Newt Limb Research Articles

Retinoids in patterning: Chimeras win by a knockout

Recent studies on the regenerating newt limb, using cells transfected with chimeric retinoic acid receptors that can be activated by thyroid hormone, have provided unique insights into the function of specific retinoic acid receptor isoforms.

Cloning and characterization of cDNAs for matrix metalloproteinases of regenerating newt limbs.

Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.

Receptor isoform specificity in a cellular response to retinoic acid.

The effects of retinoic acid on cell proliferation, differentiation and patterning are thought to be mediated by the various retinoic acid receptors. Different receptor types are encoded by distinct genes (alpha, beta, and gamma), whereas various isoforms within each type are encoded by splicing variants resulting from the use of alternative promoters. The only region that differs between isoforms is the N-terminal A region containing a transcriptional activating domain. It has been proposed that these alternative A regions confer distinct activities on the receptors, thus allowing each to mediate specific effects of retinoic acid, but it has been difficult to demonstrate such isoform specificity as most cells express a number of different retinoic acid receptors. In an attempt to test whether different isoforms can mediate distinct biological effects we are focusing on retinoic-acid-dependent growth inhibition of newt limb cells. We have constructed chimaeric receptors in which the retinoic acid binding domain of each of five newt retinoic acid receptors has been replaced with a thyroid hormone (T3) binding domain. These constructs were introduced individually into cells whose growth rate was then measured in the presence of T3. The chimaeric alpha 1 receptor mediated T3-dependent inhibition of proliferation that was comparable to that given by retinoic acid, whereas the alpha 2 isoform had no activity in this assay, nor did the delta 1A, delta 1B and Delta 2 receptors. When the A region was deleted from the alpha 1 chimaera it remained a potent T3-dependent transcriptional activator, but no longer mediated T3-dependent growth inhibition. In contrast, when the A region of alpha 1 was transferred to a delta chimaeric receptor, the resulting molecule was fully active in T3-dependent growth inhibition. This is the first direct evidence for isoform specificity in a biological response to retinoic acid, and demonstrates that the specificity of this response is confined to the A region.

Semi-automated positional analysis using laser scanning microscopy of cells transfected in a regenerating newt limb.

Limb regeneration in urodele amphibians such as the newt is a key system for investigating the positional identity of cells. The regenerate arises locally from blastemal cells, mesenchymal progenitors that normally give rise to structures distal to the amputation plane but which can be respecified (proximalized) by treatment with retinoic acid (RA) such that proximal structures are formed. To establish an assay for positional identity, cells of distal and RA-treated distal blastemas are labeled by transfection with an alkaline phosphatase marker gene using particle bombardment (biolistics). After grafting the distal blastema to a proximal stump, a context known as intercalary regeneration, the proximodistal distribution of labeled cells in the resulting regenerate is an index of positional identity. We use enzyme-labeled fluorescence (ELF) in conjunction with laser scanning microscopy to detect transfected cells within a section of the entire regenerate. A semi-automated analysis of the positional distribution of marked cells along the proximal-distal axis demonstrates that cells from both distal and RA-treated blastemas contribute to the regenerate. This procedure provides an efficient and accurate tool for positional analysis of transfected cells, and should be applicable for studying genes that play a role in specifying cell position during morphogenesis.

Stabilizing role of the basement membrane and dermal fibers during newt limb regeneration

Following amputation of a newt limb, tissues at the amputation site undergo histolysis to give rise to a growth bud, or blastema, but they also provide a base on which the regenerate is constructed. Studies suggest that dermal tissues may differentially resist histolysis. To examine stability of tissues at the amputation site, more than 80 preblastemal staged regenerating limbs were examined histologically. Initially, all soft tissues not attached to bone retracted and were covered by migrating epithelium. The dermis was seen to be stable during the first week postamputation. Muscle dedifferentiated and was heavily stained with anti-tenascin antibodies, but the intact overlying dermis was unstained. Fiber bundles, revealed by staining with phosphotungstic acid hematoxylin, isolated the dermis from dedifferentiating deeper tissues during the first week postamputation, but partially broke down during the second week. However, the basement membrane (BM) remained as the distalmost intact structure at the amputation site in all limbs examined. The BM was the foundation for new BM synthesis which preceded dermis synthesis in the base of the blastema during the second week, even while undifferentiated cells were accumulating centrally. We suggest that the dermis resists histolysis long enough for new BM to form in continuity with that of the stump. Dermis formation (dermogenesis) distal to the amputation plane begins early as in mammalian healing but is not completed until after blastema formation. Thus, factors that inhibit dermal closure appear to distinguish regenerating from non-regenerating appendages.

Activation of a single retinoic acid receptor isoform mediates proximodistal respecification

Background: The regenerating limb of urodele amphibians is an important system for evaluating the effects of retinoic acid (RA) on pattern formation. Regeneration proceeds by local formation of the blastema, a mesenchymal growth zone which normally only gives rise to structures distal to its level of origin. RA can respecify proximodistal identity in amphibian limb regeneration, and this activity of RA on the blastema is observed in two contexts. First, exposure to RA proximalizes a distal blastema resulting in duplication of structures proximal to the level of amputation. Second, after transplantation of a distal blastema to a proximal stump, the transplanted cells normally make only a minor contribution to the intercalary regenerate, but if transplanted cells are exposed to RA they occupy positions proximal to their level of origin and contribute to the regeneration of the intermediate tissue. Multiple isoforms of RA receptors (RARs) are expressed in the newt limb and are thought to mediate the respecification of positional identity.Results To identify which receptor(s) mediates proximodistal respecification, we have used the biolistics (particle bombardment) technique to transfect the blastemal mesenchyme with plasmids encoding chimeric proteins containing partial amino-acid sequences of the various newt RAR isoforms fused to a partial sequence of the thyroid hormone (3,5,3′-triiodothyronine; T3) receptor. We then used T3 treatment to selectively activate individual RAR isoforms in vivo. By analyzing the distributions of transfected cells in regenerates derived from distal-to-proximal transplantation we find that activation of a single RAR isoform, δ 2, specifically mediates proximalization.Conclusion These results demonstrate that the ability of RA to respecify proximodistal identity is mediated by a specific RAR isoform, δ 2. Activation of the RA pathway in individual cells indicates that positional respecification can be cell-autonomous. RA can respecify axial identity in several contexts in vertebrate development, but this is the first case where the RAR mediating respecification has been identified.

Stabilizing role of the basement membrane and dermal fibers during newt limb regeneration

Background Following amputation of a newt limb, tissues at the amputation site undergo histolysis to give rise to a growth bud, or blastema, but they also provide a base on which the regenerate is constructed. Studies suggest that dermal tissues may differentially resist histolysis. Methods and Results To examine stability of tissues at the amputation site, more than 80 preblastemal staged regenerating limbs were examined histologically. Initially, all soft tissues not attached to bone retracted and were covered by migrating epithelium. The dermis was seen to be stable during the first week postamputation. Muscle dedifferentiated and was heavily stained with anti-tenascin antibodies, but the intact overlying dermis was unstained. Fiber bundles, revealed by staining with phosphotungstic acid hematoxylin, isolated the dermis from dedifferentiating deeper tissues during the first week postamputation, but partially broke down during the second week. However, the basement membrane (BM) remained as the distalmost intact structure at the amputation site in all limbs examined. The BM was the foundation for new BM synthesis which preceded dermis synthesis in the base of the blastema during the second week, even while undifferentiated cells were accumulating centrally. Conclusions We suggest that the dermis resists histolysis long enough for new BM to form in continuity with that of the stump. Dermis formation (dermogenesis) distal to the amputation plane begins early as in mammalian healing but is not completed until after blastema formation. Thus, factors that inhibit dermal closure appear to distinguish regenerating from non-regenerating appendages. © 1996 Wiley-Liss, Inc.

Stage-dependent genetically-based deformities of the regenerating newt limb from 4-nitroquinoline-N-oxide mutagenesis: Potential embryonic regulation of cancer

The effect of the mutagenic carcinogen 4-nitroquinoline-N-oxide (4NQO) on limb regeneration was studied in adult Triturus cristatus newts. Delayed limb regeneration was observed when a 10- to 15-μg crystal of 4NQO was implanted at the late dedifferentiation or late bud stage. Additionally, 4NQO administration at these stages caused developmental deformities in skeletal elements of the subsequent regenerates. In contrast, 4NQO implanted at the wound healing stage did not affect skeletal morphogenesis. These critical stages of limb regeneration affected by carcinogen exposure (but not other manipulations) represent mechanisms distinct from disruption of basement membrane deposition. Secondary regeneration, initiated by reamputation of the 4NQO-treated abnormal regenerates 4–5 mm proximal to the site of carcinogen implantation, produced normal regenerates, setting a limit on the diffusion distance of 4NQO in the limb. Distal reamputation through regenerates at the level of the abnormality resulted in regeneration of the original bone deformity, suggesting that the teratogenic effect of 4NQO is mediated via heritable mutational events in blastema cells. These results extend a previously published conclusion that 4-NQO is mutagenic in non-regenerative tissue to include regenerative tissue as well. However, in spite of mutagenic activity, squamous carcinomas were not induced by 4NQO exposure at any stage of regeneration. Therefore, the resistance of regenerative tissue to 4-NQO carcinogenesis is not due to resistance to mutations, but rather to other mechanisms perhaps similar to those which regulate malignant cells in murine embryos.

Conservation of Ligand Specificity between the Mammalian and Amphibian Fibroblast Growth Factor Receptors

We have previously cloned and sequenced a newt keratinocyte growth factor receptor (KGFR) cDNA which exhibited a unique spatial and temporal expression pattern in the regenerating newt limb. In this report, we further characterize the biochemical and functional properties of this newt KGFR. A stable Chinese hamster ovary transfectant overexpressing the newt KGFR was capable of binding both 125I-fibroblast growth factor-1 (FGF-1) and 125I-FGF-7 but not 125I-FGF-2, indistinguishable from the human KGFR. Scatchard analysis and cross-linking studies further support the conclusion that FGF-1 and FGF-7 are the ligands for the newt KGFR. In addition to their ability to bind to FGFs, both the human and the newt KGFR are also capable of repressing differentiation in mouse MM14 myoblasts. MM14 cells express FGFR1 and are repressed from differentiation by FGF-1, FGF-2, and FGF-4 but not FGF-7. Co-transfection of MM14 cells with either a human or newt KGFR expression construct conferred a response to FGF-7 as determined by a human alpha-cardiac actin/luciferase reporter construct. The response to FGF-7 was similar to the endogenous FGF response as FGF-7 prevented MM14 myoblasts from undergoing terminal differentiation. Thus, both the human and the newt KGFRs transduce signals similar to those transduced via the endogenous mouse FGFR1. Together these data indicate that this newly isolated newt KGFR is a functional receptor as it binds two FGF family members with high affinity and mediates signaling in skeletal muscle myoblasts. Because the binding pattern of the newt KGFR is similar to the pattern observed for its mammalian counterpart, it emphasizes the strict conservation that this ligand/receptor system has undergone through evolution.

Synthesis and release of 9-cis retinoic acid by the urodele wound epidermis

ABSTRACT The wound epidermis is a transient secretory epithelium that apposes the mesenchymal blastema of a regenerating urodele limb, and is required for regeneration. Previous studies have shown that the positional identity of the blastema is respecified by retinoic acid (RA; Maden, M. (1982)Nature 295, 672-675), that the blastema contains RA (Scadding, S. R. and Maden, M. (1994) Dev. Biol. 162, 608617), and that an RA-reporter gene introduced into the blastema is differentially activated along the proximodistal axis (Brockes, J. P. (1992)Proc. Natl. Acad. Sci. USA 89, 11386-11390). The newt limb wound epidermis has been explanted with minimal mesenchymal contamination and cultured under conditions where it retains expression and inducibility of marker antigens. We have assayed for the release of retinoids from the wound epidermis by coculture with cells transfected with an RA-responsive reporter gene. The reporter was activated to a level corresponding to stimulation by 0.1–1 nM RA, and this activation was substantially conferred by medium conditioned by the wound epidermis. No significant activation was observed for cells transfected with mutated reporter plasmids and analysed in parallel co-cultures. Wound epidermis from contralateral proximal and distal blastemas were compared for reporter activation, and gave a P/D activation ratio significantly greater than 1.Wound epidermis explants were cultured in the presence of tritiated retinol, and extracts were analysed by HPLC on three different columns. Radioactivity was detected in peaks corresponding to didehydroretinol, 9-cis RA and other unidentified metabolites. Analysis of conditioned media samples, some after pulse chase experiments, detected significant release of retinol, 9-cis RA and other metabolites. Although all-trans RA was detectable, the predominant acidic metabolite was 9-cis RA. These experiments establish the wound epidermis as a source of RA for local cellular interactions in the blastema.

Monoclonal Antibody MT2 Identifies the Urodele α1 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 α1 chain of the chicken type XII collagen cDNA (α1[XII]), and spans a third of the chicken α1[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 α1[XII]. A Northern blot showed a 10-kb band that corresponds to the size of the chicken α1[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 β-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 α1[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.

Nerve Extracts and Substance P Activate the Phosphatidylinositol Signaling Pathway and Mitogenesis in Newt Forelimb Regenerates

We investigated the inositol phospholipid transmembrane signaling pathway as a possible mediator of neurotrophic (mitogenic) signals in the newt limb regeneration blastema. Blastema mesoderm tissues were prelabeled with myo-[ 3H]inositol, treated with 10 m M LiCl and then exposed to substance P or to extracts of spinal ganglia, brain, or spinal cord. Stimulation with substance P resulted in a rapid dose-dependent reduction of [ 3H]phosphatidylinositol 4,5-bisphosphate and [ 3H]phosphatidylinositol 4-phosphate, correlated with a rapid accumulation of inositol 1,4,5-triphosphate. This effect was inhibited when the blastema tissue was treated with neomycin, a known inhibitor of inositol phospholipid turnover. In addition, substance P stimulated the incorporation of [ 3H]thymidine into DNA of blastema mesoderm cells, and this effect was also suppressed by neomycin, at a dose corresponding to that required to inhibit inositol phosphate accumulation. Extracts of neural tissues, especially spinal ganglia, induced the formation of inositol phosphates and extract activity was attenuated following treatment with heat or trypsin. These findings suggest a role for mitogen-activated inositol phospholipid signaling, initiating events that ultimately lead to cell proliferation.

Mononuclear leukocytes in the newt limb blastema: in vitro behavior.

Since the precise interactions of the various newt limb blastema cell types (pleiomorphic, bipolar, signet and multinucleated cells) with one another are difficult to ascertain in vivo, in this study we describe the in vitro behavior and interactions of these cell types with one another. The data show that the mononuclear signet cells interact (fuse) with other signet cells or fuse directly with the multinucleated cells. Further, they indicate that the signet cells are in fact mononuclear leukocytes and the giant cells are osteoclasts. Therefore, we conclude that these two cell types are not formed by dedifferentiation but are of hematopoietic origin and may play minor roles in blastema production.

Pantropic Retroviral Vector-Mediated Gene Transfer, Integration, and Expression in Cultured Newt Limb Cells

Limb regeneration is a unique developmental phenomenon restricted to certain urodeles in which limb cells dedifferentiate and produce the blastema and then redifferentiate into the tissues that compose the missing part. Genetic modification of the blastema cells would greatly facilitate understanding the programmed gene expression that results in the reconstitution of the limb. To test whether pantropic retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein could mediate gene transfer into blastema cells, we infected a stable newt limb cell line and demonstrated integration and expression of the provirus. Thus, pantropic retroviral vectors offer a new tool for the study of limb regeneration and other developmental phenomena in amphibia.

Reversibility of the mononucleate-to-multinucleate myogenic transition during amphibian limb regeneration.

Muscle differentiation involves the fusion of mononucleate myoblasts to form multinucleate syncytial myotubes. In order to assess reversibility of the mononucleate-to-multinucleate transition in urodele limb regeneration, myotube formation was induced in cultured newt limb blastemal cells. Myotubes were purified, replated at low density and injected with a cytoplasmic lineage tracer. In some cases nuclei of myotubes were labelled by incorporation of tritiated thymidine. Labelled myotubes were stable in culture for 6-8 weeks and no transfer to mononucleate cells was observed. The myotubes were implanted under the wound epidermis of a hindlimb blastema. Labelled mononucleate cells were observed 1 week after implantation and such cells could derive both the cytoplasmic lineage tracer and the nuclear marker. The number of such cells increased by 2-3 weeks after implantation. These results provide strong support for the reversibility of muscle differentiation during urodele limb regeneration, and raise questions about the mechanism of such a reversal.

Isoform-specific induction of a retinoid-responsive antigen after biolistic transfection of chimaeric retinoic acid/thyroid hormone receptors into a regenerating limb.

Retinoic acid (RA) induces secretory differentiation in the wound epidermis of a regenerating amphibian limb. We investigated the role of individual RA receptor (RAR) types in the newt wound epidermis by introducing chimaeric RA/thyroid hormone (T3) receptors (chi alpha 1 and chi delta 1) that can be activated by T3. A biolistic particle delivery system was employed to transfect cells in the wound epidermis of a regenerating limb and approximately 10% of the cells in targeted surface areas expressed marker genes. Both chi alpha 1 and chi delta 1 were comparable in their ability to stimulate transcription of a synthetic reporter construct through a RA response element after activation with T3 in situ. This activation was also comparable to that obtained by the endogenous complement of RARs in the RA-treated, transfected wound epidermis. The RA-inducible WE3 antigen, a marker for secretory differentiation, which distinguishes the wound epidermis from normal skin (Tassava, R. A., Johnson-Wint, B. and Gross, J. 1986, J. Exp. Zool. 239, 229-240), was used to assess the functional role of chi alpha 1 and chi delta 1. Chimaeric receptors were transfected with an alkaline phosphatase marker gene, activated with T3, and the expression of both the marker and WE3 was analyzed by double-label immunofluorescence. Newt limbs transfected with chi delta 1 showed many double-labelled cells dependent on the presence of T3, whereas contralateral limbs transfected with an alkaline phosphatase marker lacking chimaeric receptor sequences did not.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Chimeric retinoic acid/thyroid hormone receptors implicate RAR-alpha 1 as mediating growth inhibition by retinoic acid.

Retinoic acid (RA) affects the growth and differentiation of cells in culture, usually to decrease the growth rate. In amphibian limb regeneration RA has the remarkable ability to affect pattern formation by changing positional identity, but its initial action on the limb is to inhibit division of the blastemal progenitor cells. Newt limb blastemal cells also show this inhibition in culture. In order to investigate the role of different RA receptors (RARs) in the RA response, the hormone binding domain of the newt RARs alpha 1 and delta 1 was replaced with the corresponding region from the Xenopus thyroid hormone receptor-alpha (TR-alpha). In COS cells transfected with each of the chimeras, transcription was activated after exposure to thyroid hormone (T3). Their profile of activity on three different response elements was indicative of RAR specificity and not TR specificity. After transfection of cultured newt blastemal cells with a DNA particle gun, the chimeras were equally active in stimulating T3-dependent transcription of two different synthetic reporter genes. Blastemal cells were transfected with chimeras or control plasmids along with a marker plasmid expressing beta-galactosidase, exposed to RA or T3 and labelled with [3H]thymidine followed by autoradiography. The alpha 1 chimera gave T3-dependent inhibition of growth, comparable to the effect exerted by RA itself, whereas the delta 1 chimera and control plasmids were inactive. The results imply that RAR-alpha 1 mediates the effects of RA on blastemal cell growth.

Reversal of muscle differentiation during urodele limb regeneration.

Recent studies suggest that maintenance of the differentiated state requires continuous regulation. Limb regeneration in urodele amphibians provides a context in which to address this issue, as limb regeneration may involve the dedifferentiation of multinucleate myotubes to yield mononucleate blastemal cells, which then proliferate and contribute to regenerate tissues. To evaluate this possibility, cultured newt limb myotubes were selectively microinjected with the lineage tracer rhodamine-dextran and introduced into regenerating limbs. In culture, such labeled myotubes were stable after 6-8 weeks, and transfer of the tracer to mononucleate cells was not observed. In contrast, after implantation of labeled myotubes under the wound epidermis of limb blastemas, strongly labeled mononucleate cells were observed after 1 week. These cells could be double-labeled with the cytoplasmic lineage tracer and [3H]thymidine that had been incorporated into the nuclei of implanted myotubes. The number of labeled mononucleate cells increased significantly by 2-3 weeks after implantation, indicating that these cells proliferated. Although the fate of these cells at later times was uncertain, we provide evidence consistent with their subsequent differentiation. These results demonstrate reversal in the mononucleate-to-multinucleate transition of vertebrate myogenesis.