Mammalian postmitotic nuclei reenter the cell cycle after serum stimulation in newt/mouse hybrid myotubes.

Cell cycle reentry and dedifferentiation of postmitotic cells are important aspects of the ability of an adult newt and other urodele amphibians to regenerate various tissues and appendages [1]. In contrast to their mammalian counterparts, newt A1 myotubes are able to reenter S phase after serum stimulation of a pathway leading to phosphorylation of the retinoblastoma protein, pRb [2]. The activity in serum is not due to mitogenic growth factors but is generated indirectly by the activation of thrombin and subsequent proteolysis [3]. In this paper we describe the formation of interspecies hybrid (heterokaryon) myotubes by the fusion of mouse C2C12 [4] and newt A1 [5, 6] myogenic cells. The C2C12 nuclei reenter the cell cycle upon serum stimulation of the hybrids, while C2C12 homokaryon myotubes remain arrested under these conditions. These findings indicate that the postmitotic arrest of the mouse nuclei is undermined by the pathway activated in the newt cytoplasm. The hybrid myotubes provide a new model for the manipulation of the postmitotic arrest in both mammalian and newt differentiated cells.

Regeneration as an evolutionary variable.

Regeneration poses a distinctive set of problems for evolutionary biologists, but there has been little substantive progress since these issues were clearly outlined in the monograph of T. H. Morgan (1901). The champions at regeneration among vertebrates are the urodele amphibians such as the newt, and we tend to regard urodele regeneration as an exceptional attribute. The ability to regenerate large sections of the body plan is widespread in metazoan phylogeny, although it is not universal. It is striking that in phylogenetic contexts where regeneration occurs, closely related species are observed which do not possess this ability. It is a challenge to reconcile such variation between species with a conventional selective interpretation of regeneration. The critical hypothesis from phylogenetic analysis is that regeneration is a basic, primordial attribute of metazoans rather than a mechanism which has evolved independently in a variety of contexts. In order to explain its absence in closely related species, it is postulated to be lost secondarily for reasons which are not understood. Our approach to this question is to compare a differentiated newt cell with its mammalian counterpart in respect of the plasticity of differentiation.

The expression pattern of tomoregulin-1 in urodele limb regeneration and mouse limb development.

Tomoregulin-1 (TMEFF1) was first identified as a gene implicated in pituitary secretion in Xenopus laevis. The predicted structure of TMEFF1 is that of a transmembrane protein with a highly conserved cytoplasmic tail, two follistatin domains and one modified EGF domain in its extracellular region. We report the cloning of the newt orthologue, and show that the expression of TMEFF1 is upregulated in the blastema during limb regeneration, and is also expressed in mouse embryonic limb development.

Plasticity of retrovirus-labelled myotubes in the newt limb regeneration blastema.

Two important indices of myogenic differentiation are the formation of syncytial myotubes and the postmitotic arrest from the cell cycle, both of which occur after fusion of mononucleate cells. We show here that these indices are reversed in the environment of the urodele limb regeneration blastema. In order to introduce an integrated (genetic) marker into newt myotubes, we infected mononucleate cells in culture with a pseudotyped retrovirus expressing human placental alkaline phosphatase (AP). After fusion the myotubes expressed AP and could be purified by sieving and micromanipulation so as to remove all mononucleate cells. When such purified retrovirus-labelled myotubes were implanted into a limb blastema they gave rise to mononucleate progeny with high efficiency. Purified myotubes labelled with fluorescent lipophilic cell tracker dye also gave rise to mononucleate cells; myotubes which were double labelled with the tracker dye and a nuclear stain gave rise to double-labelled mononucleate progeny. Nuclei within retrovirus-labelled myotubes entered S phase as evidenced by widespread labelling after injection of implanted newts with BrdU. The relation between the two aspects of plasticity is a critical further question.

Generation of mononucleate cells from post-mitotic myotubes proceeds in the absence of cell cycle progression.

The remarkable regenerative ability of adult urodele amphibians depends in part of the plasticity of differentiated cells at the site of injury. Limb regeneration proceeds by formation of a mesenchymal growth zone or blastema under the wound epidermis at the end of the stump. Previous work has shown that when cultured post-mitotic newt myotubes are introduced into the blastema, they re-enter the cell cycle and undergo conversion to mononucleate cells which divide and contribute to the regenerate [11, 13]. In order to investigate the interdependence of these two aspects of plasticity, we have blocked cell cycle progression of the myotubes either by X-irradiation or by transfection of the CDK4/6 inhibitor p16. In each case, the efficacy of the block was evaluated in culture after activation of S phase re-entry by serum stimulation. The experimental myotubes were implanted into limb blastemas along with a differentially labelled control population of myotubes containing an equivalent number of nuclei. X-irradiated myotubes gave rise to mononucleate cells in the limb blastema, and the progeny were blocked in respect of S phase entry. Comparable results were obtained with the p16-expressing myotubes. We conclude that progression through S or M phase is not required for generation of mononucleate cells and suggest that such cells may arise by budding from the muscle syncytium.

Topics in prion cell biology.

Recent studies of a transmembrane form of the prion protein (PrP) have indicated its importance for neuropathogenesis in certain contexts, and have analysed the transacting factors at the endoplasmic reticulum and the mutations within PrP that regulate its appearance. A significant focus for our understanding of the normal role of PrP has emerged from its interaction with copper ions. Studies on two yeast prions have analysed the structure and phenotype of the aggregated conformers underlying the prion state, as well as the interactions regulating their formation and turnover within a dividing cell.

Thrombin regulates S-phase re-entry by cultured newt myotubes.

BACKGROUND: Adult urodele amphibians such as the newt have remarkable regenerative ability, and a critical aspect of this is the ability of differentiated cells to re-enter the cell cycle and lose their differentiated characteristics. Unlike mammalian myotubes, cultured newt myotubes are able to enter and traverse S phase, following serum stimulation, by a pathway leading to phosphorylation of the retinoblastoma protein. The extracellular regulation of this pathway is unknown. RESULTS: Like their mammalian counterparts, newt myotubes were refractory to mitogenic growth factors such as the platelet-derived growth factor (PDGF), which act on their mononucleate precursor cells. Cultured newt myotubes were activated to enter S phase by purified thrombin in the presence of subthreshold amounts of serum. The activation proceeded by an indirect mechanism in which thrombin cleaved components in serum to generate a ligand that acted directly on the myotubes. The ligand was identified as a second activity present in preparations of crude thrombin and that was active after removal of all thrombin activity. It induced newt myotubes to enter S phase in serum-free medium, and it acted on myotubes but not on the mononucleate precursor cells. Cultured mouse myotubes were refractory to this indirect mechanism of S-phase re-entry. CONCLUSIONS: These results provide a link between reversal of differentiation and the acute events of wound healing. The urodele myotube responds to a ligand generated downstream of thrombin activation and re-enters the cell cycle. Although this ligand can be generated in mammalian sera, the mammalian myotube is unresponsive. These results provide a model at the cellular level for the difference in regenerative ability between urodeles and mammals.

A target of thrombin activation promotes cell cycle re-entry by urodele muscle cells.

A key early event of newt limb regeneration is the local dedifferentiation of cells to form dividing progenitor cells. This involves the plasticity of differentiation and the ability to re-enter the cell cycle. In culture, differentiated newt myotubes are able to re-enter S-phase in response to serum stimulation. Here, we analyzed the intracellular and extracellular requirements for this process. Cell cycle re-entry depends on the phosphorylation of the retinoblastoma protein, which is a key regulator of the G1-S transition. This is in contrast to mammalian myotubes, which are refractory to serum stimulation and cannot phosphorylate retinoblastoma protein in response to serum. The serum factor responsible for this phosphorylation appears to be distinct from common polypeptide growth factors and is enriched in crude preparations of bovine thrombin. Fractionation and analysis of this preparation indicate that the factor is regulated by thrombin and plasmin proteolysis. These results indicate that factors involved in acute responses to wounding such as clotting may be important initiators of the regenerative response.

Identification of newt connective tissue growth factor as a target of retinoid regulation in limb blastemal cells.

In order to analyse target genes regulated by retinoic acid in urodele limb regeneration, we have used pseudotyped retroviruses to obtain stably transfected newt limb blastemal (progenitor) cells in culture which express chimeric retinoic acid/thyroid hormone receptors delta1 or delta2. After treatment with thyroid hormone to activate the chimeric receptors, we used a polymerase chain reaction (PCR)-based subtraction method to identify target genes which are retinoid regulated. Newt connective tissue growth factor, a secreted protein recognised in several vertebrates, has been identified in this way and found to be expressed in the limb blastema and regulated by retinoic acid. This approach should permit a systematic analysis of retinoid target genes in limb regeneration.

Hedgehog family member is expressed throughout regenerating and developing limbs.

Members of the hedgehog family have been shown to play a key role in many developmental processes, including limb patterning and chondrogenesis. We have therefore investigated whether members of this family are also expressed during regeneration of the adult urodele limb and are regulated by retinoic acid (RA), since this derivative induces proximodistal duplications in regenerating limbs, and has been shown to regulate sonic hedgehog (shh) in the developing limbs of birds and mammals. We report here that a newt homologue of Xenopus banded hedgehog, called N-bhh, is uniformly expressed by mesenchymal blastemal cells from the initial stages of regeneration and is up-regulated by RA. In addition, we show that N-bhh is uniformly expressed in the early limb bud of the newt embryo. Since bhh has not been detected in developing limbs of higher vertebrates, its expression in developing and regenerating newt limbs may be related to the regenerative capability of urodeles.

Immortalization of rat embryo fibroblasts by a 3'-untranslated region.

We have exploited a cross-species expression screen to search for cellular immortalizing activities. A newt blastemal cDNA expression library was transfected into rat embryo fibroblasts and immortal cell lines were selected. This identified a 1-kb cDNA fragment which has a low representation in the cDNA library and is derived from the 3'-UTR of an alpha-glucosidase-related mRNA. Expression of this sequence in rat embryo fibroblasts has shown that it is active in promoting colony formation and immortalization. It is also able to cooperate with an immortalization-defective deletion mutant of SV40 T antigen, indicating that it can exert its growth-stimulatory activity in the pathway activated by a viral immortalizing oncogene. This is the first example of an immortalizing activity mediated by an RNA sequence, and further analysis of its mechanism should provide new insights into senescence and immortalization.

Amphibian limb regeneration: rebuilding a complex structure.

The ability to regenerate complex structures is widespread in metazoan phylogeny, but among vertebrates the urodele amphibians are exceptional. Adult urodeles can regenerate their limbs by local formation of a mesenchymal growth zone or blastema. The generation of blastemal cells depends not only on the local extracellular environment after amputation or wounding but also on the ability to reenter the cell cycle from the differentiated state. The blastema replaces structures appropriate to its proximodistal position. Axial identity is probably encoded as a graded property that controls cellular growth and movement through local cell interactions. The molecular basis is not understood, but proximodistal identity in newt blastemal cells may be respecified by signaling through a retinoic acid receptor isoform. The possibility of inducing a blastema on a mammalian limb cannot be discounted, although the molecular constraints are becoming clearer as we understand more about the mechanisms of urodele regeneration.

Newt myotubes reenter the cell cycle by phosphorylation of the retinoblastoma protein.

Withdrawal from the cell cycle is an essential aspect of vertebrate muscle differentiation and requires the retinoblastoma (Rb) protein that inhibits expression of genes needed for cell cycle entry. It was shown recently that cultured myotubes derived from the Rb-/- mouse reenter the cell cycle after serum stimulation (Schneider, J.W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. Science (Wash. DC). 264:1467-1471). In contrast with other vertebrates, adult urodele amphibians such as the newt can regenerate their limbs, a process involving cell cycle reentry and local reversal of differentiation. Here we show that myotubes formed in culture from newt limb cells are refractory to several growth factors, but they undergo S phase after serum stimulation and accumulate 4N nuclei. This response to serum is inhibited by contact with mononucleate cells. Despite the phenotypic parallel with Rb-/- mouse myotubes, Rb is expressed in the newt myotubes, and its phosphorylation via cyclin-dependent kinase 4/6 is required for cell cycle reentry. Thus, the postmitotic arrest of urodele myotubes, although intact in certain respects, can be undermined by a pathway that is inactive in other vertebrates. This may be important for the regenerative ability of these animals.

Is retinoic acid an endogenous ligand during urodele limb regeneration?

The effects of retinoids on a regenerating urodele limb make them interesting candidates for endogenous ligands during regeneration. We review the evidence for considering this possibility. This includes analysis of retinoids and retinoic acid receptors in the regenerate, and studies on activation of retinoid reporter genes. Recent work has provided evidence that the wound epidermis is a source of 9-cis retinoic acid, and may be a favorable model for studying the synthesis and release of this modulator.

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.

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, delta 2, specifically mediates proximalization. CONCLUSIONS: These results demonstrate that the ability of RA to respecify proximodistal identity is mediated by a specific RAR isoform, delta 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.

Retinoid signalling and retinoid receptors in amphibian limb regeneration.

Retinoic acid (RA) is able to respecify the positional identity of a regenerating urodele amphibian limb. The regenerate arises from a mesenchymal growth zone or blastema, composed of mesenchymal progenitor cells. Is RA an endogenous ligand and does it act to respecify axial identity? Two recent lines of evidence in favour of an endogenous role are the activation of an RA reporter gene after transfection into the blastema, and the demonstration that the wound epidermis at the end of the regenerate is a source of RA. To analyse the mechanism of action at the receptor level, we have constructed chimaeric newt RA receptor/Xenopus thyroid hormone receptor molecules that allow a single RA receptor to be activated by thyroid hormone.

Expression and activity of the newt Msx-1 gene in relation to limb regeneration.

The Msx-1 homeobox gene is expressed in various contexts during vertebrate development, including the progress zone of the avian and mouse limb bud. Expression of mouse Msx-1 in a cultured myogenic cell line conferred a transformed phenotype and inhibited fusion into myotubes. It has been proposed that Msx-1 expression is required to maintain certain cells in a proliferating and undifferentiated state and may be associated with the ability to regenerate limbs. Urodele amphibians such as the newt regenerate their limbs by formation of a growth zone or blastema, and we have isolated and sequenced newt Msx-1 (NvMsx-1) from a limb blastemal cDNA library. NvMsx-1 expression was detectable in RNA preparations from both limb and tail and their regeneration blastemas, although cultured cells established from limb blastemal mesenchyme gave negative results. When either COS cells or cultured newt blastemal cells were cotransfected with an expression vector for NvMsx-1 and reporter plasmids containing multiple homeobox protein binding sites, NvMsx-1 repressed reporter expression. If NvMsx-1 was expressed together with a marker enzyme in cultured newt blastemal cells, no significant difference in DNA synthesis was observed relative to control transfectants. When myogenic mononucleate cells were transfected with NvMsx-1 and subsequently exposed to low serum to promote fusion, the fraction of Msx-1 positive cells in myotubes was comparable to a control transfected population analysed in the same culture. These results indicate that although Msx-1 expression could be important for limb regeneration, it does not exert a cell-autonomous effect on proliferation or myogenic differentiation of cultured blastemal cells.