Conserved and novel functions of programmed cellular senescence during vertebrate development.

Cellular senescence, a form of stable cell cycle arrest that is traditionally associated with tumour suppression, has been recently found to occur during mammalian development. Here, we show that cell senescence is an intrinsic part of the developmental programme in amphibians. Programmed senescence occurs in specific structures during defined time windows during amphibian development. It contributes to the physiological degeneration of the amphibian pronephros and to the development of the cement gland and oral cavity. In both contexts, senescence depends on TGFß but is independent of ERK/MAPK activation. Furthermore, elimination of senescent cells through temporary TGFß inhibition leads to developmental defects. Our findings uncover conserved and new roles of senescence in vertebrate organogenesis and support the view that cellular senescence may have arisen in evolution as a developmental mechanism.

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.

Recurrent turnover of senescent cells during regeneration of a complex structure.

Cellular senescence has been recently linked to the promotion of age-related pathologies, including a decline in regenerative capacity. While such capacity deteriorates with age in mammals, it remains intact in species such as salamanders, which have an extensive repertoire of regeneration and can undergo multiple episodes through their lifespan. Here we show that, surprisingly, there is a significant induction of cellular senescence during salamander limb regeneration, but that rapid and effective mechanisms of senescent cell clearance operate in normal and regenerating tissues. Furthermore, the number of senescent cells does not increase upon repetitive amputation or ageing, in contrast to mammals. Finally, we identify the macrophage as a critical player in this efficient senescent cell clearance mechanism. We propose that effective immunosurveillance of senescent cells in salamanders supports their ability to undergo regeneration throughout their lifespan.

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.

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.

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.

A comparative study of gland cells implicated in the nerve dependence of salamander limb regeneration.

Limb regeneration in salamanders proceeds by formation of the blastema, a mound of proliferating mesenchymal cells surrounded by a wound epithelium. Regeneration by the blastema depends on the presence of regenerating nerves and in earlier work it was shown that axons upregulate the expression of newt anterior gradient (nAG) protein first in Schwann cells of the nerve sheath and second in dermal glands underlying the wound epidermis. The expression of nAG protein after plasmid electroporation was shown to rescue a denervated newt blastema and allow regeneration to the digit stage. We have examined the dermal glands by scanning and transmission electron microscopy combined with immunogold labelling of the nAG protein. It is expressed in secretory granules of ductless glands, which apparently discharge by a holocrine mechanism. No external ducts were observed in the wound epithelium of the newt and axolotl. The larval skin of the axolotl has dermal glands but these are absent under the wound epithelium. The nerve sheath was stained post-amputation in innervated but not denervated blastemas with an antibody to axolotl anterior gradient protein. This antibody reacted with axolotl Leydig cells in the wound epithelium and normal epidermis. Staining was markedly decreased in the wound epithelium after denervation but not in the epidermis. Therefore, in both newt and axolotl the regenerating axons induce nAG protein in the nerve sheath and subsequently the protein is expressed by gland cells, under (newt) or within (axolotl) the wound epithelium, which discharge by a holocrine mechanism. These findings serve to unify the nerve dependence of limb regeneration.

A single-cell analysis of myogenic dedifferentiation induced by small molecules.

An important direction in chemical biology is the derivation of compounds that affect cellular differentiation or its reversal. The fragmentation of multinucleate myofibers into viable mononucleates (called cellularization) occurs during limb regeneration in urodele amphibians, and the isolation of myoseverin, a trisubstituted purine that could apparently activate this pathway of myogenic dedifferentiation in mammalian cells, generated considerable interest. We have explored the mechanism and outcome of cellularization at a single-cell level, and we report findings that significantly extend the previous work with myoseverin. Using a panel of compounds, including a triazine compound with structural similarity and comparable activity to myoseverin, we have identified microtubule disruption as critical for activation of the response. Time-lapse microscopy has enabled us to analyze the fate of identified mononucleate progeny, and directly assess the extent of dedifferentiation.

Motif-grafted antibodies containing the replicative interface of cellular PrP are specific for PrPSc.

Prion diseases are closely associated with the conversion of the cellular prion protein (PrPC) to an abnormal conformer (PrPSc) [Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. USA 95, 13363-13383]. Monoclonal antibodies that bind epitopes comprising residues 96-104 and 133-158 of PrPC potently inhibit this process, presumably by preventing heterodimeric association of PrPC and PrPSc, and suggest that these regions of PrPC may be critical components of the PrPC-PrPSc replicative interface. We reasoned that transplanting PrP sequence corresponding to these regions into a suitable carrier molecule, such as an antibody, could impart specific recognition of disease-associated forms of PrP. To test this hypothesis, polypeptides containing PrP sequence between residues 89-112 or 136-158 were used to replace the extended heavy chain complementarity-determining region 3 of an IgG antibody specific for the envelope glycoprotein of HIV-1. Herein the resulting engineered PrP-IgGs are shown to bind specifically to infective fractions of PrP in mouse, human, and hamster prion-infected tissues, but not to PrPC, other cellular components, or the HIV-1 envelope. PrPSc reactivity was abolished when the sequence of the PrP 89-112 and 136-158 grafts was mutated, scrambled, or N-terminally truncated. Our findings suggest that residues within the 89-112 and 136-158 segments of PrPC are key components of one face of the PrPC-PrPSc complex. PrPSc-specific antibodies produced by the approach described may find widespread application in the study of prion biology and replication and in the detection of infectious prions in human and animal materials.

Heterogeneous proliferative potential in regenerative adult newt cardiomyocytes.

Adult newt cardiomyocytes, in contrast to their mammalian counterparts, can proliferate after injury and contribute to the functional regeneration of the heart. In order to understand the mechanisms underlying this plasticity we performed longitudinal studies on single cardiomyocytes in culture. We find that the majority of cardiomyocytes can enter S phase, a process that occurs in response to serum-activated pathways and is dependent on the phosphorylation of the retinoblastoma protein. However, more than half of these cells stably arrest at either entry to mitosis or during cytokinesis, thus resembling the behaviour observed in mammalian cardiomyocytes. Approximately a third of the cells progress through mitosis and may enter successive cell divisions. When cardiomyocytes divided more than once, the proliferative behaviour of sister cells was significantly correlated, in terms of whether they underwent a subsequent cell cycle, and if so, the duration of that cycle. These observations suggest a mechanism whereby newt heart regeneration depends on the retention of proliferative potential in a subset of cardiomyocytes. The regulation of the remaining newt cardiomyocytes is similar to that described for their mammalian counterparts, as they arrest during mitosis or cytokinesis. Understanding the nature of this block and why it arises in some but not other newt cardiomyocytes may lead to an augmentation of the regenerative potential in the mammalian heart.