Several well-characterized extracellular matrix (ECM) components have been localized to the amphibian limb regenerate, but the identification and characterization of novel ECM molecules have received little attention. Here we describe, using mAb MT1 and immunocytochemistry, an ECM molecule expressed during limb regeneration and limb development. In limb stumps, mAb MT1 reactivity was restricted to tendons, myotendinous junctions, granules in the basal layers of epidermis, periosteum (newts) and perichondrium (axolotls). In regenerating limbs, reactivity in the distal limb stump was first detected 5 days and 1 day after amputation of newt and axolotl limbs, respectively. In both species, mAb MT1 recognized what appeared to be an abundant blastema matrix antigen, localized in both thin and thick cords between and sometimes closely associated with blastema cells. Reactivity was generally uniform throughout the blastema except for a particularly thick layer that was present immediately beneath the wound epithelium. During redifferentiation stages, mAb MT1 reactivity persisted among blastema cells and redifferentiating cartilage but was lost proximally in areas of muscle and connective tissue differentiation. During the entire period of embryonic limb development, mAb MT1 reactivity was seen in the ECM of the mesenchyme and in a layer beneath the limb bud ectoderm, similar to its distribution during regeneration. Considerable mAb MT1 reactivity was also associated with the developing somites. The reactivity of mAb MT1 in blastema and limb bud was similar if not identical to that of a polyclonal Ab against tenascin (pAbTN), a large, extracellular matrix glycoprotein implicated in growth control, inductive interactions, and other developmental events. This pAbTN effectively competed against mAb MT1 binding on blastema sections. In immunoblots, both mAb MT1 and pAbTN recognized a very high molecular weight (approximately Mr 1000 × 103) protein in blastema extracts of both newts and axolotls. mAb MT1 immunoprecipitated a protein of Mr 1000K size which reacted to both mAb MT1 and pAbTN in immunoblots. These data show that tenascin is in the matrix of the urodele blastema and limb bud, and suggest that mAb MT1 identifies urodele tenascin.
The interaction between embryonic cells and extracellular matrix (ECM) is emerging as a critical parameter involved in dictating cell fates, proliferative behavior, morphogenetic potential, and other developmental events (Hynes, 1981; Toole, 1981; Chiquet-Ehrismann et al. 1986). To better define the roles of the ECM during amphibian limb regeneration, the distribution and timing of appearance of several major ECM/cell surface components have been investigated. Collagen (Grillo etal. 1968), hyaluronic acid (Toole and Gross, 1971), fibronectin and laminin (Repesh et al. 1982; Gulati et al. 1983) and neural cell adhesion molecule (N-CAM; Maier et al. 1986) have all been shown to be developmentally regulated during regeneration. Little is known of the role of these molecules in regeneration, although hyaluronic acid synthesis has been implicated to be directly or indirectly dependent upon the neurotrophic action of nerves (Smith et al. 1975; Mescher and Munaim, 1986), and based on antibody inhibition studies, Maier et al. (1986) have suggested an involvement of N-CAM in blastema growth. Further studies of these and other matrix components should reveal greater insights into their involvement in the major reparative and developmental events which characterize limb regeneration, including wound healing, dedifferentiation, cell proliferation and morphogenesis.
During a search for molecules of developmental significance to regeneration, we obtained a monoclonal antibody (mAb MT1; formerly referred to as mAb 4G3; Goldhamer and Tassava, 1986; Tassava, 1988) reactive to the extracellular matrix of the blastema. Based on immunohistochemical observations, the distribution of the MT1 antigen in unamputated and regenerating limbs did not correspond with previously described molecules such as fibronectin, laminin, hyaluronic acid, collagens, or N-CAM (Smith et al. 1975; Gulati et al. 1983; Maier et al. 1986), thus raising the possibility that mAb MT1 recognizes a novel ECM molecule (Goldhamer and Tassava, 1986; Tassava, 1988).
We report here experiments designed to determine the identity of this matrix component and its temporal and spatial distribution in regeneration and embryonic development. The results show that MT1 is an early appearing antigen which is abundant in the blastema and limb bud matrix, but is restricted mainly to tendons and skeletal element sheaths in mature limb tissues. Comparison of mAb MT1 reactivity with that of polyclonal antibody to tenascin demonstrated a striking degree of similarity in regenerating and developing limbs. Results from a series of tests, including immunoblotting and immunoprecipitation, are consistent with the view that mAb MT1 recognizes urodele tenascin.
Materials and methods
Adult newts (Notophthalmus viridescens) were collected from ponds in southern Ohio. Axolotl (Ambystoma mexicanum) larvae and embryos were obtained from the Indiana University axolotl colony. Care, feeding, and surgical operations for both species have been described (Kelly and Tassava, 1973; Mescher and Tassava, 1975). Axolotl larvae of 35, 45, and 75 mm snout-tail tip length were utilized. All operations were performed while animals were anesthetized with neutralized MS 222 (ethyl m-aminobenzoate methanesulfonate; Sigma). Newt embryos were obtained as previously described (Tas-sava and Acton, 1989).
mAb MT1 (matrix 1; formerly called 4G3; Goldhamer and Tassava, 1986; Tassava, 1988) was obtained by immunizing mice to homogenates of mid- and late-bud blastemas of adult newts. The immunization protocol and survey method for putative mAbs were similar to those described for mAb WE3 (Tassava et al. 1986). mAb MT1 was precipitated with ammonium sulfate from hybridoma culture medium, dissolved in phosphate-buffered saline (PBS), dialyzed against PBS, and stored frozen as a stock solution. The stock solution could be diluted over 1000 X before loss of reactivity in tissue sections. We determined that mAb MT1 is an IgM antibody (Isotyping kit; Calbiochem). Preliminary data on mAb MT1 reactivity have been reported (Goldhamer and Tassava, 1986; Tassava, 1988).
The rabbit polyclonal antiserum against chicken tenascin (pAbTN) was a kind gift from Dr R. Chiquet-Ehrismann, and its production and specificity have been reported previously (Chiquet-Ehrismann et al. 1986). This antiserum has been shown to react to tenascin from different species, including mammals and amphibians (Aufderheide et al. 1987; Epperlein et al. 1988; Riou et al. 1988). The rabbit polyclonal antiserum (IgG fraction) against axolotl plasma fibronectin (pAbFN) was a kind gift from Dr Y. Thouveny and Dr J. C. Boucaut, and its production and specificity have been described previously (Boucaut and Darribere, 1983). This antiserum does not react to tenascin, laminin, or entactin (Thouveny and Boucaut, personal communication), but reacts to both plasma and cell surface fibronectin of amphibians (Boucaut and Darribere, 1983).
Immunofluorescence methods were similar to those described (Tassava et al. 1986; Goldhamer et al. 1989). In brief, freshly dissected unamputated limbs, regenerating limbs and embryos were frozen in OCT compound (Miles) in a slurry of dry ice and isopropyl alcohol. Tissues were sectioned at 10pm on a cryostat and sections were allowed to dry at room temperature for 2h or overnight. Sections were used immediately or stored at — 20°C. mAb MT1 was applied to sections as a lOx dilution of the stock solution in PBS. After 1 h of incubation, slides were washed in PBS first with 0.05 % Triton X-100 (three washes of 3 min each) and then without Triton (one wash of 3 min). Secondary Ab (rhodamine labeled goat antimouse IgM; Cappel) was then applied for 1 h, after which slides were washed as above and coverslipped. Controls were included as previously described (Tassava et al. 1986).
For immunofluorescence staining with pAbTN and pAbFN, sections were first blocked with 5 % non-fat dry milk in PBS for 30 min, then incubated in diluted pAbTN (1:10), or pAbFN (1:10) for lh. The following steps were identical to those for mAb MT1 except that the secondary Ab was affinity-purified rhodamine-labeled goat anti-rabbit IgG (Cappel).
Newt and axolotl embryos were sampled for mAb MT1 reactivity at all stages of forelimb development; axolotl larvae were additionally sampled at various stages of hindlimb development. Both frontal and transverse cryostat sections, including limb buds and various other body organs and tissues, were examined for reactivity to mAb MT1. Sections through limb buds were also examined for pAbTN reactivity.
Reactivity to mAb MT1 was examined in distal regions of amputated axolotl limbs at 12 h, 24 h, and 4 days after amputation, and at 2 day intervals thereafter through digit stages of regeneration. Regenerating newt limbs were examined at 3, 5, 7, 10, and 14 days after amputation (pre-blastema stages) and at early-bud, mid-bud, late-bud, palette, and digit stages of regeneration (Iten and Bryant, 1973). Regenerates were always sampled so that a 1-2 mm portion of stump tissue was included.
To test whether mAb MT1 might be reacting to tenascin or fibronectin, three different tests were performed. First, we compared the reactivity patterns of mAb MT1, pAbTN, and pAbFN in adjacent cryostat sections of newt blastemas and limb buds. Second, a double labeling experiment was carried out on the same sections of newt and axolotl blastemas. Sections were incubated sequentially in mAb MT1, FITC-labeled goat anti-mouse IgG (Cappel), pAbTN and rhoda-mine-labeled goat anti-rabbit IgG (affinity purified; Bio-Rad). Each step consisted of a lh incubation followed by washes as described. The specificity of secondary antibodies and of optical filters was confirmed by including controls in which only a single pair of Abs was applied. Double-labeled sections were viewed by indirect immunofluorescence with the appropriate filter combinations so that F1TC and rhoda-mine could be examined independently. Third, a competition experiment on tissue sections was designed in which the ability of pAbTN and pAbFN to inhibit binding of mAb MT1 was tested. Sections of newt blastemas were first incubated in 5 % non-fat dry milk in PBS for 30 min. Either pAbTN (1:10 dilution in PBS) or pAbFN (no dilution) was next applied to separate sections and allowed to react for 1 h after which mAb MT1 was applied to the same sections without intervening washes. After a Ih incubation, and subsequent washes, secondary Ab specific to mAb MT1, rhodamine-conjugated goat anti-mouse IgM, was then applied as above, and sections were examined for immunoreactivity. Controls in which the secondary Ab was applied without prior incubation in mAb MT1 showed no detectable reactivity against either pAbTN or pAbFN.
Both MT1 antigen and tenascin could be extracted from various tissues using a high pH buffer (Riou et al. 1988; Crossin et al. 1986). Briefly, mid- to late-bud blastemas of axolotls were homogenized in a frosted glass homogenizer with 10 volumes of 30 mM diethylamine (pH 11.5), 1 DIM EDTA, 2mM phenylmethylsulphonyl fluoride (PMSF), and 2μM leupeptin and extracted on ice for 6h with shaking. The extracts were cleared by centrifugation at 10000g for 10 min at 4°C, dialyzed against dd H2O and aliquoted for immediate use or for storage at -80°C.
Extracts were mixed with an equal volume of 2x SDS sample buffer (Laemmli, 1970) with or without /S-mercaptoethanol. Samples were separated on 5% (reducing conditions) or 3 % (nonreducing conditions) discontinuous SDS-polyacrylamide gels with 2.5 % stacking gels (Laemmli, 1970). After gel electrophoresis, proteins were transferred to nitrocellulose (Towbin et al. 1979) and then blocked for 30 min with 5% non-fat dry milk in Tris-buffered saline (TBS). Strips of nitrocellulose were incubated separately in pAbTN (1:500), pAbFN (1:500), nonimmune rabbit serum (1:100), nonimmune mouse serum (1:100), or mAb MT1 (1:100), diluted with 5% non-fat dry milk in TBS, overnight at room temperature with gentle agitation. Strips were washed in TBS with 0.05 % Tween-20 three times before incubation in appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG for pAbTN, pAbFN, and nonimmune rabbit serum; goat anti-mouse IgG for nonimmune mouse serum; goat anti-mouse IgM (for mAb MT1). After washing as above, bands were visualized by developing the strips in a solution of 4-chloro-l-naphthol containing H2O2, and photographed after color development. Molecular weight standards were myosin (MT 440 X103 nonreduced, Mr 205 xlO3 reduced), β-galactosidase (Mr 116 X103), phosphorylase b (Mr 97 xlO3), and bovine plasma albumin (Mr 66 xlO3).
Immunoadsorbents were prepared by coupling mAb MT1 to the activated ester agarose matrix, Affi-Gel 15 (BioRad), at approximately 10 mg antibody per 1ml of beads. To an aliquot of extract used for immunoblotting, sodium phosphate (dibasic), NaCl and EDTA were added to a final concentration of 10mM phosphate, 150mM NaCl, and ImM EDTA, pH7.4 (PBS-EDTA). Extract was first incubated with quenched Affi-Gel 15 for 1 h and centrifuged to remove any proteins nonspecifically bound to the beads. The preadsorbed extract was incubated with the mAb MIT coupled Affi-Gel 15 overnight at 4°C with gentle shaking. Immunoprecipitates were collected by centrifugation, washed three times with PBS-EDTA, and resuspended in SDS sample buffer without β-mercaptoethanol, and boiled for 10 min. The beads were pelleted by centrifugation and proteins in the supernatant were separated by SDS-PAGE as above. For reduced conditions, β-mercaptoethanol was added to the supernatant to a final concentration of 10% before SDS-PAGE. Immuno-reactivities to mAb MT1, pAbTN and pAbFN of the immuno-precipitates were assessed by immunoblotting as above.
Distribution of MT1 antigen in the limb stump
In the limb stumps of axolotls and adult newts, mAb MT1 reacted to tendons, myotendinous junctions, perichondrium/periosteum, a thin layer at the junction between integumentary glands and epidermis (in newts), and to granules within the basal layers of the epidermis (Fig. 1A-C, 2C, D; Table 1). Since reactivity patterns to mAb MT1 were similar in axolotls of all three sizes, only data from 35 mm larvae will be reported here. Reactivity in the limb stump of the axolotl was similar to that of newts except that the perichondrium of axolotls stained more strongly than the periosteum of newts; also, the granular reactivity in axolotl epidermis was not as abundant as in newts (Table 1; see newt epidermal reactivity in Fig. 2C, D). While the present results do not exclude intracellular reactivity, the majority of mAb MT1 reactivity in the unamputated limb appeared to be extracellular. In newts the reactivity linking glands to the epidermis (Fig. 2C, D) and also the majority of reactivity of tendons (Fig. 1C) was most likely extracellular. In sections of both axolotl and newt epidermis which were examined both by epifluorescence (Fig. 1B) and Nomarski optics (not shown), the mAb MT1 reactive granules appeared in most cases to outline cells and therefore might largely be extracellular.
Expression of MT1 antigen during limb regeneration
In the regenerating limb, changes in mAb MT1 reactivity appeared early, already at 1 day after amputation in 35 mm axolotls and 5 days after amputation in the adult newt (Table 2). Except for timing of appearance and disappearance, reactivity in axolotls and newts was similar. Staining first appeared as a fine band under the wound epithelium and as fibers and cords among muscle and connective tissues in the area of dedifferentiation (Fig. 2A, C). Reactivity became stronger as the number of blastema cells increased, so that through early-, mid-, and late-bud blastema stages, mAb MTI reactivity was very strong and relatively uniformly distributed throughout the blastema matrix and in a thick band under the wound epithelium (Fig. 2B-D), During pre-blastema and blastema stages of regeneration, the band of MTl-reactive material under the wound epithelium ended abruptly at the level of amputation (Fig. 2A, C, D). The thick band of reactive material subjacent to the wound epithelium, as well as the majority of staining throughout the blastema, was extracellular, but a low level of intracellular reactivity cannot be ruled out (Fig. 2B, D). At differentiation stages, MT1 reactivity remained high in mesenchyme of the distal tip of the regenerate, under the distal wound epithelium at the tips of digits, and in the perichon-drium, and was somewhat less intense but present within the area of differentiating chondrocytes; reactivity decreased in differentiating muscle and connective tissues (Fig. 2E), except in tendons, where reactivity persisted (not shown). As differentiation proceeded, the reactive band under the wound epithelium was lost proximally near the level of amputation (Fig. 2E).
Throughout regeneration, the wound epithelium was less reactive to mAb MT1 than skin epidermis in both newts and axolotls but some granular reactivity was nevertheless present (Fig. 2B, C). In some sections, a diffuse reactivity was sometimes seen within some cells of the wound epithelium nearest the distal mesenchyme (Fig. 2C), especially at early blastema stages, but this was not consistent and requires further study.
Colocalization of tenascin with MT1 antigen
A polyclonal Ab to chicken tenascin (pAbTN) reacted to sections of both axolotls and newts in a fashion similar to that of mAb MT1. It was of interest therefore to determine whether mAb MT1 might in fact be reacting to urodele tenascin. Very similar patterns were seen when the reactivities of mAb MT1 and pAbTN were compared on adjacent sections (Fig. 3A, B). Reactivities of both mAb MT1 and pAbTN were seen among the blastema mesenchyme and in a layer underneath the wound epithelium. Both Abs exhibited a granular reactivity in skin epidermis (to a lesser degree in wound epithelium) and a fibrous reactivity in periosteum/perichondrium (not shown). These results show that tenascin is present in both newt and axolotl blastemas and limb stumps and that the MT1 antigen is distributed in an almost identical fashion. Unlike pAbTN, fibronectin polyclonal Ab (pAbFN) reacted more diffusely in the blastema and was weakly reactive to the layer underneath the wound epithelium (not shown). In the stump, pAbFN reactivity was more distinct from the reactivity of mAb MT1 and pAbTN. It reacted to basement membranes of muscle and epidermis, dermis, and perineurium of nerves (not shown), as previously described (Repesh et al. 1982; Gulati et al. 1983).
Double labeling on the same section revealed near identical distributions of mAb MT1 (FITC-labeled secondary Ab) and pAbTN (rhodamine-labeled secondary Ab) reactivity, even to some of the finest strands of reactive matrix (Fig. 4A, B). Finally, a competition experiment further suggested that the MT1 antigen might be tenascin. Prior incubation of sections with pAbTN almost totally eliminated subsequent binding of mAb MT1; however, prior incubation with undiluted pAbFN did not decrease subsequent binding of mAb MT1 (Fig. 5A, B, C).
Distribution of MT1 antigen and tenascin in developing limbs
Since tenascin exhibits a spatially restricted distribution in axolotl embryos (Epperlein etal. 1988), we examined both axolotl and newt embryos for mAb MT1 reactivity. Embryos of newts and axolotls exhibited similar patterns of reactivity to mAb MT1 (Fig. 6) and both resembled closely the reactivity reported for pAbTN (Epperlein et al. 1988), although the latter study did not include limb bud. Reactivity of mAb MT1 was uniformly present amongst the mesenchyme throughout the forelimb and hindlimb buds, and in a layer under the distal ectoderm (Fig. 6A). The developing limb bud matrix, including the layer under the ectoderm, was reactive at all stages examined, beginning at the time the limb bud was first recognizable as an outgrowth. mAb MTI reactivity was also seen outlining the notochord and the neural tube, in association with the somites, and in the connective tissue of the dorsal fin (Fig. 6B). Confirmation of other reactivities in the embryo will require more study. Finally, a double Ab test of mAb MTI and pAbTN on limb bud sections (as above for blastemas) revealed essentially identical patterns of reactivity for the two Abs (not shown).
Immunoblot analysis of blastema extracts
In order to determine the molecular nature of the MTI antigen and to compare it with the molecule identified by pAbTN in newts and axolotls, immunoblot analyses of both newt and axolotl blastema extracts were conducted. Only data from axolotls are shown (Fig. 7) since identical results were obtained when newt blastema extract was analyzed.
We observed previously that MTI reactivity is lost when newt or axolotl blastema extracts were treated with reducing reagents such as )3-mercaptoethanol (Goldhamer and Tassava, unpublished data). This result was confirmed here by immunoblotting of axolotl blastema extract under reducing conditions (Fig. 7A, lane 2). The same sample stained with pAbTN showed a series of strongly reactive bands of Mr 210-250X103, and less intense 105X103 and 80X103 bands (Fig. 7A, lane 1). These results indicate that disulfide bonds are essential for the maintenance of antigenicity to mAb MTI but not to pAbTN. The range of subunit molecular weights from 210-250K are in good agreement with the subunits of tenascin from chicken (Chiquet and Fambrough, 19846), Xenopus laevis (Epperlein et al. 1988), and Pleurodeles waltlii (Riou et al. 1988). Since crude extract of blastema prepared directly in SDS sample buffer did not show 105K and 80K bands (not shown), they are likely proteolytic fragments of the above subunits due to the prolonged extraction protocol (Fig. 7A, lane 1). pAbFN reacted to three distinct bands of Mr 250X103, 235 x103, and 220X103 (Fig. 7A, lane 3). 220K is the reported relative molecular weight of the fibronectin subunit, which has almost an identical migration rate on SDS gels as the major subunit of chicken tenascin (Chiquet and Fambrough, 19846). The origin of the two larger bands reactive to pAbFN is currently unknown; however, based on reported variations of fibronectin subunit mobilities on SDS gels (Hynes, 1985), it is possible that the two larger bands reflect heterogeneity of blastema fibronectin. Neither nonimmune rabbit nor mouse serum showed reactive bands corresponding to tenascin or fibronectin subunits (not shown).
Nonreduced MT1 antigen was sufficiently resolved in a 3 % polyacrylamide gel so as to detect two distinct bands which migrated into the gel only a short distance (Fig. 7B, lane 2). Precise molecular weight determination of these nonreduced molecules is difficult. How-ever, from comparison to available molecular weight standards, the larger band corresponds to roughly Mr 1000x103, which is the estimated molecular weight of the intact oligomer of tenascin (Vaughan et al. 1987). In fact, bands of similar molecular weight reacted to pAbTN (Fig. 7B, lane 1), Heterogeneity of subunit composition has been described for tenascin (Chiquet and Fambrough, 1984a,b). The two high molecular weight polypeptides revealed by mAb MT1 may represent tenascin heterogeneity among the blastema cell population. Alternatively, the smaller band could simply be a proteolytic fragment of the larger one. In addition to the high molecular weight oligomer, pAbTN identified several smaller polypeptides which did not react to mAb MT1. The sizes of these bands correspond to dimeric and monomeric forms of tenascin (Riou et al. 1988). The result can be interpreted as either proteolysis occurring during the extraction as suggested by Riou et al. (1988) or existence of incompletely assembled molecules of tenascin in the tissues. pAbFN reacted to MT 500 X103, 440 x103, and 235 X103 bands of the nonreduced sample (Fig. 7B, lane 3). Again, presence of a 500K band, which is larger than the expected dimer of 440K, may be related to subunit size heterogeneity in blastema fibronectin. Neither of the control sera reacted to MT1, TN, or FN specific bands (not shown).
Immunoprecipitation of tenascin using mAb MT1
As shown in Fig. 8A, mAb MT1 precipitated proteins that react to pAbTN (lane 1) but not to pAbFN (lane 3) after reduction. Under nonreducing conditions (Fig. 8B), the immunoprecipitates were recognized by both pAbTN and mAb MT1 but not by pAbFN. The patterns of the reactivities were essentially identical to the results of the immunoblots with crude extract except that there was no detectable fibronectin present in the precipitate. The presence of the smaller pAbTN reactive bands which did not react to mAb MT1 indicates that these peptides were recognized by mAb MT1 during immunoprecipitation but not after SDS-PAGE. A possible explanation of this apparently inconsistent result is that some molecules of tenascin exist as an oligomeric complex compatible to mAb MTI recognition without complete disulfide bonds covalently linking each subunit. Such complexes could be immunoprecipitated by mAb MTI, and after SDS treatment they would dissociate into smaller subunits. Thus, these data, together with immunoblot results, suggest that mAb MTI recognizes intact oligomeric tenascin in the blastema, and that the epitope recognized by mAb MTI is lost upon reduction.
MTI antigen expression during limb regeneration
The present results are consistent with the view that mAb MTI identifies a molecule that is important to limb regeneration in newts and axolotls. In both species, the MTI antigen is first expressed early in regeneration, just prior to or concomitant with the start of cell cycling (Chalkley, 1954; Kelly and Tassava, 1973; Mescher and Tassava, 1975). The MTI antigen is at its greatest abundance during blastema growth, where it is associated with a fibrous/cord-like component in the blastema matrix and to a relatively thick acellular layer of material immediately under the wound epithelium. During differentiation stages, mAb MTI reactivity persists in association with mesenchyme at the tips of the still growing digits and with newly differentiating cartilage. Thus, the spatial and temporal distribution of the MTI antigen during regeneration make it a candidate molecule relevant in matrix-blastema cell interactions and wound epithelium-mesenchyme interactions. The observation that mAb MTI reacts to the developing limb bud in a pattern resembling that of reactivity in the blastema suggests that the MTI antigen is relevant to both limb development and regeneration.
It is of interest to compare the timing of appearance and location of the MTI antigen with the 22/18 and WE3 antigens. Reactivity of mAb MTI appears very early, concomitantly with that of mAb 22/18 (Gordon and Brockes, 1987), an Ab that identifies an intracellular antigen of a sub-population of mesenchymal cells within the newt regenerate (Kintner and Brockes, 1984). Both mAbs MTI and 22/18 exhibit reactivity prior to mAb WE3, which begins to react to the wound epithelium during the 2nd week post-amputation in newts (Tassava et al. 1986). It may be significant that those wound epithelial cells that react most strongly to mAb WE3 reside at the base of the epithelium (Tassava et al. 1986), adjacent to the strongly reactive MT1 layer between the wound epithelium and mesenchyme. The correspondence between the timing and distribution of reactivity to mAb MT1 and those of other mAbs reactive during regeneration is an area worthy of further study.
Restricted expression of MT1 antigen in the limb stump
The MT1 antigen has a limited distribution in the limb stump, where its presence is indicated by mAb MT1 reactivity to perichondrium and periosteum, tendons, myotendinous junctions, gland-epidermis junctions and epidermis. It could be speculated that the MT1 antigen acts as an adhesive agent, holding tendons to muscle and glands to epidermis. In the epidermis, mAb MT1 reacts to a granular component, present largely between the cells within the non-differentiated, basal layers. The role for the MT1 antigen in epidermis is at present unknown.
Immunological relationship of MT1 antigen with tenascin
The possibility was considered that mAb MT1 was reacting to one of the extracellular matrix components previously described in regenerating limbs (Tassava, 1988). However, the reactivity patterns of mAb MT1 in the unamputated limb and in the blastema were not what would be expected of the commonly known extracellular matrix components such as collagens, laminin, fibronectin and hyaluronic acid, all of which have wider distributions (Repesh et al. 1982; Gulati et al. 1983; Mescher and Munaim, 1986). On the other hand, tenascin, a relatively recently characterized extracellular matrix glycoprotein (Vaughan et al. 1987), became a strong candidate based on comparison of its temporal and spatial distribution with that of mAb MT1 reactivity. Consider for example that tenascin has been shown to be present in tendons, myotendinous junctions and perichondrium (Chiquet and Fambrough, 1984a), each of which is also reactive to mAb MT1. Based strictly on Ab reactivity patterns, it was reasonable to further investigate whether the MT1 antigen is tenascin or is an antigen closely associated with tenascin. Since tenascin is known to partially colocalize with fibronectin in vivo (Chiquet and Fambrough, 1984a), and copurify in cell-surface fibronectin preparations (Chiquet-Ehrismann et al. 1986), all the experiments performed to identify the MT1 antigen included tests for fibronectin. All the results indicate that the MT1 antigen is distinct from fibronectin.
Evidence that the MT1 antigen is tenascin was provided from immunocytochemical comparisons of mAb MT1 reactivity with that of pAbTN. In embryos, mAb MT1 reacted to somites, to a layer surrounding the neural tube, and to the matrix of the developing tail fin. This pattern of reactivity resembled that reported for tenascin in chicken and amphibian embryos (Epper-lein et al. 1988). In adjacent sections of blastemas, the two Abs exhibited very similar reactivity patterns within the blastema and the limb stump. Double labeling with mAb MT1 and pAbTN on the same blastema section also revealed essentially identical reactivity patterns. Furthermore, pAbTN competed specifically with mAb MT1 binding to tissue sections whereas pAbFN did not.
Biochemical characterization provided further evidence that the MT1 antigen corresponds to urodele tenascin. Tenascin is a large, six-armed molecule, held together by disulfide bonds (Vaughan et al. 1987). On immunoblots, mAb MT1 and pAbTN reacted to high molecular weight bands of identical size under nonreducing conditions, but with reduced samples, mAb MT1 reactivity was lost. Thus, the MT1 antigen also contains disulfide bonds, and a comformation dependent antigen is lost upon reduction. Finally, mAb MT1 precipitated a high molecular weight protein corresponding in size to oligomeric tenascin, which was recognized by both pAbTN and mAb MT1, but not pAbFN. Under reducing conditions, this immunoprecipitate lost reactivity to mAb MT1 but maintained reactivity to pAbTN.
Based on the immunocytochemical data, immunoblot analyses, and immunoprecipitation results, it seems reasonable to suggest that mAb MT1 reacts to the intact molecule of newt and axolotl tenascin and that tenascin is an important component of the blas-tema matrix. Formal proof that MT1 antigen is urodele tenascin requires complete biochemical characterization and elucidation of its gene sequence. In this regard, purifying the MT1 antigen from axolotl tissue extracts by immunoaffinity chromatography and screening a newt blastema cDNA library with a chicken tenascin cDNA probe will add further data relevant to the identity of the MT1 antigen.
Functional roles of tenascin during regeneration
Strong immunoreactivity to pAbTN shows that tenascin is abundant in the blastema and limb bud of newts and axolotls. While tenascin has not previously been investigated during regeneration or early development of limbs, a variety of evidence has implicated this large matrix glycoprotein in muscle/tendon morphogenesis (Chiquet and Fambrough, 1984a, b), epithelialmesenchymal interactions during kidney and mammary development (Inaguma et al. 1988), cartilage and bone differentiation (Mackie et al. 1987), and mammary tumor development (Inaguma et al. 1988). In cell culture, tenascin is mitogenic for mammary tumor cells (Chiquet-Ehrismann et al. 1986) and inhibits cell attach-ment to fibronectin (Chiquet-Ehrismann et al. 1988). Finally, fetal bovine serum and transforming growth factor β (TGF β) individually stimulate synthesis of tenascin by human and chick embryo fibroblasts (Pear-son et al. 1988). In light of these findings, and consider-ing the suggestion of Pearson et al. (1988) that tenascin may be important to wound healing and regeneration (see Mustoe et al. 1987), it is worth investigating possible roles for tenascin in limb regeneration. The distribution of tenascin, near or in contact with essentially every mesenchymal cell within the blastema, could mean it is mitogenic or assists somehow in the mitogenic action of other molecules. The layer of tenascin under the wound epithelium could be relevant to the interaction between wound epithelium and mesenchyme.
Previous studies imply strongly that tenascin is synthesized strictly by mesenchyme (Inaguma et al. 1988). We show here that both mAb MT1 and pAbTN exhibit a granular reactivity in both newt and axolotl epidermis and to a lesser extent in the wound epithelium; however, we do not know the cellular origin of this granular reactivity. In preliminary studies (Tassava, unpublished data), mAb MT1 has been shown to react more strongly to basal layers of specialized epidermis, such as the nuptial pads of the male newt hindlimb, compared to the same region of female newts, or to other areas of epidermis of the male. These studies are being extended and may provide clues as to what role tenascin might have in the epidermis, whether the granules are extra- or intracellular, and their cellular origin.
Given the many developmental phenomena occurring during regeneration, including dedifferentiation, cellular proliferation, epithelial-mesenchymal interactions, nerve ingrowth and pattern formation, it is likely that many different matrix components play interactive roles. Not surprisingly then, previous studies have shown that fibronectin (Repesh et al. 1982; Gulati et al. 1983), laminin (Gulati et al. 1983), and hyaluronic acid (Smith et al. 1975; Mesher and Munaim, 1986; Mescher and Cox, 1988), might be important to regeneration. In this regard, Mescher and Cox (1988) found a correlation between hyaluronate accumulation, re-inner-vation, and blastema formation in axolotls. On the other hand, Maden and Keeble (1987), in examining retinoic acid (RA) induced distal-proximal duplication, found no alteration in fibronectin distribution or synthesis compared to control regenerates. We have begun studies with mAb MT1 and other matrix-reactive mAbs to determine to what extent expression of these matrix antigens are influenced by RA, nerves and/or hormones.
The mAb MT1 was obtained while R.A.T. was a senior research fellow in the Developmental Biology Laboratory, Massachusetts General Hospital, Dr Jerome Gross, Director. Efforts at the MGH were supported by NIH grant AM3564 and EY2252 to J. Gross and senior research fellowship award F33HD06675-1 to R. Tassava. Studies at The Ohio State University were supported by NIH grant HD22024 to R.A.T. D.J.G. and H.O. were supported by fellowships from The Ohio State University during a portion of this work.