Msx1‐2 immunolocalization in the regenerating tail of a lizard but not in the scarring limb suggests its involvement in the process of regeneration

The immunolocalization of the muscle segmental homoeobox protein Msx1‐2 of 27–34 kDa in the regenerating tail blastema of a lizard shows prevalent localization in the apical ependyma of the regenerating spinal cord and less intense labelling in the wound epidermis, in the apical epidermal peg (AEP), and in the regenerating segmental muscles. The AEP is a micro‐region of the regenerating epidermis located at the tail tip of the blastema, likely corresponding to the AEC of the amphibian blastema. No immunolabelling is present in the wound epidermis and scarring blastema of the limb at 18–21 days of regeneration, except for sparse repairing muscles. The presence of a proximal–distal gradient of Msx1‐2 protein, generated from the apical ependyma, is suggested by the intensity of immunolabelling. The AEP and the ependyma are believed to induce and maintain tail regeneration, and this study suggests that Msx1‐2 proteins are components of the signalling system that maintains active growth of the tail blastema. The lack of activation and production of Msx1‐2 protein in the limb are likely due to the intense inflammatory reaction following amputation. This study confirms that, like during regeneration in fishes and amphibians, also the blastema of lizards utilizes common signalling pathways for maintaining regeneration.     

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.     

Temporal distribution of 5BrdU-labelled cells suggests that most injured tissues contribute proliferating cells for the regeneration of the tail and limb in lizard

After tail and limb amputation in lizard, injection of 5BrdU for 6 days produces immunolabelled cells in most tissues of tail and limb stumps. After further 8 and 16 days, and 14 and 22 days of regeneration, numerous 5BrdU-labelled cells are detected in regenerating tail and limb, derived from most stump tissues. In tail blastema cone at 14 days, sparse-labelled cells remain in proximal dermis, muscles, cartilaginous tube and external layers of wound epidermis but are numerous in the blastema. In apical regions at 22 days of regeneration, labelled mesenchymal cells are sparse, while the apical wound epidermis contains numerous labelled cells in suprabasal and external layers, indicating cell accumulation from more proximal epidermis. Cell proliferation dilutes the label, and keratinocytes take 8 days to migrate into corneous layers. In healing limbs, labelled cells remain sparse from 14 to 22 days of regeneration in wound epidermis and repairing tissues and little labelling dilution occurs indicating low cell proliferation for local tissue repair but not distal growth. Labelled cells are present in epidermis, intermuscle and peri-nerve connectives, bone periosteum, cartilaginous callus and sparse fibroblasts, leading to the formation of a scarring outgrowth. Resident stem cells and dedifferentiation occur when stump tissues are damaged.     

Cell proliferation in the amputated limb of lizard leading to scarring is reduced compared to the regenerating tail

After amputation, the tail of lizards regenerates while the limb forms a short scarring outgrowth. Using phospho‐histone‐H3 immunohistochemistry the mitotic activity of limb tissues at 12–25 days after amputation has been studied, when a limb outgrowth of 0.5–2 mm in length is covered by wound epidermis and the underlying connective is turning into a dense scar. In comparison with a regenerating tail of 3–5 mm in length, the number of dividing cells is reduced of 40–70% in different tissues of the scarring limb 1–2 mm in length at 18 days postamputation. Dividing cells are still present at 12–25 days postamputation in the cartilaginous epiphyses of the transected tibia and fibula and of the untransected femur. Also, the injured muscles present at the base of the scarring outgrowth still contain sparse dividing cells after 25 days postamputation of the limb. Together previous studies, the present observations suggest that after the initial proliferation of fibroblasts deriving from the injured tissues, especially from the dermis and intermuscle connectives during the initial 7–15 days postinjury, these cells cover the injured tissues underneath the wound epidermis, but rapidly produce high levels of collagen turning the initial blastema into a scar.     

Immunolocalization of a p53/p63‐like protein in the regenerating tail of the wall lizard (Podarcis muralis) suggests it is involved in the differentiation of the epidermis

During tail regeneration in lizards, the epidermis forms new scales comprising a hard beta‐layer and a softer alpha‐layer. Regenerated scales derive from a controlled folding process of the wound epidermis that gives rise to epidermal pegs where keratinocytes do not invade the dermis. Basal keratinocytes of pegs give rise to suprabasal cells that initially differentiate into a corneous wound epidermis and later in corneous layers of the regenerated scales. The immunodetection of a putative p53/63 protein in the regenerating tail of lizards shows that immunoreactivity is present in the nuclei of basal cells of the epidermis but becomes mainly cytoplasmic in suprabasal and in differentiating keratinocytes. Sparse labelled cells are present in the regenerating blastema, muscles, cartilage, ependyma and nerves of the growing tail. Ultrastructural observations on basal and suprabasal keratinocytes show that the labelling is mainly present in the euchromatin and nucleolus while labelling is more diffuse in the cytoplasm. These observations indicate that the nuclear protein in basal keratinocytes might control their proliferation avoiding an uncontrolled spreading into other tissues of the regenerating tail but that in suprabasal keratinocytes the protein moves from the nucleus to the cytoplasm, a process that might be associated to keratinocyte differentiation.     

Effects on adult newt limb regeneration of partial and complete skin flaps over the amputation surface.

The influence of the wound epithelium on the cellular events preceding blastema formation was examined by comparing dedifferentiation, DNA labeling indices, and mitotic indices of the distal mesodermal tissues in control regenerating newt forelimbs and in amputated forelimbs covered with a flap of full thickness skin. Three kinds of results were seen following the skin-flap graft operations. Epidermal migration across the amputation surface was completely inhibited in 22% (8) of the cases and these limbs repaired the amputation wound but did not form regeneration blastemas. In 11% (4) of the experimental limbs, essentially normal wound epithelia displaced the skin flaps and the limb stumps formed blastemas and regenerated. The majority of the skin grafts (67%) exhibited epidermal migration restricted to the free edges of the flaps. These limbs formed eccentric blastemas on the ventral side of the limb next to the dermis-free epidermis and regenerated laterally in that direction.     

Immunohistochemical localization of a proto-cadherin fat tumour-suppressor homolog in the regenerating tail of lizard suggests a role in apical growth control

The present immunohistochemical and western blotting study evaluates the localization of a proto-cadherin which gene is overexpressed in the regenerating blastema of the lizard Podarcis muralis. Bioinformatic analysis suggests that the antibody recognizes FAT1/2 proteins. Western blot indicates a main band around 50 kDa, a likely fragment derived from the original membrane-bound large protein. Immunofluorescence shows main labelling in differentiating wound keratinocytes, lower in ependyma, mesenchyme and extracellular matrix of the blastema. The apical epidermal peg contains keratinocytes with labelled peripheral cytoplasm, as confirmed using ultrastructural immunogold that also reveals most labelling located along the cell surface of mesenchymal cells. Myoblasts and differentiating myotubes of regenerating muscles are less intensely labelled. The regenerating cartilaginous tube contains sparse labelled chondroblasts, especially in external and internal perichondria. In regenerating scales, differentiating beta-cells appear immunofluorescent mainly along the cell perimeter. In more differentiated muscle, cartilage and connective tissues of the new tail, the labelling lowers or disappears. The observations indicate that FAT1/2 proto-cadherins are present in the apical blastema where an intense remodelling takes place for the growth of the new tail but where also a tight control of cell division and migration is active and may regulate potential tumorigenic process.     

Nerve-induced ectopic limb blastemas in the Axolotl are equivalent to amputation-induced blastemas

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.     

Expression of Regeneration-Associated Antigens in Normal and Retinoid-Treated Regenerating Limbs of Ambystoma mexicanum

The expression of two regeneration-associated antigens in the blastemas of normal and retinoid-treated regenerating limbs of axolotl ( Ambystoma mexicanum ) was examined.
One antigen, 55C12, which was similar to tenascin in expression pattern and molecular weight profile, was weakly expressed in the perichondrium and tendon of normal limbs. In the regenerating limbs, the amount of 55C12 antigen increased near the amputation site within 7 days and almost all cells of the blastema mesenchyme came to be positive to the antigen at 20 days, although those of epidermis and most stump tissues were negative. When the regenerating limbs were treated with Am80, a synthetic retinoid, which induced proximo-distal duplication, the expression of 55C12 antigen in the blastema became weak temporarily and was reactivated in the anterior region of the blastema. This expression pattern suggests that the duplicated limb is formed by the preferential growth of this 55C12-positive anterior blastema region.
The other antigen, 117C1, was faintly expressed in the epidermis, dermis, muscle, perichondrium and cartilage of normal limbs, and intensely expressed in the blastema mesenchyme and wound epidermis. The Am80 treatment, however, induced no changes in the expression pattern of 117C1.
These results suggest that these antigens may distinguish two different regions of the blastema in normal regeneration and retinoid-induced duplication.     

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.     

Immunolocalization of the telomerase‐1 component in cells of the regenerating tail,testis, and intestine of lizards

Using an antibody against a lizard telomerase‐1 component the presence of telomerase has been detected in regenerating lizard tails where numerous cells are proliferating. Immunoblots showed telomerase positive bands at 75–80 kDa in normal tissues and at 50, 75, and 90 kDa in those regenerating. Immunofluorescence and ultrastructural immunolocalization showed telomerase‐immunoreactivity in sparCe (few/diluted) mesenchymal cells of the blastema, early regenerating muscles, perichondrium of the cartilaginous tube, ependyma of the spinal cord, and in the regenerating epidermis. Clusters of gold particles were detected in condensing chromosomes of few mesenchymal and epithelial cells in the regenerating tail, but a low to undetectable labeling in interphase cells. Telomerase‐immunoreactivity was intense in the nucleus and sparCe (few/diluted) in the cytoplasm of spermatogonia and spermatocytes and drastically decreased in early spermatids where some nuclear labeling remains. Some intense immunoreactivity was seen in few cells near the basal membrane of intestinal enterocytes or in leukocytes (likely lymphocytes) of the intestine mucosa. In spermatogonia, spermatids and in enterocytes part of the nuclear labeling formed cluster of gold particles in dense areas identified as Cajal Bodies, suggesting that telomerase is a marker for these stem cells. This therefore suggests that also the sparCe (few/diluted) telomerase positive cells detected in the regenerating tail may represent sparCe (few/diluted) stem cells localized in regenerating tissues where transit amplifying cells are instead preponderant to allow for tail growth. This observation supports previous studies indicating that few stem cells are present in the stump after tail amputation and give rise to transit amplifying cells for tail regeneration. J. Morphol. 276:748–758, 2015. © 2015 Wiley Periodicals, Inc.     

Wound keratins in the regenerating epidermis of lizard suggest that the wound reaction is similar in the tail and limb

The keratin cytoskeleton of the wound epidermis of lizard limb (which does not regenerate) and tail (which regenerates) hase been studied by qualitative ultrastructural, immunocytochemical, and immunoblotting methods. The process of re-epithelialization is much shorter in the tail than in the limb. In the latter, a massive tissue destruction of bones, and the shrinkage of the old skin over the stump surface, delay wound closure, maintain inflammation, reduce blastemal cell population, resulting in inhibition of regeneration. The expression of special wound keratins found in the newt epidermis (W6) or mammalian epidermis (K6, K16, and K17) is present in the epidermis of both tail and limb of the lizard. These keratins are not immunolocalized in the migrating epithelium or normal (resting) epidermis but only after it has formed the thick wound epithelium, made of lacunar cells. The latter are proliferating keratinocytes produced during the cyclical renewal or regeneration of lizard epidermis. W6-immunolabeled proteic bands mainly at 45-47 kDa are detected by immunoblotting in normal, regenerating, and scarring epidermis of the tail and limb. Immunolabeled proteic bands at 52, 62-67 kDa (with K6), at 44-47, 60, 65 kDa (with K16), and at 44-47 kDa (with K17) were detected in normal and regenerating epidermis. It is suggested that: (1) these keratins constitute normal epidermis, especially where the lacunar layer is still differentiating; (2) the wound epidermis is similar in the limb and tail in terms of morphology and keratin content; (3) the W6 antigen is similar to that of the newt, and is associated with tonofilaments; (4) lizard K6 and K17 have molecular weights similar to mammalian keratins; (5) K16 shows some isoforms or degradative products with different molecular weight from those of mammals; (6) K17 increases in wound keratinocytes and localizes over sparse filaments or small bundles of short filaments, not over tonofilaments joined to desmosomes; and (7) failure of limb regeneration in lizards may not depend on the wound reaction of keratinocytes.     

Differences in blastema-associated proteins according to position along body axis in regenerating planarians

To test the hypothesis that neoblasts in different positions in regenerating pieces of planarians may be differentially responsive to diffusible stimuli through differentially expressed membrane receptors, we compared membrane surface proteins in blastemas induced at various positions along an anterior-posterior axis in Dugesia gonocephala (Dugès). The proteins were biotinylated and identified by molecular weight in SDS-PAGE (sodium-dodecylsulfate polyacrilamide gel electrophoresis). This SDS-PAGE pattern was then compared with that of N-glycosylated proteins incorporating ³H-mannose. One 20 kDa glycoprotein present in all blastemas at 3 d was absent from more anterior blastemas at 6 d irrespective of whether those blastemas were at the cephalic or the caudal end of the regenerating piece. The expression of this protein appears to be determined by the position of the blastema along the body axis rather than by its prospective fate.     

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.