Regulation of p53 is critical for vertebrate limb regeneration.

Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during limb regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration.

Mechanisms underlying vertebrate limb regeneration: lessons from the salamander.

Limb regeneration in adult salamanders proceeds by formation of a mound of progenitor cells called the limb blastema. It provides several pointers for regenerative medicine. These include the role of differentiated cells in the origin of the blastema, the role of regenerating axons of peripheral nerves and the importance of cell specification in conferring morphogenetic autonomy on the blastema. One aspect of regeneration that has received less attention is the ability to undergo multiple episodes without detectable change in the outcome, and with minimal effect of aging. We suggest that, although such pointers are valuable, it is important to understand why salamanders are the only adult tetrapod vertebrates able to regenerate their limbs. Although this remains a controversial issue, the existence of salamander-specific genes that play a significant role in the mechanism of regeneration provides evidence for the importance of local evolution, rather than a purely ancestral mechanism. The three-finger protein called Prod1 is discussed in the present article as an exemplar of this approach.

The aneurogenic limb identifies developmental cell interactions underlying vertebrate limb regeneration.

The removal of the neural tube in salamander embryos allows the development of nerve-free aneurogenic limbs. Limb regeneration is normally nerve-dependent, but the aneurogenic limb regenerates without nerves and becomes nerve-dependent after innervation. The molecular basis for these tissue interactions is unclear. Anterior Gradient (AG) protein, previously shown to rescue regeneration of denervated limbs and to act as a growth factor for cultured limb blastemal cells, is expressed throughout the larval limb epidermis and is down-regulated by innervation. In an aneurogenic limb, the level of AG protein remains high in the epidermis throughout development and regeneration, but decreases after innervation following transplantation to a normal host. Aneurogenic epidermis also shows a fivefold difference in secretory gland cells, which express AG protein. The persistently high expression of AG in the epithelial cells of an aneurogenic limb ensures that regeneration is independent of the nerve. These findings provide an explanation for this classical problem, and identify regulation of the epidermal niche by innervation as a distinctive developmental mechanism that initiates the nerve dependence of limb regeneration. The absence of this regulation during anuran limb development might suggest that it evolved in relation to limb regeneration.

Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration.

The GPI-anchor is an established determinant of molecular localisation and various functional roles have been attributed to it. The newt GPI-anchored three-finger protein (TFP) Prod1 is an important regulator of cell behaviour during limb regeneration, but it is unclear how it signals to the interior of the cell. Prod1 was expressed by transfection in cultured newt limb cells and activated transcription and expression of matrix metalloproteinase 9 (MMP9) by a pathway involving ligand-independent activation of epidermal growth factor receptor (EGFR) signalling and phosphorylation of extracellular regulated kinase 1 and 2 (ERK1/2). This was dependent on the presence of the GPI-anchor and critical residues in the α-helical region of the protein. Interestingly, Prod1 in the axolotl, a salamander species that also regenerates its limbs, was shown to activate ERK1/2 signalling and MMP9 transcription despite being anchorless, and both newt and axolotl Prod1 co-immunoprecipitated with the newt EGFR after transfection. The substitution of the axolotl helical region activated a secreted, anchorless version of the newt molecule. The activity of the newt molecule cannot therefore depend on a unique property conferred by the anchor. Prod1 is a salamander-specific TFP and its interaction with the phylogenetically conserved EGFR has implications for our view of regeneration as an evolutionary variable.

Tig1 regulates proximo-distal identity during salamander limb regeneration.

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

The newt ortholog of CD59 is implicated in proximodistal identity during amphibian limb regeneration.

The proximodistal identity of a newt limb regeneration blastema is respecified by exposure to retinoic acid, but its molecular basis is unclear. We identified from a differential screen the cDNA for Prod 1, a gene whose expression in normal and regenerating limbs is regulated by proximodistal location and retinoic acid: Prod 1 is the newt ortholog of CD59. Prod 1/CD59 was found to be located at the cell surface with a GPI anchor which is cleaved by PIPLC. A proximal newt limb blastema engulfs a distal blastema after juxtaposition in culture, and engulfment is specifically blocked by PIPLC, and by affinity-purified antibodies to two distinct Prod 1/CD59 peptides. Prod 1 is therefore a cell surface protein implicated in the local cell-cell interactions mediating positional identity.

Positional identity of adult stem cells in salamander limb regeneration.

Limb regeneration in larval and adult salamanders proceeds from a mound of mesenchymal stem cells called the limb blastema. The blastema gives rise just to those structures distal to its level of origin, and this property of positional identity is reset to more proximal values by treatment with retinoic acid. We have identified a cell surface protein, called Prod1/CD59, which appears to be a determinant of proximodistal identity. Prod1 is expressed in an exponential gradient in an adult limb as determined by detection of both mRNA and immunoreactive protein. Prod1 protein is up-regulated after treatment of distal blastemas with RA and this is particularly marked in cells of the dermis. These cells have previously been implicated in pattern formation during limb regeneration.

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.

Distinctive expression of Myf5 in relation to differentiation and plasticity of newt muscle cells.

Regeneration in urodele amphibians such as the newt reflects the local plasticity of differentiated cells. Newt myotubes and myofibres undergo S phase re-entry and cellularisation in the limb blastema, and we have analysed the regulation of Myf5 in relation to these events. Surprisingly, Myf5 was expressed after fusion in cultured newt myotubes and in myofibers of the adult limb, in contrast to its familiar expression in myoblasts in other vertebrates. Its expression was markedly down regulated in cultured newt myotubes after S phase re-entry induced by serum stimulation, as well as by exposure to the trisubstituted purine called myoseverin which induces cellularisation. We have attempted to relate this striking difference from other vertebrates to the requirement for multinucleate urodele muscle cells to contribute to the regeneration blastema.

An orphan gene is necessary for preaxial digit formation during salamander limb development.

Limb development in salamanders differs from other tetrapods in that the first digits to form are the two most anterior (preaxial dominance). This has been proposed as a salamander novelty and its mechanistic basis is unknown. Salamanders are the only adult tetrapods able to regenerate the limb, and the contribution of preaxial dominance to limb regeneration is unclear. Here we show that during early outgrowth of the limb bud, a small cohort of cells express the orphan gene Prod1 together with Bmp2, a critical player in digit condensation in amniotes. Disruption of Prod1 with a gene-editing nuclease abrogates these cells, and blocks formation of the radius and ulna, and outgrowth of the anterior digits. Preaxial dominance is a notable feature of limb regeneration in the larval newt, but this changes abruptly after metamorphosis so that the formation of anterior and posterior digits occurs together within the autopodium resembling an amniote-like pattern.

Identification of the orphan gene Prod 1 in basal and other salamander families.

BACKGROUND: The urodele amphibians (salamanders) are the only adult tetrapods able to regenerate the limb. It is unclear if this is an ancestral property that is retained in salamanders but lost in other tetrapods or if it evolved in salamanders. The three-finger protein Prod 1 is implicated in the mechanism of newt limb regeneration, and no orthologs have been found in other vertebrates, thus providing evidence for the second viewpoint. It has also been suggested that this protein could play a role in salamander-specific aspects of limb development. There are ten families of extant salamanders, and Prod 1 has only been identified in two of them to date. It is important to determine if it is present in other families and, particularly, the basal group of two families which diverged approximately 200 MYA. FINDINGS: We have used polymerase chain reaction (PCR) to identify Prod 1 in a Chinese hynobiid species Batrachuperus longdongensis. We obtained an intestinal transcriptome of the plethodontid Aneides lugubris and, from this, identified a primer which allowed PCR of two Prod 1 genes from this species. All known Prod 1 sequences from nine species in four families have been aligned, and a phylogenetic tree has been derived. CONCLUSIONS: Prod 1 is found in basal salamanders of the family Hynobiidae, and in at least three other families, so it may be present in all extant salamanders. It remains a plausible candidate to have been involved in the origins of limb regeneration, as well as the apomorphic aspects of limb development.

Sustained ERK activation underlies reprogramming in regeneration-competent salamander cells and distinguishes them from their mammalian counterparts.

In regeneration-competent vertebrates, such as salamanders, regeneration depends on the ability of various differentiated adult cell types to undergo natural reprogramming. This ability is rarely observed in regeneration-incompetent species such as mammals, providing an explanation for their poor regenerative potential. To date, little is known about the molecular mechanisms mediating natural reprogramming during regeneration. Here, we have identified the extent of extracellular signal-regulated kinase (ERK) activation as a key component of such mechanisms. We show that sustained ERK activation following serum induction is required for re-entry into the cell cycle of postmitotic salamander muscle cells, partially by promoting the downregulation of p53 activity. Moreover, ERK activation induces epigenetic modifications and downregulation of muscle-specific genes such as Sox6. Remarkably, while long-term ERK activation is found in salamander myotubes, only transient activation is seen in their mammalian counterparts, suggesting that the extent of ERK activation could underlie differences in regenerative competence between species.

The Meis homeoprotein regulates the axolotl Prod 1 promoter during limb regeneration.

During limb regeneration in salamanders the blastemal cells give rise only to structures distal to the level of amputation. This proximodistal identity can be regulated by ectopic expression of Meis homeoproteins or the three finger protein Prod 1 which acts at the cell surface. It has been suggested that Meis acts by regulating the transcription of Prod 1. We have sequenced the axolotl Prod 1 promoter and selected two candidate sites for binding Meis homeoproteins. The sites were mutated in various combinations in promoters expressing a luciferase reporter gene. The promoter activity was assayed by nucleofecting AL1 cells, a cultured axolotl limb cell line that expresses both Prod 1 and Meis 1 and 2. The activity of the promoter was inhibited by 60% after mutation at Meis site 1, but not at Meis site 2. The promoter constructs were electroporated into axolotl limb blastemas and the wild type promoter was more active in a proximal blastema than in a contralateral distal blastema. The wild type promoter was significantly more active than a (site 1+site 2) mutant promoter in contralateral proximal blastemas, but the promoters were equivalent in contralateral distal blastemas. The separate site 1 or site 2 mutants were not significantly different from wild type in contralateral proximal blastemas, thus contrasting with the site 1 results in AL1 cells. These data provide strong support for the hypotheses that the Prod 1 promoter is regulated on the proximodistal axis, and that Meis homeoproteins directly regulate the promoter on this axis during limb regeneration in addition to cultured cells.

Solution structure and phylogenetics of Prod1, a member of the three-finger protein superfamily implicated in salamander limb regeneration.

BACKGROUND: Following the amputation of a limb, newts and salamanders have the capability to regenerate the lost tissues via a complex process that takes place at the site of injury. Initially these cells undergo dedifferentiation to a state competent to regenerate the missing limb structures. Crucially, dedifferentiated cells have memory of their level of origin along the proximodistal (PD) axis of the limb, a property known as positional identity. Notophthalmus viridescens Prod1 is a cell-surface molecule of the three-finger protein (TFP) superfamily involved in the specification of newt limb PD identity. The TFP superfamily is a highly diverse group of metazoan proteins that includes snake venom toxins, mammalian transmembrane receptors and miscellaneous signaling molecules. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of identifying potential orthologs of Prod1, we have solved its 3D structure and compared it to other known TFPs using phylogenetic techniques. The analysis shows that TFP 3D structures group in different categories according to function. Prod1 clusters with other cell surface protein TFP domains including the complement regulator CD59 and the C-terminal domain of urokinase-type plasminogen activator. To infer orthology, a structure-based multiple sequence alignment of representative TFP family members was built and analyzed by phylogenetic methods. Prod1 has been proposed to be the salamander CD59 but our analysis fails to support this association. Prod1 is not a good match for any of the TFP families present in mammals and this result was further supported by the identification of the putative orthologs of both CD59 and N. viridescens Prod1 in sequence data for the salamander Ambystoma tigrinum. CONCLUSIONS/SIGNIFICANCE: The available data suggest that Prod1, and thereby its role in encoding PD identity, is restricted to salamanders. The lack of comparable limb-regenerative capability in other adult vertebrates could be correlated with the absence of the Prod1 gene.

Molecular basis for the nerve dependence of limb regeneration in an adult vertebrate.

The limb blastemal cells of an adult salamander regenerate the structures distal to the level of amputation, and the surface protein Prod 1 is a critical determinant of their proximodistal identity. The anterior gradient protein family member nAG is a secreted ligand for Prod 1 and a growth factor for cultured newt blastemal cells. nAG is sequentially expressed after amputation in the regenerating nerve and the wound epidermis-the key tissues of the stem cell niche-and its expression in both locations is abrogated by denervation. The local expression of nAG after electroporation is sufficient to rescue a denervated blastema and regenerate the distal structures. Our analysis brings together the positional identity of the blastema and the classical nerve dependence of limb regeneration.