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.     

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.     

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.     

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.     

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.     

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.     

The cell cycle during amphibian limb regeneration.

The duration of the cell cycle in the blastema of regenerating limbs of axolotls has been measured by means of [3H]thymidine pulse labelling and autoradiography. A chase was required to define the pulse period. An average cell cycle at 20 degrees C takes 53 h, S-phase takes 38 h; including parts of mitosis, G1 is 10 h and G2 is 5 h long. The protracted cycle and S-phase are consonant with the large genome in axolotis and other urodeles. The rapidly growing blastema probably contains a steady population of about 5000 proliferating cells, as there is a regular withdrawal of differentiating cells from the population. The kinds of determination which exist in this population of cells, or are exerted on it, are briefly considered.     

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.     

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.     

Nerves and Proliferation of Progenitor Cells in Limb Regeneration

Nerves, in conjunction with the apical epidermal cap (AEC), play an important role in the proliferation of the mesenchymal progenitor cells comprising the blastema of regenerating urodele amphibian limbs. Reinnervation after amputation requires factors supplied by the forming blastema, and neurotrophic factors must be present at or above a quantitative threshold for mitosis of the blastema cells. The AEC forms independently of nerves, but requires nerves to be maintained. Urodele limb buds are independent of nerves for regeneration, but innervation imposes a regenerative requirement for nerve factors on their cells as they differentiate. There are three main ideas on the functional relationship between nerves, AEC, and blastema cells: (1) nerves and AEC produce factors with different roles in maintaining progenitor status and mitosis; (2) the AEC produces the factors that promote blastema cell mitosis, but requires nerves to express them; (3) blastema cells, nerves, and AEC all produce the same factor(s) that additively attain the required threshold for mitosis.     

Blood vessel formation during tail regeneration in the leopard gecko (Eublepharis macularius): The blastema is not avascular

Unique among amniotes, many lizards are able to self‐detach (autotomize) their tail and then regenerate a replacement. Tail regeneration involves the formation of a blastema, an accumulation of proliferating cells at the site of autotomy. Over time, cells of the blastema give rise to most of the tissues in the replacement tail. In non‐amniotes capable of regenerating (such as urodeles and some teleost fish), the blastema is reported to be essentially avascular until tissue differentiation takes place. For tail regenerating lizards less is known. Here, we investigate neovascularization during tail regeneration in the leopard gecko (Eublepharis macularius ). We demonstrate that the gecko tail blastema is not an avascular structure. Beginning with the onset of regenerative outgrowth, structurally mature (mural cell supported) blood vessels are found within the blastema. Although the pattern of blood vessel distribution in the regenerate tail differs from that of the original, a hierarchical network is established, with vessels of varying luminal diameters and wall thicknesses. Using immunostaining, we determine that blastema outgrowth and tissue differentiation is characterized by a dynamic interplay between the pro‐angiogenic protein vascular endothelial growth factor (VEGF) and the anti‐angiogenic protein thrombospondin‐1 (TSP‐1). VEGF‐expression is initially widespread, but diminishes as tissues differentiate. In contrast, TSP‐1 expression is initially restricted but becomes more abundant as VEGF‐expression wanes. We predict that variation in the neovascular response observed between different regeneration‐competent species likely relates to the volume of the blastema. J. Morphol. 278:380–389, 2017. © 2017 Wiley Periodicals, Inc.     

Autoradiographic observations on developing and growing claws of reptiles

Alibardi, L. 2010. Autoradiographic observations on developing and growing claws of reptiles. —Acta Zoologica (Stockholm) 91 : 233–241 The present qualitative autoradiographic analysis aims to present the main features of morphogenesis and growth of claws in reptiles. Lizard embryos treated with tritiated thymidine reveal that epidermal cell proliferation in terminal digits is prevalent in the dorsal side and gives origin to the curved unguis of the claw. Less proliferation occurs in the ventral side of the digit tip where the concave sub‐unguis is derived. Adult claws of a turtle show that thymidine‐labelled cells are present along most of the epidermis of the claw, especially at the claw tip. Also, injection of tritiated histidine and proline, indicating active protein synthesis, confirm autoradiographic labelling along most of the epidermis of claws, in particular at the apical tip. The present study indicates that proximal matrix regions, as have been described in mammalian nails, are absent in reptiles. This pattern of claw growth probably derives from that of terminal digital scales. In fact reptilian (and avian) claws are formed from a modification of scales, a different condition from that present in mammals.     

Formation of the peripheral nervous system during tail regeneration in urodele amphibians: ultrastructural and immunohistochemical studies of the origin of the cells.

In the regenerating newt tail, epimorphic regeneration--which recapitulates morphologically normal embryonic development--proceeds along a rostrocaudal differentiation gradient. Innervation of the new myomeres results from the spinal roots of segments rostral to the amputation plane and from ventral roots emerging from the lateroventral region of the regenerating spinal cord, in which motor neurons are differentiating. Electron microscopy and an indirect immunofluorescence study with anti-glial fibrillary acid protein (GFAP) confirm that the ventrolateral part of the regenerated ependymal tube gives rise to cells of the ventral root sheath and the spinal ganglia. Anti-GFAP and anti-neurofilament antibodies showed that ependymoglial cells and Schwann cells may play a role in neuronal pathfinding by helping guide and stabilize pioneering axons as they extend toward the myomeres. The carbohydrate epitope NC-1 is expressed in the spinal cord, in sheath cells of the spinal ganglia and in the non-myelin-forming Schwann cells of the peripheral nervous system. L1, a Ca++ independent neural cell adhesion molecule, was detected in the axonal compartments of the regenerating spinal cord, on immature and/or non-myelin-forming Schwann cells within the peripheral nervous system (PNS), and on nerve fibers within the regenerate. These immunohistochemical observations collectively support the hypothesis that Schwann cells already present in the blastema could be involved in organizing neural pathways.