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.     

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.     

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.     

Immunolabelling for RhoV and actin in early regenerating tail of the lizard Podarcis muralis suggests involvement in epithelial and mesenchymal cell motility

Immunolabelling for RhoV and actin in early regenerating tail of the lizard Podarcis muralis suggests involvement in epithelial and mesenchymal cell motility. Acta Zoologica, Stockolm. Immunolabelling for RhoV and α‐smooth muscle actin, genes that are highly expressed in the regenerating tail of lizards, shows that a main protein band immunolabelled for RhoV is seen at 65–70 kDa and only a weak band at 22–24 kDa. This suggests that alteration occurred during extraction or is due to biochemical processing of the protein. RhoV immunolabelled cells are present in apical and proximal regenerating epidermis during scale neogenesis. The apical ependyma is labelled but labelling fades and disappears in medial‐proximal regions, near the original spinal cord. Differentiating muscles and cartilage show low labelling. Ultrastructural immunolocalization of RhoV in wound keratinocytes shows labelling in regions containing actin filaments that associate with tonofilaments and desmosomes while a low labelling is present in mesenchymal cells. Filamentous regions of the nucleus, nuclear membrane and the nucleolus are immune‐labelled for RhoV. Similar localization is seen for actin that is present along the perimeters of keratinocytes associated with tonofilaments, in elongations of mesenchymal cells, in muscle satellite cells, endothelial and pericytes of blood vessels. It is suggested that RhoV and actin are associated in the dynamic cytoskeleton needed for the movements of epidermal and mesenchymal cells and in endothelial cells forming new blood vessels.     

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 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.     

Immunolocalization of c‐myc‐positive cells in lizard tail after amputation suggests cell activation and proliferation for tail regeneration

After tail amputation in lizard, a regenerative response is elicited leading to the formation of a new tail. The stimulation of the proliferation process may involve the proto‐oncogene c‐myc. The immunocytochemical analysis detects the c‐myc protein few days after wound in free cells accumulating over the injured tissues of the tail stump. Western blot detects a protein band at 68–70 kDa that is more intense in the regenerating blastema than in normal tail tissues. Nuclei positive for the c‐myc protein are seen in mesenchymal‐like cells located among muscles, connectives and fat tissues of the tail stump 4 days postamputation. Proliferating cells labelled for 5BrdU are seen at 4 days postamputation and are sparse in the mesenchyme of the regenerating blastema formed at 12 days postamputation. Fine immunolocalization of the c‐myc protein shows it is mainly located over euchromatin or poorly condensed chromatin to indicate gene activation. The study correlates the detection of the c‐myc protein with activation of cell division in the injured tissues leading to the formation of the regenerative blastema. The lizard c‐myc protein probably activates a controlled proliferation process through a mechanism that can give information on the uncontrolled process occurring in cancer.     

Fine observation on nerves colonizing the regenerating tail of the lizard Podarcis sicula

During the regeneration of lizard tail, nerves sprouting from ganglia and the spinal cord invade the blastema as far as the apical epidermis. Electron microscopical observations reveal axons storing dense granules (dg) and dense core vesicles (dcv) which are concentrated in nerve terminals or in axoplasmatic regions. In the regenerating spinal cord (SC) these terminals resemble aminergic-peptidergic endings and grow as far as the distal portion of the SC, which is made up of irregularly arranged ependymal cells. Some axons storing dcv contact blastematic cells and other nerve terminals show a plasma membrane incomplete or broken. Whether this latter aspect is due to fixation artifacts or physiological rupture is unknown. Nerves containing dcv and a few dg also originate from spinal ganglia innervating the regenerating tail. The accumulation of material into these endings is probably slow and a possible trophic influence on the regeneration of lizard tail is discussed.     

Tail regeneration in the gecko Sphaerodactylus argus shows that the formation of an axial elastic skeleton is functional for the new tail

Tail regeneration in the gecko Sphaerodactylus argus shows that the formation of an axial elastic skeleton is functional for the new tail (Acta Zoologica, Stockolm). The present autoradiographic and immunohistochemical study describes tail regeneration and formation of the axial skeleton in early regenerating tails of the Jamaican red-tailed gecko, Sphaerodactylus argus. Cell proliferation, studied by tritiated thymidine, shows intense labelling mainly in forming scales and differentiating cartilaginous, muscle and ependymal cells of the regenerating spinal cord, while the labelling is more diffuse in the apical blastema and proximal connective tissues. The slow apical proliferation maintains the tail front growing while in more proximal regions, cells initiate differentiation, losing thymidine-labelling. Cell proliferation is maximal at the beginning of scales, muscles and cartilage formation. Scales are regenerated following migration into the dermis of tritiated thymidine-labelled keratinocytes to form epithelial pegs that later split and give rise new scales. Differentiation of new corneous layers begins underneath the external corneous epidermis, starting with a shedding layer followed by a beta-layer that accumulates corneous beta proteins. Intense proliferation of apical myoblasts gives rise to long myotubes and segmented muscles. The vertebral column is substituted with a cartilaginous tube made of turgid chondrocytes accumulating chondroitin sulphate proteoglycan and elastin. Therefore, the regenerated tail remains flexible and capable of curling to maintain efficient the climbing ability in these geckos.     

The regenerating tail of lizard transits through a tumour-like stage represented by the regenerative blastema

Review. The regenerating tail of lizard transits through a tumour-like stage represented by the regenerative blastema. Acta Zoologica (Stockolm). Molecular studies on lizard tail regeneration indicate that the blastema stage is a tumour-like outgrowth capable of self-regulate to produce a new tail. Various oncogenes and tumour suppressors are expressed, and their proteins are localized in specific regions of the growing blastema. SnoRNAs are exclusively overexpressed in the tail blastema suggesting changes in ribosome translation efficiency in blastema cells, like in cancer. Blastema cells secrete high levels of hyaluronate and adopt an anaerobic metabolism (Warburg effect). These studies indicate that the lizard blastema represents a unique case among terrestrial vertebrates of physiological tumour remission. Mesenchymal cells and fibroblasts forming the blastema are turned within 1–2 months into a functional organ, the tail. In vitro studies on isolated mesenchymal cells from the regenerative blastema shows that these cells do not undergo contact inhibition but continue proliferation after confluence, and contain nestin, vimentin and K17. After 2–3 weeks they stratify into 5–7 layers forming a pellicle of loose connective tissue. Future molecular studies on genes and proteins that allow the control of growth in the lizard blastema may help to determine how lizards turn a tumour into a new organ with numerous differentiated and functional tissues, providing clues on cancer growth regulation.     

Epidermal Growth Factor and EGF Receptors are mainly expressed in the wound epidermis and proliferating ependyma of the regenerating tail of lizards

The presence of EGF and its receptor during tail regeneration in lizard has been assessed by immunoblotting and immunofluorescence to test whether this growth factor may be involved in the process. Immunolabelled bands at 8 and 42–46 kDa for EGF are detected in the regenerating tail. A main band at 45–50 kDa and other weaker bands at lower or higher molecular weight for the EGF receptor are also present. The results indicate that degraded forms of the protein are present although the specific nature of the different bands could not be determined. Immunofluorescence indicates that EGF-labelled cells and EGF receptor are especially seen in the wound epidermis and in the cytoplasm of ependymal cells. Numerous basal keratinocytes of the wound epidermis and apical epidermal peg contain labelled nuclei for EGFR, suggesting that activated receptor stimulates intense cell proliferation of the wound epidermis. Blastema and labelled myoblasts are occasionally detected in early differentiating muscles, but almost no labelled chondroblasts are present in the differentiating cartilaginous tube. The study indicates that EGF and its receptor are mainly present in epithelial cells in a form that allows them to regulate proliferation during tail regeneration.     

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.     

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.     

Prostaglandin Metabolism during Cell Aggregation in the Regenerating Vertebrate Appendage

Prostaglandin metabolism during cell aggregation period was studied in the regenerating tail of the house lizard. On the basis of scanning electron microscopy it was observed that similar kinds of cells in the blastema aggregate to form promuscie aggregate and procartilage aggregate on the 13th day of tail regeneration. In order to understand the prostaglandin metabolism the following parameters were analysed. Fatty acid composition of phospholipids and free fatty acids analysed by gas chromatography. The activity of two rate limiting enzymes-phosholipase A and C, and the activity of the enzymes which are responsible for the oxygenation of polyunsaturated fatty acids-lipoxygenase and cycloxygenase were also estimated. The characterization of the endogenous prostaglandins were carried out by high performance liquid chromatography. On the basis of the above investigations, we observed an increase in phospholipase C activity and resultant increase in free arachidonic acid level. High activity of cycloxygenase and presence of prostaglandin E2 (PGE2) were also observed PGE2 was reported to stimulate cAMP production and resultant cell differentiation. These observations suggest the involvement of prostaglandin metabolism during cell aggregation period in the regenerating blastema and resultant cytodifferentiation of blastemal cells.     

Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

Background

Epimorphic regeneration results in the restoration of lost tissues and structures from an aggregation of proliferating cells known as a blastema. Among amniotes the most striking example of epimorphic regeneration comes from tail regenerating lizards. Although tail regeneration is often studied in the context of ecological costs and benefits, details of the sequence of tissue-level events are lacking. Here we investigate the anatomical and histological events that characterize tail regeneration in the leopard gecko, Eublepharis macularius.

Results

Tail structure and tissue composition were examined at multiple days following tail loss, revealing a conserved pattern of regeneration. Removal of the tail results in a consistent series of morphological and histological events. Tail loss is followed by a latent period of wound healing with no visible signs of regenerative outgrowth. During this latent period basal cells of the epidermis proliferate and gradually cover the wound. An additional aggregation of proliferating cells accumulates adjacent to the distal tip of the severed spinal cord marking the first appearance of the blastema. Continued growth of the blastema is matched by the initiation of angiogenesis, followed by the re-development of peripheral axons and the ependymal tube of the spinal cord. Skeletal tissue differentiation, corresponding with the expression of Sox9, and muscle re-development are delayed until tail outgrowth is well underway.

Conclusions

We demonstrate that tail regeneration in lizards involves a highly conserved sequence of events permitting the establishment of a staging table. We show that tail loss is followed by a latent period of scar-free healing of the wound site, and that regeneration is blastema-mediated. We conclude that the major events of epimorphic regeneration are highly conserved across vertebrates and that a comparative approach is an invaluable biomedical tool for ongoing regenerative research.