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.     

Development of Regenerative Ability in the Lizard, Lacerta vivipara

Among animals which regenerate, it is usually observed thatyounger forms show greater powers than older ones. It becamepossible to investigate this correlation in lizards after thedevelopment of a culturing technique for the eggs of ovoviviparousreptiles (Panigel, 1956). Moffat and Bellairs (1964) amputatedthe tails of lizard embryos at various stages and examined themat hatching. Only the embryos amputated at near-hatching stagesshowed any regeneration. However, subsequent experiments onthe younger stages have shown that most of the embryos becomeconstricted and even amputated by the healing amniotic and allantoicmembranes. This younger group was therefore re-examined forregenerative ability under conditions where the formation ofconstrictions could be controlled. The results showed that smallasymmetrical outgrowths sometimes arise from a fraction of thestump area. These unusual regenerates were never seen to growlonger than one millimeter, or to differentiate cartilage ormuscle. The results suggest that normal regeneration is a processwhich cannot be elicited until a certain degree of maturityof the tail tissues has been attained, and that it is not necessarilybetter in young individuals or in tissues which are less welldifferentiated.     

Dopamine antagonist speeds up tail regeneration in lizards exposed to continuous darkness: evidence for prolactin involvement

In earlier studies, we demonstrated that continuous light (LL:LD, 24:0) stimulated tail regeneration whereas continuous darkness (DD:LD, 0:24) and pinealectomy depressed the same in the Gekkonid lizard, Hemidactylus flaviviridis, and, furthermore, exogenous prolactin significantly enhanced the regeneration process in lizards kept in 0:24 LD. However, the regeneration process in animals exposed to 24:0 LD was unaffected by the dopamine agonist, bromocriptine. This study with pimozide, an antipsychotic drug, and a potent dopamine receptor antagonist was conducted to ascertain whether the dopaminergic regulation of prolactin release is operative in lizards, as in mammals, and to provide further evidence for prolactin involvement in regenerative growth. Once daily intraperitoneal injection of 50 micrograms/kg pimozide to H. flaviviridis, 5 days prior to tail autotomy and 50 days thereafter, stimulated the regeneration process in lizards exposed to 0:24 LD. The initiation of regeneration, the total length of new growth (regenerate) produced by Day 50, and the total percentage replacement of the lost (autotomized) tails at the end of 50 days of experimentation were all significantly enhanced in pimozide-treated animals as compared with their counterparts injected with 0.6% sterile saline; in fact, better than saline-injected controls exposed to 24:0 LD of 638 lux intensity. The daily growth rate was also enhanced in pimozide-treated lizards. Interestingly, the pattern of regeneration as well as the final regenerate of pimozide-treated lizards were similar to those observed earlier in ovine prolactin-treated animals exposed to similar experimental photoperiodic schedules.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Amnio-allantoic constriction bands in lizard embryos and their effects on tail regeneration

The formation of constriction bands after amputation of the tail has been studied in embryos of the Common lizard ( Lacerta vivipara ) after the eggs have been removed from the mother and placed in culture. The constrictions are formed from the amnion and the inner wall of the allantois; they usually develop within two days after operation. They compress the tail stump and cause necrosis and detachment of its distal portion, simulating the effect of the experimental injury. Similar constriction of the tail followed by auto-amputation occurs if the embryonic membranes are incised but the tail is left intact. In neither case does the tail regenerate after such auto-amputation. Small outgrowths resembling regenerates were formed, however, in certain cases where the tail was amputated under circumstances in which constrictions could not develop. Although amputation of the tail in very late embryos was followed by the appearance of constrictions, these failed to compress the tissues sufficiently to cause subsequent auto-amputation; regeneration of the stump normally took place. The constrictions described are comparable with the amniotic bands alleged to cause congenital amputation of the extremities in man.     

Effects of hypophysectomies and thyroxine replacement upon the initiation of tail regeneration in the lizard, Anolis carolinensis

A histological evaluation of the effects of hypophysectomy and throxine therapy in young tail regenerates was carried out in the small iguanid lizard, Anolis c. carolinensis. Hypophysectomy caused a delay but did not inhibit blastema formation. The growth of the ependyma into the wound region was delayed in hypophysioprivic regenerates by about a week. Growth and differentiation of hypophysioprivic regenerates after blastema formation was variable, ranging from virtually no growth to the formation of a differentiated but very small protuberance. However, actual tail elongation was inhibited by hypophysectomy. In those hypophysioprivic regenerates that did show signs of differentiation, muscle groups were poorly defined, scanty in appearance and not as well differentiated as the cartilage tube. Thyroxine treatment in the young hypophysioprivic regenerates stimulated normal growth and normal appearance and differentiation of promuscle and procartilage aggregates as well as the growth of the ependymal tube into the blastema.     

The vascularization,innervation and myogenesis of early regenerated tail in Gekko japonicus

Journal of Molecular Histology - Many species of lizards are capable of tail regeneration. There has been increased interest in the study of lizard tail regeneration in recent years as it is an...     

Reduced tail regeneration in the Common Lizard, Lacerta vivipara, parasitized by blood parasites

1. Many lizards will lose their tail through autotomy as an antipredator device even though there must be significant costs during tail regeneration.
2. Parasites are energetically costly to the host, and may reduce the rate of cell regeneration. The relation between the presence of haemogregarines (phylum Sporozoa) and the rate of tail regeneration in the Common Lizard Lacerta vivipara (Jacquin) was examined.
3. Experimentally induced autotomy in parasitized lizards resulted in a significantly reduced rate of tail regeneration compared with non-parasitized lizards. On the other hand, tail loss was not associated with an abnormal increase of parasite load, suggesting that the physiological stress (induced by tail loss) did not cause a decrease in parasite defence.     

Role of the lizard Teira dugesii as a potential host for Ixodes ricinus tick-borne pathogens

PCR screening of ticks and tissue samples collected from 151 Teira dugesii lizards seems to indicate a potential role of this lizard species in the maintenance and transmission cycle of some Ixodes ricinus tick-borne agents, such as Rickettsia monacensis, Rickettsia helvetica, and Borrelia lusitaniae, that are circulating on Madeira Island.     

Some morphological and ultrastructural changes in the ependyma of the amputation stump during early regeneration of the tail in the lizard,Anolis carolinensis

Light and electron microscope studies indicate that the old ependyma just proximal to the plane of amputation in early lizard tail regenerates shows a sequence of morphological changes which suggests that it as well as the new ependyma growing into the regenerate may play an active role in the initiation and maintenance of early tail regeneration. The old ependyma close to the plane of amputation undergoes hypertrophy and/or hyperplasia causing a partial closure of the central canal and pseudostratification. Its nuclei shift from an original apical position to a basal one. The ependymal processes become more prominent and extend to the pia, a condition not found more rostrally. There is also a significant increase in the amount of Golgi substance and a moderate increase in the rough endoplasmic reticulum. These observations lead to the thought that these cellular changes may be an expression of enhanced secretion and other activities in the old and new ependyma just proximal or distal to the plane of amputation.     

Appendage regeneration in anamniotes utilizes genes active during larval-metamorphic stages that have been lost or altered in amniotes: The case for studying lizard tail regeneration

This review elaborates the idea that organ regeneration derives from specific evolutionary histories of vertebrates. Regenerative ability depends on genomic regulation of genes specific to the life-cycles that have differentially evolved in anamniotes and amniotes. In aquatic environments, where fish and amphibians live, one or multiple metamorphic transitions occur before the adult stage is reached. Each transition involves the destruction and remodeling of larval organs that are replaced with adult organs. After organ injury or loss in adult anamniotes, regeneration uses similar genes and developmental process than those operating during larval growth and metamorphosis. Therefore, the broad presence of regenerative capability across anamniotes is possible because generating new organs is included in their life history at metamorphic stages. Soft hyaluronate-rich regenerative blastemas grow in submersed or in hydrated environments, that is, essential conditions for regeneration, like during development. In adult anamniotes, the ability to regenerate different organs decreases in comparison to larval stages and becomes limited during aging. Comparisons of genes activated during metamorphosis and regeneration in anamniotes identify key genes unique to these processes, and include thyroid, wnt and non-coding RNAs developmental pathways. In the terrestrial environment, some genes or developmental pathways for metamorphic transitions were lost during amniote evolution, determining loss of regeneration. Among amniotes, the formation of soft and hydrated blastemas only occurs in lizards, a morphogenetic process that evolved favoring their survival through tail autotomy, leading to a massive although imperfect regeneration of the tail. Deciphering genes activity during lizard tail regeneration would address future attempts to recreate in other amniotes regenerative blastemas that grow into variably completed organs.     

Lesion and regeneration in the medial cerebral cortex of lizards.

The cerebral cortex of Squamate reptiles (lizards and snakes) may be regarded as an archicortex or "reptilian hippocampus". In lizards, one cortical area, the medial cortex, may be considered as a true "fascia dentata" on grounds of its anatomy, connectivity and cyto- chemo-architectonics of its main zinc-rich axonal projection. Moreover, its late ontogenesis and postnatal development support this view. In normal conditions, it shows delayed postnatal neurogenesis and growth during the lizard's life span. Remnant neuroblasts in the medial cortical ependyma of adult lizards seasonally proliferate. The late-produced immature neurocytes migrate to the medial cortex cell layer where they differentiate and give off zinc-containing axons directed to the rest of cortical areas. This results in a continuous growth of the medial cortex and its zinc-rich axonal projection. Perhaps the most important characteristic of the lizard medial cortex is that it can regenerate after having been almost completely destroyed. Recent experiments in our laboratory have shown that chemical lesion of its neurons (up to 95%) results in a cascade of events; first, those related with massive neuronal death and axonal-dendritic retraction and, secondly, those related with a triggered neuroblast proliferation and subsequent neo-histogenesis, and the regeneration of an almost new medial cortex that shows itself undistinguishable from a normal undamaged one. This is the only report to our knowledge that an amniote central nervous centre may regenerate by new neuron production and neo-histogenesis. Perhaps the medial cortex of lizards may be used as a model for neuronal regeneration and/or transplant experiments in mammals or even in primates.     

Analytical study of Xenopus hindlimb regenerate with special reference to muscle regeneration

Amputated hindlimbs of Xenopus laevis, develop various types of regenerates in relation with amputation level as well as stage development. The present experiments is an attempt to study the histological characteristics of Xenopus regenerations, i.e., rational changes of tissue components along the length of the regenerated part with special emphasis on the degree of muscle regeneration. Four types of regenerates were studied viz; a 4th toe obtained from a completely restored regenerated limb at 126 days after amputation of limb at base level in stage 51. An amputated limb with no external sign of regeneration of limb at thigh level in stage 60. A spike-shaped regenerate at 96 days after amputation of limb at shank level in stage 63. A spike-shaped regenerate at about 2 years after amputation of limb at shank level in stage 60. Cross sectional areas of muscle, skin gland, epidermis and cartilage in each of the four types of regenerates were measured with Image Analyzing Apparatus (VIP 121 CH, Olympus Co.). The relative area of each tissue was expressed as a percentage of the cross sectional area of the limb. The obtained values were plotted along the length of the regenerate. Digitiform regenerates were found to be more or less similar to the control limbs, i.e., provided joints and muscle, while the heteromorphic spike or rod shaped regenerates were simply provided with cartilaginous axial core without joint formation. Muscle area were reduced rapidly near the amputation area of these heteromorphic regenerates with no more continuation in the regenerated tissue. It is interesting to mention that percentage cartilage area of about 2 years old spike regenerate was higher than that of similar 96 days regenerate. In addition muscle regeneration was completely absent even in such an aged regenerate. The area showed fairly similar ratio irrespective of the external appearance of the regenerate. In 32 regenerates of which limbs were amputated at various developmental stages ranging between stage 51 and adult stage, the histological condition of muscle at the amputation site, were well observed. In all digitated types of regenerates even in those with reduced number of toes, muscles were found grown well in the regenerates. In heteromorphic regenerates without toe formation muscle did not usually regenerate. In few cases, however, a small mass of myoblastic like cells or small aggregation of differentiated muscle cells without any structural continuation with the stump muscles, were seen to develop in the midst of the regenerate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Satellite cells of te dorsal root ganglia in neuronal hypertrophy]

Amputation of the lizard tail is followed by its complete regeneration over a period of six-eight months. The new tail is innervated only by the last three pairs of spinal nerves upstream from the plane of amputation, since no nerve cells are present in the regenerated. The corresponding dorsal root ganglia increase in volume (hypertrophic ganglia) and most of their sensory neurons become hypertrophic. Satellite cells belonging to this hypertrophic ganglia increase in number. This paper describes an autoradiographic study, after administration of tritiated thymidine, of the hypertrophic dorsal root ganglia of the lizard during tail regeneration. We evaluated the number of satellite cells which neo-synthetize DNA ("labeling index = LI%) and are therefore suitable to undergo cell division. The LI% was significatively increased in hypertrophic ganglia when compared to internal control ganglia (not directly involved in the reinnervation process) and normal ganglia (lizards with intact tails). The comparison between internal control ganglia and normal ganglia showed higher LI% values in the formers, although this difference was not statistically significative. These results are in line with those obtained by other authors and suggest that satellite cells of dorsal root ganglia can undergo cellular proliferation also in the adult, especially in particular experimental conditions.     

Morphological and biochemical analyses of original and regenerated lizard tails reveal variation in protein and lipid composition

Caudal autotomy, or voluntary self-amputation of the tail, is a common and effective predator evasion mechanism used by most lizard species. The tail contributes to a multitude of biological functions such as locomotion, energetics, and social interactions, and thus there are often costs associated with autotomy. Notably, relatively little is known regarding bioenergetic costs of caudal autotomy in lizards, though key morphological differences exist between the original and regenerated tail that could alter the biochemistry and energetics. Therefore, we investigated lizard caudal biochemical content before and after regeneration in three gecko and one skink species. Specifically, we integrated biochemical and morphological analyses to quantify protein and lipid content in original and regenerated tails. All lizards lost significant body mass, mostly protein, due to autotomy and biochemical results indicated that original tails of all species contained a greater proportion of protein than lipid. Morphological analyses of two gecko species revealed interspecific differences in protein and lipid content of regenerated lizard tails. Results of this study contribute to our understanding of the biochemical consequences of a widespread predator evasion mechanism.     

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.     

Morphology and frictional properties of scales of Pseudopus apodus (Anguidae,Reptilia)

In the lizard family Anguidae different levels of limb reduction exist up to a completely limbless body. The locomotion patterns of limbless anguid lizards are similar to the undulating and concertina movements of snakes. Additionally, anguid lizards frequently use a third mode of locomotion, called slide-pushing. During slide-pushing the undulating moving body slides on the ground, while the posterior part of the body is pressed against the substrate. Whereas the macroscopic and microscopic adaptations of snake scales to limbless locomotion are well described, the micromorphology of anguid lizard scales has never been examined. Therefore we studied the macro- and micromorphology of the scales of Pseudopus apodus, an anguid lizard with a snakelike body. In addition, we measured the frictional properties of Pseudopus scales. Our data show that the microstructures of the ventral scales of this anguid lizard are less developed than in snakes. We found, however, a rostro-caudal gradient in macroscopic structuring. Whereas the ventral side of the anterior body was nearly unstructured, the tail had macroscopic longitudinal ridges. Our frictional measurements on rough substrates revealed that the ridges provide a frictional anisotropy: friction was higher in the lateral than in the rostral direction. The observed frictional properties are advantageous for a tail-based slide-pushing locomotion, for which a tail with a high lateral friction is most effective in generating propulsion.     

Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.     

Cellular and molecular mechanisms of regeneration in Xenopus

We have employed transgenic methods combined with embryonic grafting to analyse the mechanisms of regeneration in Xenopus tadpoles. The Xenopus tadpole tail contains a spinal cord, notochord and segmented muscles, and all tissues are replaced when the tail regenerates after amputation. We show that there is a refractory period of very low regenerative ability in the early tadpole stage. Tracing of cell lineage with the use of single tissue transgenic grafts labelled with green fluorescent protein (GFP) shows that there is no de-differentiation and no metaplasia during regeneration. The spinal cord, notochord and muscle all regenerate from the corresponding tissue in the stump; in the case of the muscle the satellite cells provide the material for regeneration. By using constitutive or dominant negative gene products, induced under the control of a heat shock promoter, we show that the bone morphogenetic protein (BMP) and Notch signalling pathways are both essential for regeneration. BMP is upstream of Notch and has an independent effect on regeneration of muscle. The Xenopus limb bud will regenerate completely at the early stages but regenerative ability falls during digit differentiation. We have developed a procedure for making tadpoles in which one hindlimb is transgenic and the remainder wild-type. This has been used to introduce various gene products expected to prolong the period of regenerative capacity, but none has so far been successful.