Development 108, 657-668 (1990) 657 Printed in Great Britain ©The Company of Biologists Limited 1990 An extracellular matrix molecule of newt and axoloti regenerating limb blastemas and embryonic limb buds: immunological relationship of MT1 antigen with tenascin HIROAKI ONDA, DAVID J. GOLDHAMER* and ROY A. TASSAVAt Department of Molecular Genetics, The Ohio State University, 484 W. 12th Ave., Columbus, Ohio 43210, USA 1 Author to whom correspondence should be addressed. 2 Present address: Department of Biology, University of Virginia, Charlottesville, Va. 22901, USA. Summary Several well-characterized extracellular matrix (ECM) period of embryonic limb development, mAb MT1 components have been localized to the amphibian limb reactivity was seen in the ECM of the mesenchyme and regenerate, but the identification and characterization of in a layer beneath the limb bud ectoderm, similar to its novel ECM molecules have received little attention. Here distribution during regeneration. Considerable mAb we describe, using mAb MT1 and immunocytochem- MT1 reactivity was also associated with the developing istry, an ECM molecule expressed during limb regener- somites. The reactivity of mAb MT1 in blastema and ation and limb development. In limb stumps, mAb MT1 limb bud was similar if not identical to that of a reactivity was restricted to tendons, myotendinous junc- polyclonal Ab against tenascin (pAbTN), a large, extra- tions, granules in the basal layers of epidermis, perios- cellular matrix glycoprotein implicated in growth con- teum (newts) and perichondrium (axolotls). In regener- trol, inductive interactions, and other developmental ating limbs, reactivity in the distal limb stump was first events. This pAbTN effectively competed against mAb detected 5 days and 1 day after amputation of newt and MT1 binding on blastema sections. In immunoblots, axoloti limbs, respectively. In both species, mAb MT1 both mAb MT1 and pAbTN recognized a very high 3 recognized what appeared to be an abundant blastema molecular weight (approximately MT1000 x 10 ) protein matrix antigen, localized in both thin and thick cords in blastema extracts of both newts and axolotls. mAb between and sometimes closely associated with blastema MT1 immunoprecipitated a protein of MT 1000K size cells. Reactivity was generally uniform throughout the which reacted to both mAb MT1 and pAbTN in immu- blastema except for a particularly thick layer that was noblots. These data show that tenascin is in the matrix of present immediately beneath the wound epithelium. the urodele blastema and limb bud, and suggest that During redifferentiation stages, mAb MT1 reactivity mAb MT1 identifies urodele tenascin. persisted among blastema cells and redifferentiating cartilage but was lost proximally in areas of muscle and Key words: limb regeneration, tenascin, extracellular connective tissue differentiation. During the entire matrix, monoclonal antibody. Introduction 1982; Gulati et al. 1983) and neural cell adhesion molecule (N-CAM; Maier et al. 1986) have all been The interaction between embryonic cells and extra- shown to be developmental^ regulated during regener- cellular matrix (ECM) is emerging as a critical para- ation. Little is known of the role of these molecules in meter involved in dictating cell fates, proliferative regeneration, although hyaluronic acid synthesis has behavior, morphogenetic potential, and other develop- been implicated to be directly or indirectly dependent mental events (Hynes, 1981; Toole, 1981; Chiquet- upon the neurotrophic action of nerves (Smith et al. Ehrismann et al. 1986). To better define the roles of the 1975; Mescher and Munaim, 1986), and based on ECM during amphibian limb regeneration, the distri- antibody inhibition studies, Maier et al. (1986) have bution and timing of appearance of several major suggested an involvement of N-CAM in blastema ECM/cell surface components have been investigated. growth. Further studies of these and other matrix Collagen (Grillo etal. 1968), hyaluronicacid (Toole and components should reveal greater insights into their Gross, 1971), fibronectin and laminin (Repesh et al. involvement in the major reparative and developmental 658 H. Onda, D. J. Goldhamer and R. A. Tassava events which characterize limb regeneration, including its production and specificity have been reported previously wound healing, dedifferentiation, cell proliferation and (Chiquet-Ehrismann et al. 1986). This antiserum has been morphogenesis. shown to react to tenascin from different species, including During a search for molecules of developmental mammals and amphibians (Aufderheide et al. 1987; Epperlein et al 1988; Riou et al. 1988). The rabbit polyclonal antiserum significance to regeneration, we obtained a monoclonal (IgG fraction) against axolotl plasma fibronectin (pAbFN) antibody (mAb MT1; formerly referred to as mAb was a kind gift from Dr Y. Thouveny and Dr J. C. Boucaut, 4G3; Goldhamer and Tassava, 1986; Tassava, 1988) and its production and specificity have been described pre- reactive to the extracellular matrix of the blastema. viously (Boucaut and Darribere, 1983). This antiserum does Based on immunohistochemical observations, the dis- not react to tenascin, laminin, or entactin (Thouveny and tribution of the MT1 antigen in unamputated and Boucaut, personal communication), but reacts to both plasma regenerating limbs did not correspond with previously and cell surface fibronectin of amphibians (Boucaut and described molecules such as fibronectin, laminin, hyalu- Darribere, 1983). ronic acid, collagens, or N-CAM (Smith et al. 1975; Gulati et al. 1983; Maier et al. 1986), thus raising the Immunocytochemistry possibility that mAb MT1 recognizes a novel ECM Immunofluorescence methods were similar to those described molecule (Goldhamer and Tassava, 1986; Tassava, (Tassava et al. 1986; Goldhamer et al. 1989). In brief, freshly 1988). dissected unamputated limbs, regenerating limbs and em- bryos were frozen in OCT compound (Miles) in a slurry of dry We report here experiments designed to determine ice and isopropyl alcohol. Tissues were sectioned at 10/m\ on the identity of this matrix component and its temporal a cryostat and sections were allowed to dry at room tempera- and spatial distribution in regeneration and embryonic ture for 2h or overnight. Sections were used immediately or development. The results show that MT1 is an early stored at — 20°C. mAb MT1 was applied to sections as a lOx appearing antigen which is abundant in the blastema dilution of the stock solution in PBS. After 1 h of incubation, and limb bud matrix, but is restricted mainly to tendons slides were washed in PBS first with 0.05% Triton X-100 (three washes of 3min each) and then without Triton (one and skeletal element sheaths in mature limb tissues. wash of 3min). Secondary Ab (rhodamine labeled goat anti- Comparison of mAb MT1 reactivity with that of poly- mouse IgM; Cappel) was then applied for 1 h, after which clonal antibody to tenascin demonstrated a striking slides were washed as above and coverslipped. Controls were degree of similarity in regenerating and developing included as previously described (Tassava et al. 1986). limbs. Results from a series of tests, including immuno- For immunofluorescence staining with pAbTN and blotting and immunoprecipitation, are consistent with pAbFN, sections were first blocked with 5 % non-fat dry milk the view that mAb MT1 recognizes urodele tenascin. 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 Materials and methods affinity-purified rhodamine-labeled goat anti-rabbit IgG (Cappel). General Newt and axolotl embryos were sampled for mAb MT1 Adult newts {Notophthalmus viridescens) were collected from reactivity at all stages of forelimb development; axolotl larvae ponds in southern Ohio. Axolotl (Ambystoma mexicanum) were additionally sampled at various stages of hindlimb larvae and embryos were obtained from the Indiana Univer- development. Both frontal and transverse cryostat sections, sity axolotl colony. Care, feeding, and surgical operations for including limb buds and various other body organs and both species have been described (Kelly and Tassava, 1973; tissues, were examined for reactivity to mAb MT1. Sections Mescher and Tassava, 1975). Axolotl larvae of 35, 45, and through limb buds were also examined for pAbTN reactivity. 75 mm snout-tail tip length were utilized. All operations were Reactivity to mAb MT1 was examined in distal regions of performed while animals were anesthetized with neutralized amputated axolotl limbs at 12 h, 24 h, and 4 days after MS 222 (ethyl m-aminobenzoate methanesulfonate; Sigma). amputation, and at 2 day intervals thereafter through digit Newt embryos were obtained as previously described (Tas- stages of regeneration. Regenerating newt limbs were exam- sava and Acton, 1989). ined at 3, 5, 7, 10, and 14 days after amputation (pre-blastema stages) and at early-bud, mid-bud, late-bud, palette, and digit Antibodies stages of regeneration (Iten and Bryant, 1973). Regenerates mAb MT1 (matrix 1; formerly called 4G3; Goldhamer and were always sampled so that a 1-2 mm portion of stump tissue Tassava, 1986; Tassava, 1988) was obtained by immunizing was included. mice to homogenates of mid- and late-bud blastemas of adult To test whether mAb MT1 might be reacting to tenascin or newts. The immunization protocol and survey method for fibronectin, three different