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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Microscopical observations on the regenerating tail in the tuatara Sphenodon punctatus indicate a tendency to scarring,but also influence from somatic growth

The process of tail regeneration in the tuatara (Sphenodon punctatus) is not entirely known. Similarity to and differences from lizard tail regenerations are indicated in the present histological and ultrastructural study. Regeneration is influenced by the animal's age and ambient temperature, but in comparison to that of lizards it is very slow and tends to produce outgrowths that do not reach the length of the original tail. Although microscopically similar to lizard blastemas, the mesenchyme rapidly gives rise to a dense connective tissue that contains few muscle bundles, nerves, and fat cells. The unsegmented cartilaginous tube forming the axial skeleton is not calcified after 5 months of regeneration, but calcification in the inner region of the cartilage, present after 10 months, increases thereafter. Amyelinic and myelinic peripheral nerves are seen within the regenerating tails of 2–3 mm in length and the spinal cord forms an ependymal tube inside a cartilaginous casing. Tissues of the original tail, like muscles, vertebrae and the adipose mass, are largely replaced by dense connective tissue that occupies most of the volume of the new tail at 5 and 10 months of regeneration. It is unknown whether the differentiation of the dense connective tissue is caused by the relatively low temperature that this species lives under or stems from a genetic predisposition toward scarring as with most other amniotes. Increases of muscle and adipose tissues seen in older regenerated tails derive from somatic growth of the new tail in the years following tail loss and not from a rapid regeneration process like that in lizards.     

The growth and differentiation of the regenerating spinal cord of the lizard, Anolis carolinensis

The present work describes the ultrastructure of the spinal cord in the regenerating tail of the lizard, Anolis. The distal growing region of the tail contains the advancing ependymal tube which is relatively devoid of axons but already contains channels between ependymal cell processes which anticipate their ingrowth. More proximally, fascicles of naked axons having their origin in the stump are present in the ependymal channels. Therefore, the pattern of fiber regeneration in the spinal cord is prescribed by the ependyma and not by the growing axons. Details of the ultrastructure of proximal, intermediate, and distal regions of the regenerate are reported. Particular attention is paid to the structure and differentiation of the ependymal cells and the relation of the ependyma to other glial cells, to nerve fibers, and to meningeal tissues.