Search Results (14)

Molecules involved in the activation of regeneration in reptiles are almost unknown. MARCK-like proteins are indicated to activate regeneration in some amphibians and fish, and it would be important to know whether this is a general process also present in other vertebrates. To address this problem, the present study reports the immunolocalization of a MARCK-like protein in injured tissues of a lizard. Bioinformatics and immunofluorescence after 5BrdU administration, and detection of MARCK-like proteins, have been performed on regenerating tail and limb of the lizard Podarcis muralis. Transcriptome data indicate up-regulation of MARCKS and MARCK-like1 expression in the initial regenerating tail and limb blastemas, supporting their involvement in the activation of regeneration in both appendages. Immunofluorescence for 5BrdU shows numerous proliferating cells in the blastemas of both appendages. Immunolocalization of a MARCK-like protein, using an antibody generated against a homologous protein from the axolotl, shows that the wound epidermis, nerves, and myotubes accumulate most of the protein in the limb and tail. MARCK-like immunolabeling is also detected in the regenerating spinal cord of the tail. The study indicates that, although the limb later turns into a scar, the MARCK-like protein is also up-regulated in this appendage, like in the regenerating tail. These results indicate that the initial reaction to an injury in lizards, an amniote representative, includes some triggering processes observed in amphibians and fish (anamniotes), with the activation of MARCK-like genes and proteins. This suggests that a MARCK-like-dependant mechanism for tissue repair is likely activated during the initial phases of vertebrate wound healing. Full article

The present, brief review paper summarizes previous studies on a new interpretation of the presence and absence of regeneration in invertebrates and vertebrates. Broad regeneration is considered exclusive of aquatic or amphibious animals with larval stages and metamorphosis, where also a patterning process is activated for whole-body regeneration or for epimorphosis. In contrast, terrestrial invertebrates and vertebrates can only repair injury or the loss of body parts through a variable “recovery healing” of tissues, regengrow or scarring. This loss of regeneration likely derives from the change in genomes during land adaptation, which included the elimination of larval stages and intense metamorphosis. The terrestrial conditions are incompatible with the formation of embryonic organs that are necessary for broad regeneration. In fact, no embryonic organ can survive desiccation, intense UV or ROS exposition on land, and rapid reparative processes without embryonic patterning, such as recovery healing and scarring, have replaced broad regeneration in terrestrial species. The loss of regeneration in land animals likely depends on the alteration of developmental gene pathways sustaining regeneration that occurred in progenitor marine animals. Terrestrial larval stages, like those present in insects among arthropods, only metamorphose using small body regions indicated as imaginal disks, a terrestrial adaptation, not from a large restructuring process like in aquatic-related animals. These invertebrates can reform body appendages only during molting, a process indicated as regengrow, not regeneration. Most amniotes only repair injuries through scarring or a variable recovery healing, occasionally through regengrow, the contemporaneous healing in conjunction with somatic growth, forming sometimes new heteromorphic organs. Full article

The present brief manuscript summarizes the main points supporting recently proposed hypotheses explaining the different distributions of regenerative capacity among invertebrates and vertebrates. The new hypotheses are based on the evolution of regeneration from marine animals to the terrestrial animals derived from them. These speculations suggest that animals that were initially capable of broad regeneration in the sea underwent epigenetic modifications during terrestrial adaptation that determined the loss of their regenerative abilities in sub-aerial conditions. These changes derived from the requirements of life on land that include variable dry and UV-exposed conditions. Terrestrial conditions do not allow for organ regeneration, especially in arthropods and amniotes. Nematodes, the other main metazoan group unable of regeneration, instead evolved eutely (a fixed number of body cells), a process which is incompatible with regeneration. All these changes involved gene loss, modification and new gene interactions within the genomes of terrestrial adapting animals that gave rise to sophisticated invertebrates and vertebrates adapted to living on land but with low cellular plasticity. Full article

Here we report the immunolocalization of mucin, nestin, elastin and three glycoproteins involved in tissue mineralization in small and large juveniles of Neoceratodus forsteri. Both small and larger juvenile epidermis are mucogenic and contain a diffuse immunolabeling for nestin. Sparse PCNA-labeled cells, indicating proliferation, are found in basal and suprabasal epidermal layers. No scales are formed in small juveniles but are present in a 5 cm long juvenile and in larger juveniles. Elastin and a mineralizing matrix are localized underneath the basement membrane of the tail epidermis where lepidotriches are forming. The latter appears as “circular bodies” in cross sections and are made of elongated cells surrounding a central amorphous area containing collagen and elastin-like proteins that undergo calcification as evidenced using the von Kossa staining. However, the first calcification sites are the coniform teeth of the small juveniles of 2–3 cm in length. In the superficial dermis of juveniles (16–26 cm in length) where scales are formed, the spinulated outer bony layer (squamulin) of the elasmoid scales contains osteonectin, alkaline phosphatase, osteopontin, and calcium deposits that are instead absent in the underlying layer of elasmodin. In particular, these glycoproteins are localized along the scale margin in juveniles where scales grow, as indicated by the presence of PCNA-labeled cells (proliferating). These observations suggest a continuous deposition of new bone during the growth of the scales, possibly under the action of these mineralizing glycoproteins, like in the endoskeleton of terrestrial vertebrates. Full article

The ability to heal or even regenerate large injuries in different animals derives from the evolution of their specific life cycles during geological times. The present, new hypothesis tries to explain the distribution of organ regeneration among animals. Only invertebrates and vertebrates that include larval and intense metamorphic transformations can broadly regenerate as adults. Basically, regeneration competent animals are aquatic while terrestrial species have largely or completely lost most of the regeneration ability. Although genomes of terrestrial species still contain numerous genes that in aquatic species allow a broad regeneration (“regenerative genes”), the evolution of terrestrial species has variably modified the genetic networks linking these genes to the others that evolved during land adaptation, resulting in the inhibition of regeneration. Loss of regeneration took place by the elimination of intermediate larval phases and metamorphic transformations in the life cycles of land invertebrates and vertebrates. Once the evolution along a specific lineage generated species that could no longer regenerate, this outcome could not change anymore. It is therefore likely that what we learn from regenerative species will explain their mechanisms of regeneration but cannot or only partly be applied to non-regenerative species. Attempts to introduce “regenerative genes” in non-regenerative species most likely would disorder the entire genetic networks of the latter, determining death, teratomas and cancer. This awareness indicates the difficulty to introduce regenerative genes and their activation pathways in species that evolved genetic networks suppressing organ regeneration. Organ regeneration in non-regenerating animals such as humans should move to bio-engineering interventions in addition to “localized regenerative gene therapies” in order to replace lost tissues or organs. Full article

The adhesive digital pads in some gecko and anoline lizards are continuously utilized for movements on vertical surfaces that may determine wear and a decrease of adhesion efficiency. The pads are formed by lamellae bearing adhesive setae that are worn out following frequent usage and are replaced by new inner setae that maintain an efficient adhesion. Whether the extensive usage of adhesive setae determines a higher shedding frequency in the digital pads with respect to other body regions remains unknown. Setae replacement has been analyzed in embryos and adult lizards using autoradiography and 5BrdU-immunohistochemistry. The observation strongly suggests that during development and epidermal renewal in adult lamellae, there is a shifting of the outer setae toward the apex of the lamella. This movement is likely derived from the continuous addition of proteins in the beta- and alpha-layers sustaining the outer setae while the inner setae are forming. Ultrastructural and in situ hybridization studies indicate that the thin outer beta- and alpha-layers still contain mRNAs and ribosomes that may contribute to the continuous production of corneous beta proteins (CBPs) and keratins for the growth of the free margin at the apex of the lamella. This process determines the apical shifting and release of the old setae, while the new inner setae formed underneath becomes the new outer setae. Full article

The ability to repair injuries among reptiles, i.e., ectothermic amniotes, is similar to that of mammals with some noteworthy exceptions. While large wounds in turtles and crocodilians are repaired through scarring, the reparative capacity involving the tail derives from a combined process of wound healing and somatic growth, the latter being continuous in reptiles. When the tail is injured in juvenile crocodilians, turtles and tortoises as well as the tuatara (Rhynchocephalia: Sphenodon punctatus, Gray 1842), the wound is repaired in these reptiles and some muscle and connective tissue and large amounts of cartilage are regenerated during normal growth. This process, here indicated as “regengrow”, can take years to produce tails with similar lengths of the originals and results in only apparently regenerated replacements. These new tails contain a cartilaginous axis and very small (turtle and crocodilians) to substantial (e.g., in tuatara) muscle mass, while most of the tail is formed by an irregular dense connective tissue containing numerous fat cells and sparse nerves. Tail regengrow in the tuatara is a long process that initially resembles that of lizards (the latter being part of the sister group Squamata within the Lepidosauria) with the formation of an axial ependymal tube isolated within a cartilaginous cylinder and surrounded by an irregular fat-rich connective tissue, some muscle bundles, and neogenic scales. Cell proliferation is active in the apical regenerative blastema, but much reduced cell proliferation continues in older regenerated tails, where it occurs mostly in the axial cartilage and scale epidermis of the new tail, but less commonly in the regenerated spinal cord, muscles, and connective tissues. The higher tissue regeneration of Sphenodon and other lepidosaurians provides useful information for attempts to improve organ regeneration in endothermic amniotes. Full article

Previous studies indicated that Fibroblast Growth Factors (FGFs) are present during tail and early limb regeneration in lizards, but FGFs disappear in the limb that turns into a scar and does not regenerate at 25–40 days post-amputation. Based on these indications, the aim of the present study was to evaluate the influence of administered FGFs on limb regeneration in lizards by injections of FGF1–2 into amputated hind-limbs that were studied between 40 and 70 days post-amputation. Outgrowths of 2.0 to 3.5 mm were produced but they did not develop an autopodium during this period. The skin remained most un-scaled, resembling that of a tail blastema. Four hours before sacrifice, the animals were injected with 5BrdU to study cell proliferation using microscopic and immunofluorescent methods. Histological examination of the outgrowths at 40–70 days of regeneration showed the presence a rod of cartilage (femur), or partially or completely sub-divided into two parts likely corresponding to a tibia and fibula. The regenerated cartilage was in continuity with the transected long bones and was surrounded by a perichondrium and a dense connective tissue, sparse nerves while muscles were reduced or absent. Qualitative observations on 5BrdU-immunolabeling indicated that most proliferating cells were present in the apical wound epidermis, the apical-most perichondrium and in the regenerating scales at 40–60 days post-amputation, but decreased at 70 days. Few 5BrdU-labeled cells were seen in other tissues, including in the regenerated cartilages. The present study indicates that FGF1-2 treatment in lizards mainly stimulate cartilage regeneration and the formation of a thick epidermis with an Apical Epidermal Peg, the epidermal micro-region that favors regeneration. In summary, these results suggest that FGFs treatments on amputated limbs could also be attempted in others amniotes, including mammals. However FGFs are not capable to induce an autopodium, which requires further signaling factors for its formation. Full article

After bone damage, fracture or amputation, lizards regenerate a variable mass of cartilaginous and fibro-cartilaginous tissues, depending from the anatomical site and intensity of inflammation. Aside tail and vertebrae, also long bones and knee epiphyses can regenerate a relative large mass of cartilage after injury. Regeneration is likely related to the persistence of stem cells in growing centers of these bones, localized in the epiphyses of femur, tibia and fibula. The epiphyses form ossified secondary centers in adults but a few progenitor cells remain in the articular cartilage and growth plate, allowing a continuous growth during most lifetime of lizards. The present Review indicates that putative progenitor/stem cells, identified by long labeling retaining of 5-bromo-deoxy-uridine (5BrdU) and immunolocalization of telomerase, remain localized in the articular cartilage and growth plates of the femur and tibia. These cells are re-activated after limited epiphyses damage or amputation of the distal part of the femur or tibia-fibula, and can re-form cartilaginous epiphyses. Regenerating chondrocytes show an intense proliferation and the production of new extracellular matrix components such as collagen VI, chondroitin sulfate proteoglycan, and hyaluronate receptors. The molecular factors at the origin of the chondrogenic potential of the articular cartilage, growth plates, and the periosteum in lizard bones remain to be studied. Full article

The epiphysis of femur and tibia in the lizard Podarcis muralis can extensively regenerate after injury. The process involves the articular cartilage and metaphyseal (growth) plate after damage. The secondary ossification center present between the articular cartilage and the growth plate is replaced by cartilaginous epiphyses after about one month of regeneration at high temperature. The present study analyzes the origin of the chondrogenic cells from putative stem cells located in the growing centers of the epiphyses. The study is carried out using immunocytochemistry for the detection of 5BrdU-labeled long retaining cells and for the localization of telomerase, an enzyme that indicates stemness. The observations show that putative stem cells retaining 5BrdU and positive for telomerase are present in the superficial articular cartilage and metaphyseal growth plate located in the epiphyses. This observation suggests that these areas represent stem cell niches lasting for most of the lifetime of lizards. In healthy long bones of adult lizards, the addition of new chondrocytes from the stem cells population in the articular cartilage and the metaphyseal growth plate likely allows for slow, continuous longitudinal growth. When the knee is injured in the adult lizard, new populations of chondrocytes actively producing chondroitin sulfate proteoglycan are derived from these stem cells to allow for the formation of completely new cartilaginous epiphyses, possibly anticipating the re-formation of secondary centers in later stages. The study suggests that in this lizard species, the regenerative ability of the epiphyses is a pre-adaptation to the regeneration of the articular cartilage. Full article

Cartilage regeneration is massive during tail regeneration in lizards but little is known about cartilage regeneration in other body regions of the skeleton. The recovery capability of injured epiphyses of femur and tibia of lizard knees has been studied by histology and 5BrdU immunohistochemistry in lizards kept at high environmental temperatures. Lizard epiphyses contain a secondary ossified center of variable extension surrounded peripherally by an articular cartilage and basally by columns of chondrocytes that form the mataphyseal or growth plate. After injury of the knee epiphyses, a broad degeneration of the articular cartilage during the first days post-injury is present. However a rapid regeneration of cartilaginous tissue is observed from 7 to 14 days post-injury and by 21 days post-lesions, a large part of the epiphyses are reformed by new cartilage. Labeling with 5BrdU indicates that the proliferating cells are derived from both the surface of the articular cartilage and from the metaphyseal plate, two chondrogenic regions that appear proliferating also in normal, uninjured knees. Chondroblasts proliferate by interstitial multiplication forming isogenous groups with only a scant extracellular matrix that later increases. The high regenerative power of lizard articular cartilage appears related to the permanence of growing cartilaginous centers in the epiphyses of long bones such as those of the knee during adulthood. It is likely that these regions contain resident stem cells that give rise to new chondroblasts of the articular and metaphyseal cartilage during most of the lizard’s lifetime, but can produce an excess of cartilaginous tissues when stimulated by the lesion. Full article

The lumbar spinal cords of lizards were transected, but after the initial paralysis most lizards recovered un-coordinated movements of hind limbs. At 25-45 days post-lesion about 50% of lizards were capable of walking with a limited coordination. Histological analysis showed that the spinal cord was transected and the ependyma of the central canal formed two enlargements to seal the proximal and distal ends of the severed spinal cord. Glial and few small neurons were formed while bridge axons crossed the gap between the proximal and the distal stumps of the transected spinal cord as was confirmed by retrograde tract-tracing technique. The bridging fibers likely derived from interneurons located in the central and dorsal grey matter of the proximal spinal cord stump suggesting they belong to the local central locomotory pattern generator circuit. The limited recovery of hind limb movements may derive from the regeneration or sprouting of short proprio-spinal axons joining the two stumps of the transected spinal cord. The present observations indicate that the study on spinal cord regeneration in lizards can give insights on the permissive conditions that favor nerve regeneration in amniotes. Full article