent plaque purification and Southern blotting, six clones were determined to contain cTN8-hybridizing EcoRI inserts. The inserts of these six clones were subcloned into an EcoRI site of pBluescript (Stratagene) by standard methods. One clone, NvTN.1 (&/oto@zaZmus viridescens Tenascin.l), was further analyzed for restriction enzyme mapping and sequencing. DNA Sequencing A double-stranded cDNA fragment of NvTN.1 was sequenced using the dideoxy method and Sequenase (USB) according to the manufacturer’s instructions. For sequencing internal regions of NvTN.1, nested deletions of the insert were created with exonuclease III/mung bean nuclease (Stratagene) following the manufacturer’s suggestions. The sequence data were compiled using the DNASTAR align program; the 1.9-kb NvTN.1 sequence

was compared to the reported tenascin cDNA sequence of chicken (Spring et al., 1989; see also Jones et al., 1989), human (Gulcher et al, 1989), and mouse (Weller et al, 1991). In Situ Hybridization To produce a newt tenascin-specific riboprobe, a XhoI site in NvTN.1 was used to clone an EcoRI/XhoI fragment into EcoRI and XhoI sites of pBluescript. This plasmid DNA, after digestion with SacI, served as a template for T3 RNA polymerase (Stratagene) to produce a 350-base “antisense” riboprobe. Similarly, a Sac1 site was used to clone a SacI/EcoRI fragment into Sac1 and EcoRI sites of pBluescript. The resultant plasmid DNA, after digestion with XhoI, was used as a template for T7 RNA polymerase (Stratagene) to produce a 350base “sense” riboprobe (see Fig. 2A). Sense and antisense orientations of NvTN.1 were determined from the sequence comparison with chicken tenascin cDNA. Linearized template was transcribed with the appropriate RNA polymerase according to the manufacturer’s sug-

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FIG. 3. (A) A micrograph showing irk situ hybridization of the newt tel nascin antisense probe ulna of a n unamputated limb. No specific labeling is present in muscle (,m), nerves (n), dermis indicates ; dermal pigment granules which can be identified unambigl uously under light-field

to a longitudinal section through the midr adius/ (d), glands (g), or epidermis (ep). An open arrow microscopy. (B and D) Micrographs sh lowing

ONDAETAL.

Tena,scin

gestions in the presence of high specific activity (1250 Ci/mmole) [(u-~YS]UTP (Amersham, Arlington Heights, IL). Transcribed products were isolated by either ethanol precipitation or a Nensorb affinity column (NEN). A protocol for in situ hybridization was adapted from “Current Protocols in Molecular Biology” (Ausubel et al., 1987) with minor modifications. Regenerates at desired stages were dissected and immediately frozen in OCT compound (Miles, Elkhart, IN). For each limb stump/regenerate sample, a set of lo-@m-thick cryostat sections was placed onto precleaned poly-L-lysinecoated slides. Sections were cut through the anteriorposterior axis and sampled so that ventral, middle, and dorsal regions were represented. Prior to hybridization, sections were fixed with 4% paraformaldehyde, treated with proteinase K (10 pg/ml) for 2 min at room temperature, acetylated by immersing slides in 0.25% acetic anhydride in 0.1 M triethanolamine buffer, pH 8.0, for 10 min, and dehydrated through graded ethanol. The hybridization was carried out at 42-50°C in hybridization mix (50% formamide, 0.6 M NaCl, 10 mM Tris-HCl, 2 mM EDTA, 1X Denhardt’s solution, 1 mg/ml yeast tRNA, pH 7.5) with either sense or antisense riboprobe (at a concentration of 0.3 pg/ml/kb, which is equivalent to - 1 x lo7 cpm/ml) for 16 hr. Slides were washed in 2~ SSC for 15 min at room temperature, and twice in 50% formamide, 2~ SSC, 0.1% fl-mercaptoethanol at 5060°C and then treated with RNase A (20 pg/ml) for 30 min at 37°C. Slides were further washed twice in 50% formamide, 2~ SSC, 0.1% P-mercaptoethanol and twice in 0.1X SSC, 1% P-mercaptoethanol for 15 min each at 50-60°C. Dehydrated sections were coated with a 1:l dilution of Kodak NTB-2 photographic emulsion and exposed for 5 days at 4°C. Slides were developed with D-19 developer for 2.5 min at 15°C and fixed for 5 min with Kodak fixer. Sections were counterstained with hematoxylin and eosin to identify tissues. The silver grains were visualized using either darkfield optics alone or darkfield optics in combination with epifluorescence optics to visualize tissue morphology. Imm zcnohistochemistry Slides containing a set of sections adjacent to those used for in situ hybridization were examined for the distribution of tenascin protein by indirect immunofluorescence. Sections were reacted with monoclonal antibody (mAb) MT1 and rhodamine-labeled goat anti-

mRNA

223

in Rege?lerntes

mouse IgM secondary antibody, ah, 1990).

as described

(Onda et

RESULTS Isolation and Characterization cDNA

of the Newt Tenascin

A cDNA library constructed from mRNA of newt mid-bud blastemas was screened with a chicken tenascin cDNA, cTN8. Low stringency hybridization conditions identified 12 positive plaques of which 6 were purified, and EcoRI fragments were subcloned into a plasmid vector. One clone, NvTN.1, containing a 1.9-kb EcoRI fragment, strongly hybridized to cTN8 on a Southern blot (result not shown) and was chosen for further analysis by restriction enzyme mapping and sequencing. At the nucleotide level, NvTN.1 is 70% identical to a portion of chicken tenascin sequence (GALTNI; Spring et al., 1989). Comparable sequence homologies were also obtained by comparing the NvTN.1 sequence with sequences of human and mouse tenascin (Gulcher et al., 1989; Weller et ah, 1991). The region of homology spans the nucleotides 980 to 2930 of the chicken tenascin cDNA and codes for 11 epidermal growth factor-like (EGFL) repeats and three and a half fibronectin (FN) type III repeats (Figs. 1, 2). The alignment of the NvTN.1 sequence with the full length chicken tenascin cDNA indicated that NvTN.1 is truncated at both the 5’ and the 3’ ends (Fig. 2A). The amino acid sequence deduced from a reading frame which spans the entire length of NvTN.1 revealed striking similarities to reported amino acid sequences of chicken (Jones et al., 1989; Spring et al., 1989), human (Gulcher et ash, 1989), and mouse (Weller et al., 1991) tenascin. The sequence spans 11 EGFL repeats and three and one-half FN type III repeats which are characteristic of the modular structure of the tenascin polypeptide. The 11 cysteine-rich EGFL repeats show an invariable spacing pattern between cysteines (Fig. 2B) identical to that found in chicken, human, and mouse tenascin sequences (Jones et al., 1989; Spring et ah, 1989; Gulcher et al., 1989; Weller et al., 1991). Furthermore, the amino acid sequence in these repeats is 74% identical to chicken tenascin, and the similarity is over 90% if conservative substitution is allowed. The amino acid sequence identity between newt and mouse, or newt and human, was about 60% in the EGFL repeats.

localization of mRNA on longitudinal sections through the distal end of a regenerate at 2 days after amputation. 27~ situ hybridization with antisense probe (B) and sense probe (D) on adjacent sections. The antisense probe exhibits hybridization in the wound epithelium (we), but not in the mesodermal tissues, Bone (b) typically exhibits high background. Note that no bone remained on the section in D. (C) An immunofluorcscence micrograph probed with mAb MT1 on a section adjacent to the section in B. Strong immunoreactivity was observed only in the stump tissues. Arrows indicate the thickness of the wound epithelium. Arrowheads indicate the level of amputation. Myotendinous junction (mj); periosteum (po). Bars, (A) 45 brn; (B, C, and D) 125 Frn.

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FIG. 4 Micrographs illustrating localization of polypeptide and mR 4A in early regenerates. Longitudinal sections through regent !ra tes at 5 days (A and B) and 8 days (C and D) were hybridized with antisense I: ,obe (A and C) and adjacent sections were stained with mAb M Tl (B and

ONDAETAL.

Tenascin

The remainder of the NvTN.1 sequence codes for the first three and one-half repeats of the FN type III domain. Each repeat shows invariable tryptophan, leutine, and threonine residues (Fig. 2B), known characteristics of the tenascin polypeptide (Spring et al., 1989; Weller et ah, 1991). Overall amino acid similarity between corresponding portions of chicken, human, and mouse tenascin and the NvTN.l-deduced amino acid sequence was about 65% for each pairwise comparison. The third FN type III repeat in chicken and human tenascin contains the tripeptide sequence, RGD (Jones et al., 1988; Spring et al., 1989; Gulcher et ah, 1989), known to be important for cell attachment (Ruoslahti and Pierschbacher, 1987). The deduced sequence of NvTN.1 contained no RGD sequence. Instead, it contained an RGL sequence in the third FN type III repeat at the position of the RGD sequence in chicken and human (Fig. 2B). Tenascin mRNA

Expressirm

During

Regeneration

Preblastemal stages. No specific riboprobe hybridization was detected in unamputated limb tissues such as epidermis, gland-epidermal junctions (Fig. 3A), periosteum, tendon, and myotendinous junctions (not shown), tissues that have been shown to be immunoreactive to mAb MT1 (see Onda et al, 1990). At 1 day after amputation, wound closure was already complete, and the amputation surface was covered by a thin layer of the wound epithelium. No hybridization was seen at this stage (not shown). At 2 days after amputation, when few mesenchymal cells are present under the wound epithelium, tenascin transcripts were not yet detectable in the distal stump mesoderm. However, strong hybridization was seen in the wound epithelium; the labeling was strongest in the basal layers but was present throughout the thickness and length of the wound epithelium (Fig. 3B). At this stage, tenascin protein was detectable with mAb MT1 only in tissues normally immunoreactive in the unamputated limb, such as periosteum, myotendinous junction, and epidermal-gland junctions (Fig. 3C). No specific hybridization was observed with a sense probe (Fig. 3D). By 5 days after amputation, the labeling in the wound epithelium increased, especially in the basal cells of the central portion. The hybridization near the stump border became restricted to only basal cells (Fig. 4A). A small number of strongly labeled mesodermal cells appeared at the distal edge of the transected tissues; the distribution of the cells extended somewhat proximal from the level of amputation, among muscle and other

D). Note that no hybridization can be seen in dermis level of amputation. Open arrows in B and D point periosteum (PO). Bar, 125 pm.

mRNA

in Regenerates

225

internal connective tissues, but the exact cell type could not be determined (Fig. 4A). Tenascin protein was distributed in both mesodermal ECM and the wound epithelium; a mAb MT1 immunoreactive layer underneath the wound epithelium began to appear at this stage (Fig. 4B). The overall distribution of tenascin mRNA in both wound epithelium and mesodermal stump corresponded to the pattern of tenascin protein deposition; however, only a subset of the cells in the stump was transcribing the tenascin gene at this stage (Figs. 4A and 4B). Between 5 and 14 days after amputation, both tenascin mRNA expression and protein deposition increased dramatically in the distal stump mesoderm (Figs. 4 and 5). By 8 days after amputation, the number of cells expressing tenascin mRNA increased in all mesodermal tissues of the stump except in the dermis (Fig. 4C). The wound epithelium, especially the basal layer of the central portion, was also strongly labeled at Day 8 (Fig. 4C). During the next few days, prior to the apparent blastema1 outgrowth, mesodermal cells transcribing tenascin mRNA continued to increase in both number and concentration at the distal end of the limb stump (Fig. 5). The basal layer of wound epithelial cells continued to express tenascin mRNA but also contained a fine, diffuse, cytoplasmic material immunoreactive to mAb MTl; the cells of the outer layer showed a patchy reactivity that was likely extracellular (Figs. 5C and 5D). The antibody also reacted to characteristic fibrillar structures in the ECM surrounding the mesenchymal cells and to a thick layer of material at the base of the wound epithelium. By 14 days after amputation, almost all the mesenchyma1 cells at the distal stump were strongly labeled with the antisense probe (Figs. 6A and 6D). The mesodermal cells transcribing tenascin mRNA in the stump more proximal to the level of amputation were often distributed among dissociated muscle bundles and were sometimes closely associated with individual muscle fibers (Figs. 6B and 6E). Most of the labeling was in fibroblastlike cells outside of muscle fibers, but some labeling was present in cells closely associated with muscle fibers (Figs. 6C and 6F). Identity of these cells could not be determined unambiguously by light microscopy. In all the stages analyzed (i.e., preblastemal, blastemal, and redifferentiation stages) sense probe did not show any specific hybridization (not shown). BZastemaZ growth stages. The vast majority of mesenchymal cells in early- to late-bud blastemas and the distal stump contained tenascin mRNA, but the intensity of labeling within a single blastema and between blaste-

(d). Arrows indicate the thickness to the layer of mAb MT1 reactive

of the wound epithelium (we). Arrowheads material under the wound epithelium.

mark the Muscle (m);

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(D) in the distal end of 11 day (A and C) and 14 day ?IG. 5 Mic :rographs showing localization of tenascin mRNA (A, B, ar Id C) and polypeptide of 11 day (A and C) and 14 day (B) regenerates with ar itisense pr *obe and 1D) 1-1zgenerates. ITI situ hybridization on longitudinal sections ng under the wound epithelium (we). Note that very little 1abeling ca n be sheIwing : incr .eased labeling in the distal mesenchymal cells accumulatil

@I

ONDAETAL.

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EGFL repeats in newt tenascin. Strong conservation of the primary structure of the EGFL domain in tenascin suggests a fundamental function. The EGFL repeats have been implicated in local mitogenic activity and receptor binding based on findings of the EGFL domain in other ECM molecules (Panayotou et ab, 1989; Engel, 1989), but the exact role of this growth factor-like domain in tenascin remains unknown (Spring et al., 1989; Weller et ah, 1991). The tripeptide RGD sequence in the third FN type III repeat of chicken and human tenascin sequence (Jones et ah, 1988; Gulcher et ah, 1989) has been implicated in binding to cell-surface receptors in a manner similar to that of other known ECM proteins containing the RGD sequence (Hynes, 1987; Ruoslahti and Pierschbacher, 1987). However, subsequently, a cell-binding role of the RGD sequence in tenascin has been questioned (see Weller et al., 1991). Weller et al. (1991) found that mouse tenascin lacks an RGD sequence in the third FN type III repeat and instead has an RVD sequence. We show here that newt tenascin contains an RGL sequence at that position. Although the existence of RGD sequences outside of the NvTN.1 coding region cannot be ruled out, our present finding favors the view that tenascin-cell binding is independent of the RGD sequence (ChiquetEhrismann et ab, 1988; Spring et aZ., 1989). With a riboprobe constructed from NvTN.1, we utilized in situ hybridization to analyze the distribution of tenascin transcripts in unamputated and regenerating limb tissues. The results with unamputated limbs suggest that the tenascin molecule exhibits little turnover. DISCUSSION No transcripts could be detected in periosteum, tendons, myotendinous junctions, or epidermis, all of which show In this paper we describe the isolation and characterization of a newt tenascin partial cDNA, NvTN.1. The strong reactivity to mAb MT1 (Onda et ab, 1990). During regeneration, tenascin mRNA was first denucleotide sequence similarity between chicken tenatected in the wound epithelium. Since the wound epithescin sequence (GALTNl; Spring et al, 1989) and NvTN.1 indicates that NvTN.1 is a newt tenascin cDNA. The lium is derived directly from epidermis, it is clear that overall similarity between chicken and newt sequence is in newts, cells of epidermal origin are capable of excomparable to the homology between chicken and pressing the tenascin gene. Presumably, the mAb MTl-positive granules present in epidermis throughout mouse, or chicken and human, sequences and indicates the body (Onda et al, 1990) are the result of a low level of that the tenascin gene sequence was conserved during It is interesting that tenathe divergence of these vertebrate classes. The high de- tenascin gene transcription. scin protein is not immunologically detectable in the gree of homology between the deduced amino acid sewound epithelium of rat skin wounds (Mackie et ah, quences of NvTN.1 and chicken tenascin sequence con1988) nor of the regenerating tail of Pleurodeles waltz firms that NvTN.1 is a cDNA for newt tenascin. A re(Arsanto et ah, 1990). However, in the latter two studies, markable feature is that the unique cysteine pattern of strong antitenascin immunoreactivity was reported in a EGFL repeats, X,CX&X,CX,CX,CX&, found in tenalayer at the base of the wound epithelium, as we obscin from different sources (Spring et ah, 1989; Gulcher served during newt and axolotl limb regeneration (Onda et ah, 1989; Weller et al., 1991), is also observed in all the

mas was not uniform (Figs. 7A and 7B). In contrast, the labeling in the wound epithelium decreased and became restricted to the basal cells of the distal portion of the wound epithelium (Figs. 7A and 7B). The labeling in the wound epithelium was often strongest near the center. The distribution of tenascin mRNA in the blastema was less uniform than the distribution of the protein product in the ECM as visualized by mAb MT1 reactivity in adjacent sections (not shown; see Onda et ah, 1990). The precise distribution of tenascin mRNA in relation to the limb axes will require further analysis. Rediferentiation stages. After the blastema growth period, mesenchymal cells start to redifferentiate into various adult tissues. Starting at the palette stage, some mesenchymal cells initiate condensation to form the cartilage anlagens. During these redifferentiation stages, tenascin mRNA expression became restricted. At late-palette and early-digit stages, tenascin mRNA was mainly found in condensing mesenchyme, in cells outlining cartilage anlagens, and in mesenchymal cells at the still growing tips of digits (Figs. 7C and 7D). During these stages, ECM tenascin protein became restricted in its distribution and began to disappear from the condensed cartilage (not shown). In late-digit regenerates, tenascin mRNA was seen in the wound epithelium only at the distal tips of still growing digits; none was seen in the wound epithelium at the side of or in between the digits (Figs. 7C and 7D). Finally, when redifferentiation was almost complete, tenascin mRNA became undetectable (not shown).

found in dermis (d). (C) An enlargement of part of A showing intense labeling in the basal layer of the wound epithelium (indicated by an open arrow). Arrows indicate the thickness of the wound epithelium. (+) In A and C indicates the corresponding position in the fields. (D) An immunofluorescence micrograph stained with mAb MT1 shows granular reactivity around the outer cell layers (arrowheads) and fine diffuse reactivity within cells of the basal layer (open arrow) of the wound epithelium. Bars, (A and B) 135 Km; (C) 62 grn; (D) 40 pm.

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FIG. 6. High-magnification micrographs of in situ hybridization in 3.4-day regenerates. Micrographs of the identical fields were taken un der either combined darkfield-epifluorescence optics (A, B, and C) or d arkfield optics alone (D, E, and F). In distal mesenchyme (A 6md D) essentially all the cells hybridize to the probe. In more proximal reg ions (B and E), the hybridization was often associated with dissc bcia ted muscle bundles (mb). C and F are enlarged micrographs of parts of B ztnd E marked by the open arrow. Most of the cells showing hybridi !zat .ion

ONDAETAL.

et al., 1990). The abundance of tenascin mRN9 in the basal cells of the newt wound epithelium suggests that the immunoreactivity to mAb MT1 observed within wound epithelial cells and in a thick layer at the base of the wound epithelium represents tenascin polypeptides synthesized by the wound epithelium; previously it was suggested that this layer of tenascin is synthesized by mesodermal cells in response to an induction by the wound epithelium (Mackie et al., 1988; Arsanto et al., 1990). When an antibody and riboprobe specific to frog tenascin becomes available, it would be of interest to examine the wound epithelium of amputated limbs at regenerating and nonregenerating stages of the anuran life cycle. The spatial distribution of tenascin in blastemas suggests an important role in epithelial-mesenchymal interactions, as in developing organs (Chiquet-Ehrismann et al., 1986; Aufderheide et al., 1987; Vainio et al., 1989; Prieto et al., 1990). Whereas a role for tenascin in epidermal closure has been implicated during rat wound healing (Mackie et al., 1988), such a role for tenascin in newts is not supported by the present results. We show that wound epithelial closure is complete before transcription of tenascin mRNA begins and before mAb MT1 reactivity is seen (Fig. 3). Others suggested that tenascin may modulate cell adhesion directly by binding cell surface receptors (Friedlander et ah, 1988; Bourdon and Ruoslahti, 1989) or indirectly by inhibiting binding to other ECM molecules such as fibronectin (ChiquetEhrismann et al., 1988; Lightner and Erickson, 1990), or by a combination of both mechanisms (Spring et al., 1989). Tenascin may be involved in the cell-to-cell interaction between epithelial cells and mesenchyme or in the cell-to-matrix interactions at the distal tip of the wound epithelium during regeneration. Thus, restriction of transcription to the distal portion of the wound epithelium during blastemal stages suggests a functional differentiation of the newt wound epithelium at these stages as was implicated for the apical epithelial cap of axolotl regenerates based on the specificity of mAb 9Gl reactivity (Onda and Tassava, 1991). Examination of the transcription pattern in the mesoderm shows that the initial expression of the tenascin gene extends some distance proximal to the amputation level and involves a subset of the stump mesodermal cells. The subsequent increase in the number and the concentration of tenascin transcribing cells in the distal stump could be a result of the migration and proliferation of cells initially transcribing tenascin and/or initiation of tenascin gene transcription in additional distal stump cells. Present experiments do not provide a dis-

were located muscle fiber

outside showed

of the muscle hybridization

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Tmnscin mRNA in Regeneratrs

tinction between these possibilities. In light of the findings that tenascin may mediate cell migration during development (Chuong et al., 1987; Halfter et al., 1989; Riou et al., 1990) and that migrating fibroblasts can deposit tenascin along their migratory pathways (Halfter et al., 1990) it is possible that stump mesodermal cells transcribe the tenascin gene and then deposit tenascin protein in the ECM as they migrate distally. Most of the mesodermal cells transcribing the tenascin gene in early regenerates are found associated with muscle; some of these are among the dissociated fibers near the distal ends of transected muscle. These cells are most likely fibroblasts (or myoblasts?) resulting from dedifferentiation. In chickens, both fibroblasts and myoblasts are capable of synthesizing tenascin in vitro, and increased synthesis or accumulation of tenascin in muscle and nerves is observed at the site of peripheral nerve injury (Daniloff et al., 1989). It is possible that, in newts, the production of tenascin may be indicative of cellular dedifferentiation as a response to the injury of amputation. Dermal fibroblasts are thought to be a major contributor to the blastema in axolotls (Gardiner et al., 1986) and possibly also in newts (Tank, 1984). In this regard, it is intriguing that there is essentially no tenascin transcription in cells in the dermis of the regenerating newt limb. Since almost all blastemal cells transcribe the tenascin gene, it follows that dermal fibroblasts must first migrate out of the dermis and then begin transcription. Studies with triploid markers will be useful to examine tenascin gene transcription in cells originating from specific tissues. The synthesis of tenascin by almost all mesenchymal cells during blastemal stages is consistent with the view that tenascin is an important component of the ECM during blastemal growth. Furthermore, since the blastema is believed to be comprised of cells originating from a variety of stump tissues, a common regulatory mechanism that is also shared by basal cells of the wound epithelium must be operating. Because tenascin mRNA can vary due to alternative splicing, a possibility worth considering is that different cell types of the regenerate synthesize different tenascin subunits, as occurs in chick embryos (Prieto et al., 1990). A shift from ubiquitous to selective expression during redifferentiation stages suggests that the tenascin gene is responding in a cell-specific manner as cells differentiate. Although the present results cannot determine whether such a change is the cause of the cellular differentiation or merely a consequence of the loss of undif-

fiber (mf) as indicated by arrows in C and F; however, occasionally, a nucleus (arrow head). Bars, (A and D) 60 Frn; (B and E) 125 pm; (C and F) 30 pm.

closely

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FIG. 7. Micrographs illustrating localization of mRNA in midbud (A and B) and digit (C and D) stage regenerates. In situ hybric tenascin transcripts. The pattern and inter longitudinal sections through midbud-stage regenerates (A and B) she ,wing blastemal hybridization varied somewhat from section to section, but almost all m esenchymal cells exhibited hybridization. Note that signal in epithelium became restricted to the basal layer of the distal part. AI rowheads indicate the level of amputation. In situ hybridi’ longitudinal section through the distal tip of a digit-stage regenerate mias observed under either combined darkfield-epifluorescencc or epifluorescence optics alone (D). Intense hybridization was seen i n the distal mesenchyme (m) and cells outlining forming Hybridization in the wound epithelium (we) was restricted to the basa L cells distal to the growing digits. Arrows indicate the thick wound epithelium. Condensed cartilage (c) did not hybridize. Bars, (A) I 125 pm; (B, C and D) 110 Wm.

Szal isity the zatic ? opt digi ness

Lion on of the wound m to a its(C) ts (d). of the

ONDA ET AL.

Tenascin

ferentiated characteristics, it is clear that the tenascin gene is developmentally regulated during regeneration. The recently identified promoter for the chicken tenascin gene demonstrates a variety of upstream regulatory elements which can be activated in a cell-specific manner (Jones et ah, 1990). It is likely that expression of the tenascin gene during regeneration is controlled by a complex developmental program involving a number of different trans-acting factors which are in turn regulated by specific cell-to-cell or cell-to-ECM interactions. The challenge will be to understand these regulatory mechanisms and to elucidate the role of tenascin during regeneration. The authors thank Jeremy P. Brockes for providing the newt cDNA library, Ruth Chiquet-Ehrismann for providing the chicken tenascin cDN.4, and Amanda A. Simcox and Eric V. Yang for helpful comments on the manuscript. Supported by NIH Grant HD 22024 and Office of Naval Research Grant NO0014 to R.A.T. and by NIH Grants ROl CA 45611 and P30 CA 16058 to I.-M.C. I.-M.C. is a recipient of a Research Career Development Award (K04 CA01369) from the National Institutes of Health. REFERENCES ARSANTO, J.-P., DIANO, M., THOUVENY, Y., THIERY, J. P., and LEVI, G. (1990). Patterns of tenascin expression during tail regeneration of the amphibian urodele Pleurodeles waltl. Development 109,177-188. AUFDERHEIDE, E., CHIQUET-EHRISMANN, R., and EKBLOM, P. (1987). Epithelial-mesenchymal interactions in the developing kidney lead to expression of tenascin in the mesenchyme. J. Cell Biol. 105,599608. AUSUBEL, F. M., BRENT, R., KINGSTON, R. E., MOORE, D. D., SEIDMAN, J. G., SMITH, J. A., and STRUL, K. (Eds.) (1987). “Current protocols in Molecular Biology.” Greene Publishing Associates and Wiley, New York. BOURDON, M. A., and RUOSLAHTI, E. (1989). Tenascin mediates cell attachment through an RGD-dependent receptor. J. Cell Biol. 108, 1149-1155. CHIQUET, M., and FAMBROUGH, D. M. (1984). Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis. J. Cell Biol. 98, 1924-1936. CHIQUET-EHRISMANN, R., KALLA, P., PEARSON, C. A., BECK, K., and CHIQUET, M. (1988). Tenascin interferes with fibronectin action. Cell 53, 383-390. CHIQUET-EHRISMANN, R., MACKIE, E. J., PEARSON, C. A., and SAKAKURA, T. (1986). Tenascin: An extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis. Cell 47, 131-139. CHUONG,C.-M., CROSSIN,K. L., and EDELMAN, G. M. (1987). Sequential expression and differential function of multiple adhesion molecules during the formation of cerebellar cortical layers. J. Cell Biol. 104, 331-342. CROSSIN,K. L., HOFFMAN, S., GRUMET, M., THIERY, J. P., and EDELMAN, G. M. (1986). Site-restricted expression of cytotactin during development of the chicken embryo. J. Cell Biol. 102,1917-1930. DANILOFF, J. K., CROSSIN,K. L., PINCON-RAYMOND, M., MURAWSKY, M., RIEGER, F., and EDELMAN, G. M. (1989). Expression of cytotactin in the normal and regenerating neuromuscular system. J. Cell Bioi. 108, 625-635. ENGEL, J. (1989). EGF-like domains in extracellular matrix proteins: Localized signals for growth and differentiation? FEBS Lett. 251, l-7.

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FERGUSON, J. E., SCHOR, A. M., HOWELL, A., and FERGUSON,M. W. J. (1990). Tenascin distribution in the normal human breast is altered during the menstrual cycle and in carcinoma. Diflerentiatkm 42, 199-207. FRIEDLANDER, D. R., HOFFMAN, S., and EDELMAN, G. M. (1988). Functional mapping of cytotactin: Proteolytic fragments active in cellsubstrate adhesion. J. Cell Biol. 107, 2329-2340. GARDINER, D. M., MUNEOKA, K., and BRYANT, S. V. (1986). The migration of dermal cells during blastema formation in axolotls. Del?. Biol. 118, 488-493. GULCHER, J. R., NIES, D. E., MARTON, L. S., and STEFANSSON,K. (1989). An alternatively spliced region of the human hexabrachion contains a novel repeat of potential N-glycosylation sites. Proc. Nutl. Acad.

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HALFTER, W., CHIQUET-EHRISMANN, R., and TUCKER, R. P. (1989). The effect of tenascin and embryonic basal lamina on the behavior and morphology of neural crest cells in vitro. Dev. Biol. 132, 14-25. HALFTER, W., LIVERANI, D., and MONARD, D. (1990). Deposition of extracellular matrix along the pathways of migrating fibroblasts. Cell Tissue

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HYNES, R. 0. (1987). Integrins: A family of cell surface receptors. Cell 48,549-554. INAGUMA, Y., KUSAKABE, M., MACKIE, E. J., PEARSON, C. A., CHIQUETEHRISMANN, R., and SAKAKURA, T. (1988). Epithelial induction of stromal tenascin in the mouse mammary gland: From embryogenesis to carcinogenesis. De?? Biol. 128, 245-255. ITEN, L. E., and BRYANT, S. V. (1973). Forelimb regeneration from different levels of amputation in the newt, Notophthalmus viridescens: Length, rate and stages, Wilhelm Rouxb Arch. 173,263-282. JONES, F. S., BURGOON, M. P., HOFFMAN, S., CROSSIN, K. L., CmNINGHAM, B. A., and EDELMAN, G. M. (1988). A cDNA clone for cytotactin contains sequences similar to epidermal growth factorlike repeats and segments of fibronectin and fibrinogen. Proc. Nat/. Acad.

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JONES, F. S., HOFFMAN, S., CUNNINGHAM, B. A., and EDELMAN, G. M. (1989). A detailed structural model of cytotactin: Protein homologies, alternative RNA splicing, and binding regions. Proc Natl. Acud.

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JONES, F. S., CROSSIN,K., CUNNINGHAM, B. A., and EDELMAN, G. M. (1990). Identification and characterization of the promoter for the cytotactin gene. Proc. Natl. Acad Sci. USA 87.6497-6501. LIGHTNER, V. A., and ERICKSON,H. P. (1990). Binding of hexabrachion (tenascin) to the extracellular matrix and substratum and its effect on cell adhesion. J. Cell Sci. 95, 263-267. LIGHTNER, V. A., GUMKOWSKI, F., BIGNER, D. D., and ERICKSON, H. P. (1989). Tenascin/hexabrachion in human skin: Biochemical identification and localization by light and electron microscopy. J. Cell Biol. 108,2483-2493. MACKIE, E. J., HALFTER, W., and LIVERANI, D. (1988). Induction of tenascin in healing wounds. J. Cell Biol. 107, 2757-2767. MACKIE, E. J., THESLEFF, I., and CHIQUET-EHRISMANN, R. (1987). Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro. J. Cell Biol. 105, 2569-2579.

ONDA, H., GOLDHAMER, D. J., and TASSAVA, R. A. (1990). An extracellular matrix molecule of newt and axolotl regenerating limb blastema and embryonic limb buds: Immunological relationship of MT1 antigen with tenascin. Development 108,657-668. ONDA, H., and TASSAVA, R. A. (1991). Expression of the 9Gl antigen in the apical cap of axolotl regenerates requires nerves and mesenthyme. J. Exp. 2001. 257,336-349. PANAYOTOU, G., END, P., AUMAILLEY, M., TIMPL, R., and ENGEL, J. (1989). Domains of laminin with growth-factor activity. Cell 56,93101.

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CHIQUET-EHRISMANN, R. (1988). Tenascin: cDNA cloning and induction by TCF-B. EMBO J. 7,2977-2981. PRIETO, A. L., JONES, F. S., CUNNINGHAM, B. A., CROSSIN,K. L., and EDELMAN, G. M. (1990). Localization during development of alternatively spliced forms of cytotactin mRNA by in situ hybridization. J. Cell Biol. 111, 685-698. PROBSTMEIER, R., MARTINI, R., and SCHACHNER,M. (1990). Expression of JUTenascin in the crypt-villus unit of adult mouse small intestine: Implications for its role in epithelial shedding. Development 109,313-321.

RAGSDALE, C. W., JR., PEDOVICH, M., GATES, P. B., CHAMBON, P., and BROCKES,J. P. (1989). Identification of a novel retinoic acid receptor in regenerative tissues of the newt. Nature (Lmdm) 341,654-657. RIOU, J.-F., SHI, D.-L., CHIQUET, M., and BOUCAUT, J.-C. (1990). Exogenous tenascin inhibits mesodermal cell migration during amphibian gastrulation. Dev. Biol. 137,305-317. RUOSLAHTI, E., and PIERSCHBACHER,M. D. (1987). New perspectives in cell adhesion: RGD and intergrins. Science 238,491-497.

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SINGER, M., and SALPETER, M. M. (1961). Regeneration in vertebrates: The role of the wound epithelium. In, “Growth in Living Systems” (M. X. Zarrow, Ed.), pp. 277-311. Basic Books, New York. SPRING, J., BECK, K., and CHIQUET-EHRISMANN, R. (1989). Two contrary functions of tenascin: Dissection of the active sites by recombinant tenascin fragments. Cell 59,325-334. TANK, P. W. (1984). The influence of flank dermis on limb regeneration in the newt, Notophthalmus viridescens. J. Exp. Zool. 229,143153. THORNTON, C. S. (1968). Amphibian limb regeneration. In “Advances in Morphogenesis” (M. Abercrombie et al, Eds.), Vol. 7, pp. 205-249. Academic Press, New York. VAINIO, S., JALKANEN, M., and THESLEFF, I. (1989). Syndecan and tenascin expression is induced by epithelial-mesenchymal interactions in embryonic tooth mesenchyme. J. Cell Biol. 108,1945-1954. WELLER, A., BECK, S., and EKBLOM, P. (1991). Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis. J. Cell Biol. 112,335-362.