The urodele limb regeneration blastema

Abstract. The idea that the undifferentiated limb regeneration blastema of urodele amphibians is an undetermined and pluripotent structure is examined. A detailed review of the literature shows that this notion has no basis in fact. The data show that the morphogenetic potency of the blastema is restricted to its prospective significance and that this potency can be fully expressed when the blastema is transplanted either to neutral location or to regenerating organ of another type. Within this morphogenetic constraint, however, blastema cells have histogenetic potency that is, at least in some cases, greater than their limb cell phenotype of origin. The morphogenetic responses of the regeneration field to discontinuities suggest that its autonomous determining relationships are based on the inheritance, from parent limb cells, of graded set of mesodermal positional values specifying the pattern of the amputation plane, and single epidermal external boundary value. The dividing mesenchymal cells of the blastema change positional value to erase any discontinuity between themselves and the epidermis, and the epidermis acts as stop signal to inform the mesenchyme when the regenerate boundary has been reached. In vitro experiments suggest that changes in mesenchymal positional value in response to discontinuity can be interpreted in terms of gradients of cell-cell adhesivity, and they focus attention on the importance of molecular studies of blastema cell surfaces for our future understanding of regeneration and morphogenesis in general.     

Proximodistal patterning during limb regeneration

Regeneration is an ability that has been observed extensively throughout metazoan phylogeny. Amongst vertebrates, the urodele amphibians stand out for their exceptional capacity to regenerate body parts such as the limb. During this process, only the missing portion of the limb is precisely replaced--amputation in the upper arm results in regeneration of the entire limb, while amputation at the wrist produces a hand. Limb regeneration occurs through the formation of a local proliferative zone called the blastema. Here, we examine how proximodistal identity is established in the blastema. Using cell marking and transplantation experiments, we show that distal identities have already been established in the earliest stages of blastemas examined. Transplantation of cells into new environments is not sufficient to respecify cell identity. However, overexpression of the CD59, a cell surface molecule previously implicated in proximodistal identity during limb regeneration, causes distal blastema cells to translocate to a more proximal location and causes defects in the patterning of the distal elements of the regenerate. We suggest a model for the limb regeneration blastema where by 4 days post-amputation the blastema is already divided into distinct growth zones; the cells of each zone are already specified to give rise to upper arm, lower arm, and hand.     

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.     

Cell differentiation and cell fate during urodele tail and limb regeneration

One of the most striking natural examples of adult tissue plasticity in vertebrates is limb and tail regeneration in urodele amphibians. In this setting, amputation triggers the destabilization of cell differentiation and the production of progenitor cells that extensively proliferate and pattern themselves to recreate a perfect replica of the missing part. A precise understanding of which cells dedifferentiate and how plastic they become has recently begun to emerge. Furthermore, information on which developmental gene programs are activated upon injury is becoming better understood. These studies indicate that, upon injury, an unusual cohort of genes are co-expressed. The future challenge will be to link the systems for studying dedifferentiation with activation of gene expression to understand on a molecular level how cells are 'pushed backward' to regenerate a complex structure such as a limb or tail.     

Reduction of sodium dependent stump currents disturbs urodele limb regeneration.

We have asked the question whether the natural electric currents which leave urodele limb stumps are in any way needed for their regeneration. As an initial test, we have greatly reduced such currents in the tiger salamander, Ambystoma tigrinum, by applying 0.5 mM amiloride to the stump skin or by immersion of the animals in sodium depleted media. We have also reduced such currents in the red spotted newt, Notophthalmus viridescens, by such immersion. Limb regeneration in half of the amiloride-treated animals was either entirely blocked or grossly deficient, while the others regenerated normally. Limb regeneration in sodium depleted media was consistently inhibited for some weeks but then recovered. These results are consistent with the hypothesis that stump currents are in some way needed for normal regeneration.     

The urodele limb regeneration blastema. Determination and organization of the morphogenetic field

The idea that the undifferentiated limb regeneration blastema of urodele amphibians is an undetermined and pluripotent structure is examined. A detailed review of the literature shows that this notion has no basis in fact. The data show that the morphogenetic potency of the blastema is restricted to its prospective significance and that this potency can be fully expressed when the blastema is transplanted either to a neutral location or to a regenerating organ of another type. Within this morphogenetic constraint, however, blastema cells have a histogenetic potency that is, at least in some cases, greater than their limb cell phenotype of origin. The morphogenetic responses of the regeneration field to discontinuities suggest that its autonomous determining relationships are based on the inheritance, from parent limb cells, of a graded set of mesodermal positional values specifying the pattern of the amputation plane, and a single epidermal external boundary value. The dividing mesenchymal cells of the blastema change positional value to erase any discontinuity between themselves and the epidermis, and the epidermis acts as a stop signal to inform the mesenchyme when the regenerate boundary has been reached. In vitro experiments suggest that changes in mesenchymal positional value in response to discontinuity can be interpreted in terms of gradients of cell-cell adhesivity, and they focus attention on the importance of molecular studies of blastema cell surfaces for our future understanding of regeneration and morphogenesis in general.     

Structural specificity of beta-endorphin C-terminal tetrapeptide (MPF) in promoting urodele limb regeneration

The ability of B-endorphin to initiate limb regeneration in hypophysectomised newts is confined to the human species of the peptide and is contained in its C-terminal tetrapeptide sequence, Lys-Lys-Gly-Glu (MPF). Results with fifteen MPF analogs show that: (a) small structural change at the C-terminal Glu residue destroys activity, (b) at the Gly position, change of -NH-CH2-CO by -NH-NH-CO- (Azgly) or -NH-CHMe-CO- (Ala) also destroys the activity, but methylation of the NH results in an analog (Sar) with good activity, (c) analogs in which the Lys residues are replaced by D-Lys, Nle or Orn may retain some activity, particularly when the second Lys is replaced by D-Lys, and (d) the activity is retained or increased by N-terminal acylation. By combining 'favourable' changes, an analog acetyl-Lys-D-Lys-Sar-Glu was devised which was 1.25 times more potent than MPF, and metabolically more stable.     

Stretch receptors in urodele limb muscles

Summary Non-encapsulated, fine beaded nerve endings were found histologically on some muscle fibres in a number of limb muscles in newts and axolotls. They were present in newt muscles that had been chronically de-efferented, and in which no efferent activity survived, and were therefore likely to be sensory. They were located only on muscle fibres on or near the outside surface of the muscle. These small-diameter muscle fibres were characterised histochemically by low lipid, SDH and phosphorylase content; ultrastructurally by low glycogen content, and relatively large myofilaments poorly delimited by a sparse SR. There were many of this type (Type 1) that did not support sensory endings. A few endings occurred on another larger-diameter type of fibre (Type 2) whose properties were opposite to those listed above for Type 1. There was virtually no specialization of muscle fibre structure beneath the sensory endings.Physiological experiments involving ramp-and-hold and sinusoidal stretch applied to the muscle whilst recording single-unit afferent responses in m.ext. dig. III of axolotls showed unit responses very similar to those known from muscle spindles, particularly those of the frog.We are grateful to the M.R.C. for an equipment grant (to R.M.A.P.R.), and to Mrs. Janis Taberner for her technical help. Part of this work was done during the tenure of a Nuffield-NRC Lectureship (Q.B.) which is gratefully acknowledged, as is financial support by Prof. J.R. Nussall during a visit to the University of Alberta at Edmonton.     

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.     

Small molecules that recapitulate the early steps of urodele amphibian limb regeneration and confer multipotency

In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates to contribute new limb tissue. However, mammalian muscle cannot dedifferentiate after injury. We have developed a novel, small-molecule-based method to induce dedifferentiation in mammalian skeletal muscle. Muscle cellularization was induced by the small molecule myoseverin. Candidate small molecules were tested for the induction of proliferation in the cellulate. We observed that treatment with the small molecules BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) induced proliferation. Moreover, these proliferating cells were multipotent, as confirmed by the chemical induction of mesodermal-derived cell lineages. Microarray analysis showed that the multipotent, BIO-treated cellulate possessed a markedly different gene expression pattern than lineage-restricted C2C12 myoblasts, especially for genes related to signal transduction and differentiation. Sequential small molecule treatment of the muscle cellulate with BIO, SB203580, or SQ22536 and the aurora B kinase inhibitor, reversine, induced the formation of cells with neurogenic potential (ectodermal lineage), indicating the acquirement of pluripotency. This is the first demonstration of a small molecule method that induces mammalian muscle to undergo dedifferentiation and rededifferentiation into alternate cell lineages. This method induces dedifferentiation in a simple, stepwise approach and has therapeutic potential to enhance tissue regeneration in mammals.     

Retinoid antagonists inhibit normal patterning during limb regeneration in the axolotl, Ambystoma mexicanum

Retinoic acid (RA) has been detected in the regenerating limb of the axolotl, and exogenous RA can proximalize, posteriorize, and ventralize blastemal cells. Thus, RA may be an endogenous regulatory factor during limb regeneration. We have investigated whether endogenous retinoids are essential for patterning during axolotl (Ambystoma mexicanum) limb regeneration by using retinoid antagonists that bind to specific RAR (retinoic acid receptor) or RXR (retinoid X receptor) retinoid receptor subtypes. Retinoid antagonists (Ro41-5253, Ro61-8431, LE135, and LE540) were administered to regenerating limbs using implanted silastin blocks loaded with each antagonist. The skeletal pattern of regenerated limbs treated with Ro41-5253 or Ro61-8431 differed only slightly from control limbs. Treatment with LE135 inhibited limb regeneration, while treatment with LE540 allowed relatively normal limb regeneration. When LE135 and LE540 were implanted together, regeneration was not completely inhibited and a hand-like process regenerated. These results demonstrate that interfering with retinoid receptors can modify pattern in the regenerating limb indicating that endogenous retinoids are important during patterning of the regenerating limb.     

Spinal cord regeneration in a tail autotomizing urodele

Adult urodele amphibians possess extensive regenerative abilities, including lens, jaws, limbs, and tails. In this study, we examined the cellular events and time course of spinal cord regeneration in a species, Plethodon cinereus, that has the ability to autotomize its tail as an antipredator strategy. We propose that this species may have enhanced regenerative abilities as further coadaptations with this antipredator strategy. We examined the expression of nestin, vimentin, and glial fibrillary acidic protein (GFAP) after autotomy as markers of neural precursor cells and astroglia; we also traced the appearance of new neurons using 5‐bromo‐2′‐deoxyuridine/neuronal nuclei (BrdU/NeuN) double labeling. As expected, the regenerating ependymal tube was a major source of new neurons; however, the spinal cord cranial to the plane of autotomy showed significant mitotic activity, more extensive than what is reported for other urodeles that cannot autotomize their tails. In addition, this species shows upregulation of nestin, vimentin, and GFAP within days after tail autotomy; further, this expression is upregulated within the spinal cord cranial to the plane of autotomy, not just within the extending ependymal tube, as reported in other urodeles. We suggest that enhanced survival of the spinal cord cranial to autotomy allows this portion to participate in the enhanced recovery and regeneration of the spinal cord. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

A comparative proteomic analysis during urodele lens regeneration

To examine underlying mechanisms of urodele lens regeneration we have employed a proteomic analysis of 650 proteins involved in several signaling pathways. We compared expression of these proteins between the regeneration-competent dorsal iris and the regeneration-incompetent ventral iris in the newt. After a series of screenings we selected several proteins to evaluate their expression quantitatively on immunoblots. We then used these selected proteins to compare their expression between the dorsal iris of the newt and the iris of the axolotl, another urodele, which does not regenerate the lens. In the newt we find that most proteins are expressed in both dorsal and ventral iris, even though there is differential regulation. Moreover, several of these proteins are expressed in the axolotl iris as well and for some of them their expression is consistent with the regeneration potential.     

Punctuated cell cycling in the regeneration blastema of urodele amphibians

Abstract. We previously assumed that all cells in the regeneration blastema are randomly distributed throughout the cell cycle and actively cycling towards the next mitotic division. We now show that data from continuous labeling (³H-thymidine) experiments do not support this view and favor instead the hypothesis that the blastema cell cycle is punctuated in the g₁ phase wherein cells can enter what we term a transiently quiescent (TQ) position. We call this hypothesis the punctuated cycling (PC) hypothesis. We further propose that the relative sizes of the quiescent and actively cycling populations explain (1) variations in rates of regeneration in different sizes of urodele amphibians, (2) the rate and success of regeneration in different species, and (3) how various controlling factors, such as injury, nerves, growth factors, wound epidermis, and hormones, influence the initiation and progression of the regeneration process. This PC hypothesis is important for interpreting previous pulse labeling data, is consistent with recently obtained continuous labeling data, and is experimentally testable.     

Regeneration inducers in limb regeneration

Limb regeneration ability, which can be observed in amphibians, has been investigated as a representative phenomenon of organ regeneration. Recently, an alternative experimental system called the accessory limb model was developed to investigate early regulation of amphibian limb regeneration. The accessory limb model contributed to identification of limb regeneration inducers in urodele amphibians. Furthermore, the accessory limb model may be applied to other species to explore universality of regeneration mechanisms. This review aims to connect the insights recently gained to emboss universality of regeneration mechanisms among species. The defined molecules (BMP7 (or2) + FGF2 + FGF8) can transform skin wound healing to organ (limb) regeneration responses. The same molecules can initiate regeneration responses in some species.