Fibroblast growth factors in regenerating limbs of Ambystoma: cloning and semi-quantitative RT-PCR expression studies.

Urodele amphibians (newts and salamanders) have the ability to regenerate amputated limbs throughout their life span. Because fibroblast growth factors (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fgf4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanum, and locally collected spotted salamander, Ambystoma maculatum, two salamanders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleotide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showed that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, however, was undetectable by RT-PCR in the distal tips of regenerates, suggesting that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneurogenic and innervated limbs, suggesting that they are not the neurotrophic factors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 upregulation, suggesting Fgf pathways are downstream of nerve-dependence. These data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001.

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated, nerve-dependent newt limb blastemas by rhGGF2.

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF-like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine-rich domain (CRD-NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD-NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve-dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve-dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response.

Apical epithelial cap morphology and fibronectin gene expression in regenerating axolotl limbs.

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate limbs. The regenerated structure is often indistinguishable from the developmentally produced original. Thus, the two processes by which the limb is produced - development and regeneration - are likely to use many conserved biochemical and developmental pathways. Some of these limb features are also likely to be conserved across vertebrate families. The apical ectodermal ridge (AER) of the developing amniote limb and the larger apical epithelial cap (AEC) of the regenerating urodele limb are both found at the limb's distalmost tip and have been suggested to be functionally similar even though their morphology is quite different. Both structures are necessary for limb outgrowth. However, the AEC is uniformly smooth and thickly covers the entire limb-tip, unlike the AER, which is a protruding ridge covering only the dorsoventral boundary. Previous data from our laboratory suggest the multilayered AEC may be subdivided into separate functional compartments. We used hematoxylin and eosin (H+E) staining as well as in situ hybridization to examine the basal layer of the AEC, the layer that lies immediately over the distal limb mesenchyme. In late-stage regenerates, this basal layer expresses fibronectin (FN) message very strongly in a stripe of cells along the dorso-ventral boundary. H+E staining also reveals the unique shape of basal cells in this area. The stripe of cells in the basal AEC also contains the notch/groove structure previously seen in avian and reptilian AERs. In addition, AEC expression of FN message in the cells around the groove correlates with previous amniote AER localization of FN protein inside the groove. The structural and biochemical analyses presented here suggest that there is a specialized ridge-like compartment in the basal AEC in late-stage regenerates. The data also suggest that this compartment may be homologous to the AER of the developing amniote limb. Thus, the external differences between amniote limb development and urodele limb regeneration may be outweighed by internal similarities, which enable both processes to produce morphologically complete limbs. In addition, we propose that this basal layer of the AEC is uniquely responsible for AEC functions in regeneration, such as secreting molecules to promote mesenchymal cell cycling and dictating the direction of limb outgrowth. Finally, we include here a clarification of existing nomenclature to facilitate further discussion of the AEC and its basal layer.

Effects of peripheral nerve implants on the regeneration of partially and fully innervated urodele forelimbs.

This study addresses the cellular mechanism of the nerve requirement for regeneration of the urodele forelimb. Others have suggested that only the Schwann cell lineage of the blastema requires nerves for regeneration and that upon limb denervation, Schwann cells arrest in the cell cycle and produce a factor that inhibits the cycling of the remaining blastema cells. Our objective was to test this Schwann cell inhibitor model. First, pieces of peripheral nerve were implanted into partially denervated (one third of the nerve supply cut) axolotl forelimbs in an attempt to provide sufficient additional Schwann cells to increase the threshold nerve requirement above that provided by the remaining nerves. These limbs showed delayed regeneration in 68% of the cases and mild deformities, as seen by Victoria Blue staining, in 10% of the cases, as compared with control, partially denervated contralateral limbs that received grafts of muscle or frozen/thawed nerve. Second, when pieces of peripheral nerve were implanted into fully innervated newt limbs, blastema formation was limited, and regeneration was delayed in 80% of experimental cases when compared with control, contralateral newt limbs with muscle or frozen/thawed nerve implants. The results support the inhibition model and further link the need for nerves in regeneration to a possible specific requirement by Schwann cells.

Abnormal limb regeneration in the short-toes mutant of the axolotl, Ambystoma mexicanum: studies of age, level of amputation, and extracellular matrix.

Limb regeneration in the short-toes axolotl is impaired. Our goal was to characterize the regeneration process in this mutant by histological and immunocytochemical methods. Previous research indicates that age and a defective basement membrane may be instrumental factors in short-toes axolotl regeneration (Del Rio-Tsonis et al. [1992] Proc. Natl. Acad. Sci. U.S.A., 89:5502-5506). The present results show that limb regeneration can occur even in older (1-2-year-old) short-toes axolotls. The process was always significantly delayed, but the time required for complete regeneration varied. Even so, the basement membrane of short-toes regenerates showed no differences in thickness or shape compared with wild-type regenerates. Distally amputated short-toes limbs gave rise to more digits in the regenerate, indicating that regeneration may be somewhat dependent on the level of amputation. Since extracellular matrix (ECM) remodeling occurs extensively during regeneration, we compared the ECM of the short-toes and wild-type regenerates using monoclonal antibodies (mAbs) MT2 and ST1 (Tassava et al. [1996] Wound Rep. Reg., 4:75-81). The short-toes regenerates showed decreased reactivity to mAb MT2, which identifies type XII collagen, an ECM protein that is normally unregulated during regeneration, and increased reactivity to mAb ST1, which identifies a limb ECM component that typically undergoes breakdown in the distal stump. Thus, impaired regeneration in the short-toes axolotl is correlated with impaired ECM remodeling in the distal limb stump. This supports the view that ECM remodeling plays an important role in regeneration.

Expression of type XII collagen by wound epithelial, mesenchymal, and ependymal cells during blastema formation in regenerating newt (Notophthalmus viridescens) tails.

Previously we showed that type XII collagen (col XII) is highly upregulated in the regenerating newt (Notophthalmus viridescens) forelimb. Here, using immunohistochemistry and in situ hybridization, we studied the pattern of expression of col XII during early stages of adult newt tail regeneration. The results show that immunoreactivity of col XII is first seen as a thin layer beneath the wound epithelium (WE) at 3 days after amputation. Reactivity associated with the mesenchyme becomes obvious at day 4 and increases considerably between days 6 and 7 after amputation. In situ hybridization indicates that the early WE-associated reactivity and later mesenchymal reactivity are due to increased col XII gene expression by the WE and mesenchyme, respectively. At 7 days after tail amputation both wound epithelial and mesenchymal cells exhibit a strong riboprobe signal. Interestingly, a distinct riboprobe signal is also seen in the cells of the outgrowing ependymal tube at day 7 but little if any col XII immunoreactivity is present. The spatial pattern of col XII gene expression changes by day 14 after amputation in that transcription in mesenchyme is maintained at a high level, in the WE it is reduced, and in ependyma it ceases to be detectable. Local deprivation of the spinal cord significantly lowers the level of col XII message in the mesenchyme. Much of this decrease in transcription is due to minimal mesenchymal cell accumulation secondary to spinal cord ablation. The temporal and spatial patterns of expression of the col XII gene in the WE, mesenchyme, and ependyma during tail regeneration strongly suggest a role for col XII in regulating both spinal cord outgrowth and spinal cord-dependent tail regeneration.

Extracellular matrix protein turnover during salamander limb regeneration.

After amputation of a salamander limb, the extracellular matrix undergoes remodeling. The extracellular matrix that maintains the differentiated state of limb tissues is broken down and replaced by an extracellular matrix essential for dedifferentiation and blastema formation. We used monoclonal antibodies in immunohistochemistry methods and riboprobes in in situ hybridization to evaluate the upregulation of tenascin, type XII collagen, fibronectin, and the MT5 antigen. The Stump 1 antigen, an extracellular matrix protein that is abundant in the normal limb, is downregulated during regeneration and reappears late in regeneration as differentiation occurs. In the embryo, the Stump 1 antigen is also absent from the early limb bud and first appears during differentiation stages. Tenascin and fibronectin are also upregulated in the limb bud of the embryo, and these two extracellular matrix proteins appear to function during limb regeneration in adults and limb development in embryos. However, type XII collagen and the MT5 antigen are not found in the limb bud, indicating that type XII collagen and the MT5 antigen have roles in the regenerating limb but not in the embryo limb bud.

Monoclonal antibody MT2 identifies the urodele alpha 1 chain of type XII collagen, a developmentally regulated extracellular matrix protein in regenerating newt limbs.

We previously described the upregulation of the MT2 antigen during urodele limb regeneration and characterized the MT2 antigen as a 310- to 325-kDa chondroitin-sulfated glycoprotein with a core protein of 285-300 kDa. In this study, we screened a newt blastema cDNA library using monoclonal antibody (mAb) MT2 and obtained a 1-kb cDNA fragment, designated Isolate (IS)-1. Subsequent screening of the same library using IS-1 cDNA as a probe provided IS-2, a 2.8-kb cDNA. IS-2 overlaps IS-1 at its 5' end, is highly homologous to a portion of the alpha 1 chain of the chicken type XII collagen cDNA (alpha 1[XII]), and spans a third of the chicken alpha 1[XII] cDNA, from the last 62 amino acids of the second A domain of von Willebrand factor to the first two repeats of the fourth fibronectin type III domain. The peptide sequence deduced from cDNA IS-2 demonstrates invariable tryptophan, leucine, threonine, and tyrosine residues that are highly conserved among all the fibronectin type III domains within IS-2 and between corresponding sequences of IS-2 and chicken alpha 1[XII]. A Northern blot showed a 10-kb band that corresponds to the size of the chicken alpha 1[XII] mRNA. A fusion gene was constructed by inserting the IS-2 cDNA downstream from the malE gene of Escherichia coli, which encodes maltose-binding protein (MBP). The isopropyl beta-D-thiogalactoside-induced fusion protein had the expected molecular weight and reacted to both mAb MT2 and rabbit anti-MBP serum. We conclude that mAb MT2 identifies the urodele alpha 1[XII]. The expression pattern of the type XII collagen gene in newt limb regenerates was examined by in situ hybridization. Type XII collagen transcripts first appeared at 3 days after amputation in cells of the basal layer of the wound epithelium. At Day 10, both the basal wound epithelial cells and the distal mesenchyme cells were highly transcriptionally active. At mid-bud and late-bud blastema stages, wound epithelium expression had decreased, whereas the mesenchyme remained strongly active in transcription and showed a tendency toward distal regionalization. Condensing cartilage showed no signal. Finally, at the late digit stage, hybridization became largely restricted to the perichondrium. The in situ results suggest a developmental role for type XII collagen in regeneration.

Examination of fibronectin distribution and its sources in the regenerating newt limb by immunocytochemistry and in situ hybridization.

Using monoclonal antibodies (mAbs) reactive to newt limb regenerates, we hope to gain insight into the identity and function of regeneration significant molecules. mAb MT4 (matrix 4) identifies an extracellular matrix (ECM) protein that is strongly up-regulated first in the distal stump and then in the blastema during regeneration. Within the first 24 hr after amputation the MT4 antigen is localized to an acellular space beneath the wound epithelium, and first appears in the basal cells of the wound epithelium between days 5 and 7. At mid-bud blastema stages, the MT4 antigen is homogeneously distributed as thin fibers in the blastema ECM, and is later largely restricted to the distal tip of the blastema and the areas of cartilage condensation. After extraction and immunoblotting, the MT4 antigen was observed as three reduced species of M(r) 225, 250, and 260. Taken together, the immunoblot and immunocytochemistry results suggested that mAb MT4 recognized newt fibronectin (FN). Sequence from a cDNA (NvFN.10) obtained by screening a newt blastema cDNA expression library with mAb MT4 conclusively identified the MT4 antigen as FN. To further investigate the expression of FN in regeneration, cDNA NvFN.10 was used to construct a riboprobe and in situ hybridization was done. In the unamputated limb only a few scattered cells expressed the FN gene. Within the first 3 days after amputation strong hybridization signal was observed in the basal cells of the wound epithelium. Most of the stump cells that dedifferentiated and accumulated beneath the wound epithelium at 7 days expressed the FN gene, while the basal cells of the wound epithelium maintained their expression. At mid- and late-bud blastema stages the vast majority of the blastema cells were strongly expressing the FN gene, but the wound epithelial cells now showed only weak FN transcription. Thus initially FN comes from the plasma. Then FN is synthesized by both the wound epithelium and mesenchyme. Finally, at blastema stages FN is produced primarily by the mesenchyme. The expression pattern of FN throughout regeneration suggests that this glycoprotein has roles in wound epithelial and mesenchymal cell migration and mesenchymal cell proliferation and differentiation.

Heterogeneity in the expression of fibroblast growth factor receptors during limb regeneration in newts (Notophthalmus viridescens).

Two closely related fibroblast growth factor receptors, FGFR1 and FGFR2, have been cloned from a newt (Notophthalmus viridescens) limb blastema cDNA library. Sequence analysis revealed that we have isolated both the bek and KGFR variants of FGFR2. These two variants differ only in the second half of the last of their three Ig-like domains. The expression patterns of FGFR1 and FGFR2 during limb regeneration have been determined by in situ hybridization. During the preblastema stages of regeneration, FGFR2 expression is observed in the basal layer of the wound epithelium and in the cells of the periosteum. As regeneration progresses to the blastema stages, FGFR2 expression continues to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. From the early bud to the mid-bud blastema stage, FGFR1 expression is observed throughout the blastema mesenchyme but, unlike FGFR2, is distinctly absent from the wound epithelium. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. During these later stages of regeneration, the wound epithelium hybridization to the FGFR2 probe is no longer observed. The expression patterns of these receptors suggest that FGFR1 and FGFR2 have distinct roles in limb regeneration, despite their sharing a number of the FGF ligands. Further investigation regarding the potential sources of the FGF ligands will help establish the role that FGFs and FGFRs play in limb regeneration.

The wound epithelium of regenerating limbs of Pleurodeles waltl and Notophthalmus viridescens: studies with mAbs WE3 and WE4, phalloidin, and DNase 1.

The wound epithelium of regenerating limbs of the American newt, Notophthalmus viridescens (Nv), up-regulates a number of antigens, including those recognized by mAbs WE3 and WE4. In the present study, we show that the WE3 antigen is up-regulated in a similar fashion in the wound epithelium of the European newt, Pleurodeles waltl (Pw). mAb WE3 and WE4 reactivities to secretory/transport body cell types, including integumentary glands, perineurium, endothelium, and conjunctiva, are also similar in these two species of newt. However, mAb WE4 reacts to both the epidermis and wound epithelium in Pw, whereas in Nv, mAb WE4 reacts only to the wound epithelium. Because the WE3 antigen is cytoskeleton-associated and Western blots reveal a 43 kDa species, we compared mAb WE3 reactivity with that of rhodamine-labeled phalloidin, a known actin-binding compound. Phalloidin did not react preferentially to the wound epithelium, conjunctiva, or other cell types strongly reactive to mAb WE3. Pretreatment of sections and tissue extracts with DNAse 1, a protein known to bind to actin, nearly abolished mAb WE3 reactivity in tissue sections and both WE3 and WE4 reactivity in ELISA assays, respectively. The results lead to the hypothesis that the WE3 and WE4 antigens are actin-binding proteins unique to the wound epithelium and other secretory/transport cell types.

Monoclonal antibody ST1 identifies an antigen that is abundant in the axolotl and newt limb stump but is absent from the undifferentiated regenerate.

Monoclonal antibodies (mAb) utilized in regeneration studies to date identify antigens that are up-regulated in the blastema. We obtained a monoclonal antibody, designated ST1 (Stump 1), that is reactive to an extracellular matrix (ECM) antigen exhibiting the opposite distribution; ST1 is an abundant antigen of the limb stump soft tissues but is absent from within the blastema. The border between abundance and absence of mAb ST1 reactivity was sharp and extended as a concavity into the stump. This distinct dichotomy led to further studies relevant to understanding how this extracellular matrix antigen is modulated during regeneration. mAb ST1 reactivity decreased in the internal tissues at the distal end of the limb prior to blastema formation and remained absent until the onset of differentiation. The initial decrease in mAb ST1 reactivity was dependent on the combined effects of injury and the wound epithelium but was nerve independent. At blastema stages of regeneration, the distribution of tenascin, ascertained by mAb MT1 reactivity, closely matched the area without reactivity to mAb ST1. The spatial and temporal distribution of the ST1 antigen in unamputated limbs and during regeneration did not correspond to any previously described ECM component.

Retinoic acid enhances monoclonal antibody WE3 reactivity in the regenerate epithelium of the adult newt.

Monoclonal antibody (mAb) WE3 recognizes an antigen that is developmentally expressed in the wound epithelium during adult newt limb regeneration. Experiments were designed to determine whether retinoic acid (RA), dissolved in dimethyl sulfoxide (DMSO) and administered by intraperitoneal injection, would enhance the temporal appearance of the WE3 antigen. RA given on days 1 or 4 after amputation, when the WE3 antigen is not yet detectable, resulted in moderate reactivity to mAb 2 days after injection and strong reactivity throughout the wound epithelium 4 days after injection. DMSO alone had no enhancing effect. RA also caused limb skin epidermis to exhibit reactivity to mAb WE3, initially near the amputation level, but then also more proximally. By 4 and 6 days after RA injection, epidermis of the flank, eye lid, and unamputated hind limbs also became strongly reactive to mAb WE3. Outer layers of skin epidermis were shed, resulting in an epidermis only one or two cells thick. Epidermis of newts given DMSO alone remained non-reactive to mAb WE3. When RA was given on days 7 and 10 after amputation, when a low level of mAb WE3 reactivity is already present in the wound epithelium, a considerable enhancement of mAb WE3 reactivity occurred through the next few days. No such enhancement was seen with DMSO alone. RA also greatly increased mAb WE3 reactivity in the wound epithelium of denervated limbs, in which case the wound epithelial reactivity to mAb WE3 is normally low. Retinol palmitate also increased mAb WE3 reactivity. The results raise the possibility that the WE3 antigen is a component of most if not all retinoid target tissues in newts.

Monoclonal antibody MT2 identifies an extracellular matrix glycoprotein that is co-localized with tenascin during adult newt limb regeneration.

Using immunohistochemical techniques and mAb MT2, we describe here a novel extracellular matrix (ECM) molecule that is developmentally regulated during limb regeneration in adult newts. The MT2 antigen appears during preblastema stages, is most abundant during blastema stages, and persists, near undifferentiated cells, until digit stages. The MT2 antigen is located in an acellular layer under the wound epithelium and throughout the ECM of the undifferentiated mesenchyme as a thick, cord-like component. In unamputated limbs mAb MT2 reactivity is restricted to tendons, myotendinous junctions, periosteum and to a layer of material beneath the epidermis. In both unamputated limbs and regenerating limbs, the reactivity to mAb MT2 colocalizes closely with urodele tenascin. Immunoblot analysis of blastema extracts showed that the unreduced form of the MT2 antigen is a large, polydispersed protein of approximately the same size as tenascin. However, based upon (a) molecular weights of reduced subunits, (b) competition experiments on tissue sections, and (c) analysis of molecules immunoprecipitated by mAb MT2, we conclude that the MT2 substance is unrelated biochemically to tenascin. The results from immunoblots, enzyme digestions and DEAE-Sephacell binding studies suggest that the unreduced MT2 antigen is a large protein composed of subunits which are connected by disulfide bonds. Reduction of the MT2 antigen results in three components recognized by mAb MT2. The largest of these reduced components is a chondroitin sulfate-like glycoprotein with a molecular weight (Mr) of 310-325 x 10(3). A second component (Mr, 285-300 x 10(3)) is the core protein of the 310-325 x 10(3) glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Ganglia implantation as a means of supplying neurotrophic stimulation to the newt regeneration blastema: cell-cycle effects in innervated and denervated limbs.

Regulation of blastema cell proliferation during amphibian limb regeneration is poorly understood. One unexplained phenomenon is the relatively low level of active cell cycling in the adult newt blastema compared to that of larval axolotls. In the present study, we used ganglia implantation as a means of "superinnervating" normally innervated adult newt blastemas to test whether blastema cell subpopulations are responsive to nerve augmentation. The effectiveness of implanted ganglia to provide neurotrophic stimulation was demonstrated in denervated blastemas. Blastemas implanted with 2 dorsal root ganglia and simultaneously denervated 14 days after amputation exhibited control levels of cell cycle activity 6 days later, as measured by 3H-thymidine pulse labeling. Denervated blastemas that were sham-operated or implanted with pituitary glands exhibited cell-cycle declines similar to those of denervated blastemas without implanted ganglia. Thus, 2 implanted ganglia provide neurotrophic stimulation equivalent to that of the normal nerve supply. Dorsal root ganglia implanted into normally innervated blastemas, which should effectively double neurotrophic activity to the blastema, had no effect on cell-cycle activity, innervated blastemas implanted with ganglia for 6 days exhibited pulse labeling indices similar to those of normally innervated blastemas. These data indicate that neurotrophic stimulation is not normally limiting in innervated limbs, and that some other factor, whether extrinsic or intrinsic to blastema cells, accounts for the relatively low level of active cell cycling in the adult newt blastema.

Characterization of a newt tenascin cDNA and localization of tenascin mRNA during newt limb regeneration by in situ hybridization.

We previously showed that tenascin, a large, extracellular matrix glycoprotein, 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, initiation of cell cycling, blastema outgrowth, and cellular differentiation.

Expression of the 9G1 antigen in the apical cap of axolotl regenerates requires nerves and mesenchyme.

Monoclonal antibody 9G1 (mAb 9G1) is reactive to the wound epithelium of axolotl larvae and therefore provided the opportunity to examine the interaction between the wound epithelium, nerves, and blastemal mesenchyme during axolotl limb regeneration. In unamputated limbs, mAb 9G1 is reactive to most or all cells of the dermis, skeletal elements, blood vessels, and nerves, to a few unidentified cells in muscle, and to none in epidermis. During regeneration of axolotl limbs, mAb 9G1 reacts strongly to an intracellular antigen of the blastemal mesenchyme and of the distal-most portion of the wound epithelium, the so-called apical epithelial cap (AEC). Because this thickened wound epithelium of regenerating amphibian limbs has been suggested as functioning in a manner similar to the apical ectodermal ridge (AER) of embryonic limb buds, it was of interest to further examine the reactivity of mAb 9G1 during various stages of regeneration. Whether mAb 9G1 reactivity in the AEC depended on mesenchyme and/or nerves was also tested. Monoclonal antibody 9G1 reactivity appears in the AEC of regenerating limbs prior to outgrowth of the blastema and persists throughout blastemal stages. Apical epithelial cap reactivity to mAb 9G1 is nerve dependent during early stages of blastema development and becomes nerve-independent at later stages. When epithelium-free blastemal mesenchyme is grafted onto injured flank musculature, ectopic limb regeneration occurs and the AEC derived from flank epidermis exhibits mAb 9G1 reactivity. These results show that a mAb 9G1 reactive AEC is characteristic of regenerating limbs and that expression of the 9G1 antigen by the AEC is dependent upon underlying blastemal mesenchyme and nerves.

Expression of the WE3 antigen in newt epithelium originating from subcutaneous grafts of skin.

mAb WE3 recognizes an antigen that is developmentally regulated in the wound epithelium of regenerating newt limbs. The antigen is precociously expressed when pieces of WE3-negative wound epithelium are grafted subcutaneously (Tassava et al.: Recent Trends in Regeneration Research. New York: Plenum Publishing Co., pp. 37-49, 1989). In the present study, we investigated whether the WE3 antigen is expressed in epidermis of subcutaneous grafts of skin. Small pieces of limb skin were grafted into small tunnels in the lower jaw, limb, and tail, oriented either the same as (epidermis facing out) or opposite to (epidermis facing in) the orientation of the host skin. In most cases, the epithelium migrated from the graft along the wounded surface of the tunnel, closed onto itself, and formed a multilayered "emigrant" epithelium. Infrequently, the migrating epithelium combined with the wound epithelium of the insertion wound. In no case did the epithelium migrate over the cut edge of the grafted dermis. Reactivity to mAb WE3 was first seen at 4 days after grafting, when the migrating epithelium had almost closed over onto itself. By 6 days and thereafter, the entire emigrant epithelium was reactive to mAb WE3. While initially restricted to the emigrant epithelium, at 10 days after grafting and thereafter, reactivity was also seen in the epidermis that remained in contact with the dermis. Expression of the WE3 antigen was not influenced by the orientation of the graft nor by the graft site. The results show that, compared to amputated limbs, the epithelium originating from these grafts precociously expresses the WE3 antigen. Also, epidermis of grafted skin is capable of expressing the WE3 antigen.