An epidermal signal regulates Lmx-1 expression and dorsal-ventral pattern during Xenopus limb regeneration

The results of recent studies have supported the idea that the ability to organize the formation of axes such as the anteroposterior and proximodistal axes corresponds to limb regeneration ability in Xenopus. In this study, we investigated the mechanism by which the dorsoventral (D-V) axis of regenerating Xenopus limbs is established and the relationships between D-V patterning and regenerative ability. Transplantation experiments were performed to study which epidermis or mesenchyme is responsible for the D-V patterning in regenerating limbs. Naked mesenchyme of a donor limb was rotated and implanted on a host opposite-side limb stump to make a reversed recombination about the D-V axis. The resultant regenerates had a normal-looking D-V pattern, including Lmx-1 expression, muscle pattern, and joints, in stage 52 recombinants and a reversed D-V pattern in stage 55 recombinants. Further experiments in recombination at stage 52 and stage 55 showed that the epidermal signal is responsible for producing the D-V pattern in the regenerating blastema. These results, together with the finding that Lmx-1 expression is absent in the froglet forelimb blastema, suggest that D-V axis formation is a key step in understanding the loss of regenerative ability.     

Expression of the 9G1 antigen in the apical cap of axolotl regenerates requires nerves and mesenchyme

Monoclonal antibody 9G1 (mAb 9G1) is reactive to the wound epithelium of axolotl larvae and therefore provided the opportunity to examine the interaction between the wound epithelium, nerves, and blastemal mesenchyme during axolotl limb regeneration. In unamputated limbs, mAb 9G1 is reactive to most or all cells of the dermis, skeletal elements, blood vessels, and nerves, to a few unidentified cells in muscle, and to none in epidermis. During regeneration of axolotl limbs, mAb 9G1 reacts strongly to an intracellular antigen of the blastemal mesenchyme and of the distal-most portion of the wound epithelium, the so-called apical epithelial cap (AEC). Because this thickened wound epithelium of regenerating amphibian limbs has been suggested as functioning in a manner similar to the apical ectodermal ridge (AER) of embryonic limb buds, it was of interest to further examine the reactivity of mAb 9G1 during various stages of regeneration. Whether mAb 9G1 reactivity in the AEC depended on mesenchyme and/or nerves was also tested. Monoclonal antibody 9G1 reactivity appears in the AEC of regenerating limbs prior to outgrowth of the blastema and persists throughout blastemal stages. Apical epithelial cap reactivity to mAb 9G1 is nerve dependent during early stages of blastema development and becomes nerve-independent at later stages. When epithelium-free blastemal mesenchyme is grafted onto injured flank musculature, ectopic limb regeneration occurs and the AEC derived from flank epidermis exhibits mAb 9G1 reactivity. These results show that a mAb 9G1 reactive AEC is characteristic of regenerating limbs and that expression of the 9G1 antigen by the AEC is dependent upon underlying blastemal mesenchyme and nerves.     

Immunolocalization of Wnts in the lizard blastema supports a key role of these signaling proteins for tail regeneration

A highly upregulated gene during tail regeneration in lizards is Wnt2b, a gene broadly expressed during development. The present study examines the distribution of Wnt proteins, most likely wnt2b, by western blotting and immunofluorescence in the blastema-cone of lizards using a specific antibody produced against a lizard Wnt2b protein. Immunopositive bands at 48–50 and 18 kDa are present in the regenerative blastema, the latter likely as a degradation product. Immunofluorescence is mainly observed in the wound epidermis, including in the Apical Epidermal Peg where the protein appears localized in intermediate and differentiating keratinocytes. Labeling is more intense along the perimeter of keratinocytes, possibly as a secretory product, and indicates that the high epidermal proliferation of the regenerating epidermis is sustained by Wnt proteins. The regenerating spinal cord forms an ependymal tube within the blastema and shows immunolabeling especially in the cytoplasm of ependymal cells contacting the central canal where some secretion might occur. Also, regenerating nerves and proximal spinal ganglia innervating the regenerating blastema contain this signaling protein. In contrast, the blastema mesenchyme, muscles and cartilage show weak immunolabeling that tends to disappear in tissues located in more proximal regions, close to the original tail. However, a distal to proximal gradient of Wnt proteins was not detected. The present study supports the hypothesis that Wnt proteins, in particular Wnt2b, are secreted by the apical epidermis covering the blastema and released into the mesenchyme where they stimulate cell multiplication.     

Lipid of normal and regenerating limb tissues of the adult newt, Diemictylus viridescens: acidic and non-acidic lipids, phospholipids, and cholesterol

Cryostat-cut sections of unamputated and amputated-regenerating limbs of the adult newt were examined following the Nile blue test for acidic and non-acidic lipids, the acid hematein and plasmal tests for phospholipids, and a Schultz test for cholesterol. Triglycerides (Nile blue test) are prominent in dermis and macrophages: triglyceride droplets are scattered in epidermis, wound epithelium, and regeneration blastema. Fatty acids (Nile blue test) are present in all tissues of the normal and regenerating limb: nerve myelin contains relatively little free fatty acid, while macrophages appear to contain the least amount of this lipidic substance. Plasmalogens (plasmal test) are prominent in the myelin of nerves, and macrophages: a weak cytoplasmic reaction obtains in the epidermis, subcutaneous glands, striated muscle, tunics of blood vessels, wound epithelium, blastema cells, chondrocytes, perichondrium and periosteum. Mitochondria responding for cephalin, lecithin, and sphingomyelin (acid hematein test) are ubiquitously distributed among the cells and tissues of the normal and regenerating limb. These phosphatides are prominent in nerve myelin, macrophages, and in dermal droplets: a variable response obtains from the myofibrils of striated muscle. Cholesterol (Schultz test) was demonstrated only in nerve myelin and in macrophages associated with injured nerves. Particular attention was paid to the lipid responses of the regeneration blastema, and the conclusion was reached that not all of the lipid previously demonstrated with sudan dyes was characterized by the current series of lipid tests. A modified Nile blue sulfate test that promises greater specificity in distinguishing between acidic and non-acidic lipids is introduced.     

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.     

Evidence that the nerve controls molecular identity of progenitor cells for limb regeneration

Adult urodele amphibians can regenerate their limbs after amputation by a process that requires the presence of axons at the amputation plane. Paradoxically, if the limb develops in the near absence of nerves (the 'aneurogenic' limb) it can subsequently regenerate in a nerve-independent fashion. The growth zone (blastema) of regenerating limbs normally contains progenitor cells whose division is nerve-dependent. A monoclonal antibody that marks these nerve-dependent cells in the normal blastema does not stain the mesenchymal cells of developing limb buds and only stains the amputated limb bud when axons have reached the plane of amputation. This report shows that the blastemal cells of the regenerating aneurogenic limb also fail to react with the antibody in situ. These data suggest that the blastemal cells arising during normal regeneration have been altered by the nerve. This regulation may occur either at the time of amputation (when the antigen is expressed) or during development (when the limb is first innervated).     

Histochemical studies on cell death and histolysis during regeneration. I. Distribution of acid phosphomonoesterase activity in the normal, the regenerating and the resorbing forelimb of the larval spotted salamander, Amblystoma maculatum

Histochemical methods, especially azo dye methods for detecting acid phosphomonoesterase activity were applied to normal, regenerating and denervated, amputated limbs from larval Amblystoma maculatum. Efforts were made to control inactivation of enzymic activity and diffusion of both enzyme and reaction product. “Base-line” values for enzymic activity were determined for normal limbs. Activity appeared most intense in macrophages, less intense in epidermis and cartilage matrix. Some activity was detected in Schwann cells, peri- and endoneurium and muscle fibers form normal limbs. Enzymic activity in regenerating limbs was strongest within macrophages which appeared in increased numbers especially in early stages. Wound tissue showed little increased activity. As the blastema formed, increased enzymic activity was detected in epidermis and within increased numbers of macrophages. Chondrocytes showed increased activity especially during cartilage matrix deposition. Amputated, denervated limbs showed large numbers of active macrophages beneath and within epidermis and along muscle. As regression commenced, areas of cartilage matrix breakdown showed increased enzymic activity but, in general, greatest activity was in macrophages. The various possible roles of acid phosphomonoesterase activity in the specific biological situations dealt with are considered in light of such observations.     

Analysis of gene expressions during Xenopus forelimb regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic 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.     

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.     

Monoclonal antibody MT2 identifies an extracellular matrix glycoprotein that is co-localized with tenascin during adult newt limb regeneration

Using immunohistochemical techniques and mAb MT2, we describe here a novel extracellular matrix (ECM) molecule that is developmentally regulated during limb regeneration in adult newts. The MT2 antigen appears during preblastema stages, is most abundant during blastema stages, and persists, near undifferentiated cells, until digit stages. The MT2 antigen is located in an acellular layer under the wound epithelium and throughout the ECM of the undifferentiated mesenchyme as a thick, cord-like component. In unamputated limbs mAb MT2 reactivity is restricted to tendons, myotendinous junctions, periosteum and to a layer of material beneath the epidermis. In both unamputated limbs and regenerating limbs, the reactivity to mAb MT2 colocalizes closely with urodele tenascin. Immunoblot analysis of blastema extracts showed that the unreduced form of the MT2 antigen is a large, polydispersed protein of approximately the same size as tenascin. However, based upon (a) molecular weights of reduced subunits, (b) competition experiments on tissue sections, and (c) analysis of molecules immunoprecipitated by mAb MT2, we conclude that the MT2 substance is unrelated biochemically to tenascin. The results from immunoblots, enzyme digestions and DEAE-Sephacell binding studies suggest that the unreduced MT2 antigen is a large protein composed of subunits which are connected by disulfide bonds. Reduction of the MT2 antigen results in three components recognized by mAb MT2. The largest of these reduced components is a chondroitin sulfate-like glycoprotein with a molecular weight (Mr) of 310-325 x 10(3). A second component (Mr, 285-300 x 10(3)) is the core protein of the 310-325 x 10(3) glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ultrastructural immunolocalization of hyaluronate in regenerating tail of lizards and amphibians supports an immune‐suppressive role to favor regeneration

Hyaluronate is produced in high amount during the initial stages of regeneration of the tail and limbs of lizards, newts, and frog tadpoles. The fine distribution of hyaluronate in the regenerating tail blastemas has been assessed by ultrastructural immunolocalization of the Hyaluronate Binding Protein (HABP), a protein that indirectly reveals the presence of hyaluronate in tissues. The present electron microscopic study shows that HABP is detected in the cytoplasm but this proteins is mainly localized on the surfaces of cells in the wound epidermis and mesenchymal cells of the blastema. HABP appears, therefore, accumulated along the cell surface, indicating that hyaluronate coats these embryonic‐like cells and their antigens. The high level of hyaluronate in the blastema, aside favoring tissue hydration, cell movements, and remodeling for blastema formation and growth, likely elicits a protection from the possible immune‐reaction of lymphocytes and macrophages to embryonic‐fetal‐like antigens present on the surface of blastema and epidermal cells. Their survival, therefore, allows the continuous multiplication of these cells in regions rich in hyaluronate, promoting the regeneration of a new tail or limbs. The study suggests that organ regeneration in vertebrates is only possible in the presence of high hyaluronate content and hydration. These two conditions facilitate cell movement, immune‐protection, and activate the Wnt signaling pathway, like during development.     

Retinoic acid and its receptors in limb regeneration

The effects of retinoids on the regenerating amphibian limb are described: the mesenchymal cells of the blastema can be proximalized, posteriorized and ventralized. Ectopic limbs are also induced after retinoid treatment of regenerating tails, but not during limb development unless the limb bud is damaged. The cellular and molecular alterations induced by retinoids are reported as well as experiments which have revealed the importance of endogenous retinoids for normal limb regeneration. Various retinoic acid receptors are expressed in the regeneration blastema and the experiments which have revealed functions for individual isoforms are described. These experiments reveal that retinoids are a crucial component of the normal, regenerating limb and demonstrate the value of the regenerating limb as an experimental system for providing functional data on individual retinoic acid receptors.     

Changes in the Distribution of Fibronectin During Limb Regeneration in Newts Using Immunocytochemistry

The distribution of fibronectin in regenerating newt limbs was studied using immunocytochemistry. At appropriate intervals after the initial amputation at the elbow (10–30 days), animals were reamputated at the shoulder and processed for light microscopy. The peroxidase-antiperoxidase technique was used to localize affinity-purified antibodies to fibronectin in limb tissues. At the amputation site, fibronectin was associated with basal laminae and connective tissues adjacent to dedifferentiating limb tissues destined to form the regeneration blastema. Accumulation and growth of the blastema was accompanied by the apparent de novo synthesis of fibronectin, where it appeared randomly in the interstitium between blastemal cells. The onset of chondrogenesis was characterized by a central condensation of prechondroblasts that formed the cartilage anlagen. Fibronectin formed an amorphous network between presumptive chondroblasts. As the mature cartilage phenotype was expressed and chondrocytes became isolated in lacunae, fibronectin was greatly reduced and then disappeared. The extracellular matrix surrounding undifferentiated blastemal cells still contained fibronectin. Fibronectin was also found in high concentrations between differentiating myoblasts. A condensation of fibronectin was also observed beneath the epidermis at the distal limb tip at the onset of digit formation. These observations are consistent with the hypothesis that fibronectin may play a key role in the morphogenetic events that result in the spatial organization and subsequent differentiation of cells during pattern formation in the regenerating limb.     

Analysis of Gene Expressions during Xenopus Forelimb Regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.