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.     

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.     

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.     

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.     

Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b . We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl ( Ambystoma mexicanum ), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.     

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 δ1 or δ2. 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.     

Expression of complement 3 and complement 5 in newt limb and lens regeneration

Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.     

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.     

New regulators of vertebrate appendage regeneration

Appendage regeneration is a complex and fascinating biological process exhibited in vertebrates by urodele amphibians and teleost fish. A current focus in the field is to identify new molecules that control formation and function of the regeneration blastema, a mass of proliferative mesenchyme that emerges after limb or fin amputation and serves as progenitor tissue for lost structures. Two studies published recently have illuminated new molecular regulators of blastemal proliferation. After amputation of a newt limb, the nerve sheath releases nAG, a blastemal mitogen that facilitates regeneration. In amputated zebrafish fins, regeneration is optimized through depletion of the microRNA miR-133, a mechanism that requires Fgf signaling. These discoveries establish research avenues that may impact the regenerative capacity of mammalian tissues.     

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.     

Mechanism of Action of Secreted Newt Anterior Gradient Protein

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.     

An extracellular matrix molecule of newt and axolotl regenerating limb blastemas and embryonic limb buds: immunological relationship of MT1 antigen with tenascin.

Several well-characterized extracellular matrix (ECM) components have been localized to the amphibian limb regenerate, but the identification and characterization of novel ECM molecules have received little attention. Here we describe, using mAb MT1 and immunocytochemistry, an ECM molecule expressed during limb regeneration and limb development. In limb stumps, mAb MT1 reactivity was restricted to tendons, myotendinous junctions, granules in the basal layers of epidermis, periosteum (newts) and perichondrium (axolotls). In regenerating limbs, reactivity in the distal limb stump was first detected 5 days and 1 day after amputation of newt and axolotl limbs, respectively. In both species, mAb MT1 recognized what appeared to be an abundant blastema matrix antigen, localized in both thin and thick cords between and sometimes closely associated with blastema cells. Reactivity was generally uniform throughout the blastema except for a particularly thick layer that was present immediately beneath the wound epithelium. During redifferentiation stages, mAb MT1 reactivity persisted among blastema cells and redifferentiating cartilage but was lost proximally in areas of muscle and connective tissue differentiation. During the entire period of embryonic limb development, mAb MT1 reactivity was seen in the ECM of the mesenchyme and in a layer beneath the limb bud ectoderm, similar to its distribution during regeneration. Considerable mAb MT1 reactivity was also associated with the developing somites. The reactivity of mAb MT1 in blastema and limb bud was similar if not identical to that of a polyclonal Ab against tenascin (pAbTN), a large, extracellular matrix glycoprotein implicated in growth control, inductive interactions, and other developmental events. This pAbTN effectively competed against mAb MT1 binding on blastema sections. In immunoblots, both mAb MT1 and pAbTN recognized a very high molecular weight (approximately Mr 1000 x 10(3)) protein in blastema extracts of both newts and axolotls. mAb MT1 immunoprecipitated a protein of Mr 1000K size which reacted to both mAb MT1 and pAbTN in immunoblots. These data show that tenascin is in the matrix of the urodele blastema and limb bud, and suggest that mAb MT1 identifies urodele tenascin.     

A critical role for thrombin in vertebrate lens regeneration

Lens regeneration in urodele amphibians such as the newt proceeds from the dorsal margin of the iris where pigment epithelial cells (PEC) re-enter the cell cycle and transdifferentiate into lens. A general problem in regeneration research is to understand how the events of tissue injury or removal are coupled to the activation of plasticity in residual differentiated cells or stem cells. Thrombin, a pivotal regulator of the injury response, has been implicated as a regulator of cell cycle re-entry in newt myotubes, and also in newt iris PEC. After removal of the lens, thrombin was activated on the dorsal margin for 5-7 days. Inactivation of thrombin by either of two different inhibitors essentially blocked S-phase re-entry by PEC at this location. The axolotl, a related species which can regenerate its limb but not its lens, can activate thrombin after amputation but not after lens removal. These data support the hypothesis that thrombin is a critical signal linking injury to regeneration, and offer a new perspective on the evolutionary and phylogenetic questions about regeneration.     

Expression and biological effect of urodele fibroblast growth factor 1: relationship to limb regeneration

Fibroblast growth factors (FGFs) have been previously implicated in urodele limb regeneration. Here, we examined expression of FGF-1 by blastema cells and neurons and investigated its involvement in wound epithelial formation and function and in the trophic effect of nerves. Neurons innervating the limb and blastema cells in vivo and in vitro expressed the FGF-1 gene. The peptide was present in blastemas in vivo. Wound epithelium thickened when recombinant newt FGF-1 was provided on heparin-coated beads, demonstrating that the FGF-1 was biologically active and that the wound epithelium is a possible target tissue of FGF. FGF-1 did not stimulate accessory limb formation. FGF-1 was as effective as 10% fetal bovine serum in maintaining proliferative activity of blastema cells in vitro but was unable to maintain growth of denervated, nerve-dependent stage blastemas when provided on beads or by injection. FGF-1 had a strong stimulating effect on blastema cell accumulation and proliferation of limbs inserted into the body cavity that were devoid of an apical epithelial cap (AEC). These results show that FGF-1 can signal wound epithelium cap formation and/or function and can stimulate mesenchyme accumulation/proliferation in the absence of the AEC but that FGF-1 is not directly involved in the neural effect on blastema growth.     

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. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 150–158, 2000     

The Regenerative Plasticity of Isolated Urodele Myofibers and Its Dependence on Msx1

The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.