Amphibian Regeneration Research Articles

Retinoic acid and limb regeneration

A key problem in the study of vertebrate development is to determine the molecular basis of positional value along a developmental axis. In amphibian regeneration, retinoic acid is able to respecify positional value in a graded fashion that is dependent on its concentration. In view of the fact that retinoic acid is a naturally occurring metabolite of vitamin A, this raises the possibility that it is deployed in vivo as an endogenous morphogen. Furthermore, the recent evidence that its effects are mediated by nuclear receptors of the steroid/thyroid hormone superfamily suggests the possibility of understanding the mechanism of its graded effects on morphogenesis. Such insights would be of crucial importance for our understanding of vertebrate patterning along an axis.

Identification of a novel retinoic acid receptor in regenerative tissues of the newt

In urodele amphibians, the progenitor cells that regenerate amputated limbs (known as the blastema) normally replace only the missing structures. After systemic delivery of retinoic acid (RA), more proximal structures are also formed, indicating that RA can control position specification in the proximal-distal axis of the regenerating limb. According to dose and experimental context, retinoids can also re-specify the anteroposterior axis of the limb, induce deletions of skeletal elements, or block re-growth completely. To study the molecular basis of these morphogenetic effects, we screened complementary DNA libraries of newt regenerative tissues (limbs and tails) for hormone nuclear receptors activated by RA. Two functional retinoic acid receptors (RARs) were identified, one of which is the newt homologue of the human alpha-receptor (RAR alpha). The second receptor, called RAR delta, is novel. Sequence analysis suggests that the composite newt RAR previously reported is chimaeric, consisting of 5'RAR-beta-like and 3' RAR delta clones. We conclude that multiple RARs are expressed during limb regeneration in amphibians and suggest that receptor heterogeneity may underlie the different effects of retinoids on limb morphogenesis.

Vitamin A modification of the positional information of blastema cells during limb regeneration in the axolotl Ambystoma mexicanum

Vitamin A is known to cause proximodistal duplication of parts of the limb during limb regeneration in amphibians. The objective of this study was to investigate the nature and location of the cellular changes induced by vitamin A when it causes this duplication in the axolotl Ambystoma mexicanum. When axolotls were treated with retinol palmitate by immersion for 14 days before limb amputation, proximodistal duplications were still observed in subsequent regenerates of limbs amputated after vitamin A treatment was discontinued. This observation suggests that some characteristic of the cells is changed by vitamin A and that exogenous vitamin A need not be present while the limb is regenerating. When a limb that was induced to undergo proximodistal duplication by vitamin A was reamputated 49 days later through the original mid radius–ulna amputation plane, it regenerated a limb of normal structure. A regeneration blastema transplanted from a vitamin A treated axolotl to an untreated axolotl regenerated on the host limb stump, producing a limb with proximodistal duplication; this indicates that the blastema cells underwent some change by the early to mid cone stage, which was expressed later when the blastema redifferentiated into a new limb. Conversely, when an untreated blastema was transplanted onto a vitamin A treated axolotl from which the forelimb blastema had been removed, proximodistal duplications developed. This result is interpreted to mean that the stump cells, although morphologically of the radius–ulna level, were proximalized by the prior vitamin A treatment and still displayed proximal positional values, leading to intercalation of missing proximodistal structures. These results are consistent with the hypothesis that vitamin A brings about a temporary change in the positional information of the limb stump and blastema cells, and that when the vitamin A treatment is discontinued, there is a gradual return to normal positional values over a period of several weeks.

Actinomycin D, cycloheximide, and tunicamycin inhibit vitamin A induced proximodistal duplication during limb regeneration in the axolotl Ambystoma mexicanum

Vitamin A is known to cause proximodistal duplication of parts of the limb during limb regeneration in amphibians. The objective of this study was to investigate the cellular site of action of vitamin A when it causes this duplication in the axolotl Ambystoma mexicanum. Both forelimbs of larval axolotls were amputated through the radius–ulna, and treated with retinol palmitate by immersion for 10 days. Actinomycin D, cycloheximide, and tunicamycin were each applied to the right forelimb by implantation of a silastin block containing one of these antibiotics on days 2 and 6 postamputation. The left limb received a control silastin block at the same time. Actinomycin D and cycloheximide completely blocked the proximodistal duplication caused by retinol palmitate, and tunicamycin substantially inhibited the development of these duplications. These results suggest that retinol palmitate (i) induces duplication by switching on a gene (as it is antagonized by actinomycin D), (ii) requires protein synthesis (as it is antagonized by cycloheximide), and (iii) may effect glycoprotein synthesis (as it is antagonized by tunicamycin).

A test of the punctuated-cycling hypothesis in Ambystoma forelimb regenerates: the roles of animal size, limb innervation, and the aneurogenic condition

Abstract The punctuated-cycling (PC) hypothesis [39] predicts that the proportion of actively cycling (AC) cells within the blastema influences the rate of limb regeneration in urodele amphibians. To test this, we compared the rate of regeneration and the parameters of the PC hypothesis in small and large Ambystoma mexicanum larvae and in aneurogenic limbs of Ambystoma maculatum. Aneurogenic limbs regenerated more slowly than limbs of small axolotls, but considerably faster than limbs of large axolotls. Regardless of regeneration rates, virtually all blastema cells were in the proliferative fraction (Pf) (ranging from 92.3% +4.2% to 96.2% +mn3.4%). As predicted, in the blastemata of more rapidly regenerating small axolotls, 86% of the proliferative fraction was actively cycling, but as regeneration slowed, the proportion of the proliferative fraction that was actively cycling decreased (the AC of aneurogenic limbs being 69.5%, and that of large axolotl limbs being 57.3%) and the proportion of transiently quiescent cells increased. The parameters of the PC hypothesis were also examined in small axolotls at two different times during regeneration. During dedifferentiation and initial blastema formation, 61% of the cells in the proliferative fraction were actively cycling and 34% were transiently quiescent. During the rapid-growth phase of the blastema, 88% of the cells in the proliferative fraction were actively cycling and only 7% of the cells were transiently quiescent. It therefore appears that dedifferentiated cells do not immediately begin active cycling and that the transiently quiescent population is relatively large; however, during the period of rapid growth the proportion of transiently quiescent cells is small. In amputated/denervated limbs of small axolotls, the size of the proliferative fraction decreased as the length of the denervation interval increased. Furthermore, with prolonged denervation the total proportion of actively cycling blastema cells also declined (to about 15%). The failure of denervated limbs to regenerate was correlated with an increased nonproliferative fraction and a reduced proportion of actively cycling cells.

Chronic infusion of agents that increase cyclic AMP concentration enhances the regeneration of mammalian peripheral nerves in vivo

Our previous investigation indicates that forskolin, a robust activator of adenylate cyclase, promotes sensory nerve regeneration in amphibians. The present study was designed to determine if forskolin had a similar effect in mammals. We also wished to test the hypothesis that cyclic AMP modulates nerve regeneration by comparing the effects of chronically infused forskolin with the effects of infused dibutyryl cyclic AMP, 8-bromo cyclic AMP, and the phosphodiesterase inhibitor, theophylline. Our results indicated that all agents promoted some aspect of regeneration. The two which presumably generated the largest increase in cyclic AMP concentration, forskolin and 8-bromo cyclic AMP, had the most profound effect on axonal elongation. All agents decreased the time to sprout initiation, but theophylline produced the largest decrease and its effect was mimicked by caffiene, a methylxanthine with limited ability to inhibit phosphodiesterase. This suggests that sprout formation may be triggered by an increase in intraaxonal free Ca 2+, possibly modulated by cyclic AMP. The role of cyclic AMP in axonal elongation remains to be determined, but may be associated with stimulation of protein synthesis in the nerve cell body.

The handedness and origin of supernumerary limb structures following 180° rotation of the chick wing bud on its stump

ABSTRACT The pattern of differentiated wing structures formed following 180° rotation of the undifferentiated wing bud tip on its base was examined in detail. These analyses were performed to determine the handedness and origin of the supernumerary structures which arise. In contrast to the variable classes of symmetric and/or asymmetric limb anatomies observed following the same operation with amphibian regeneration blastemas, wings of predictable handedness were observed. Both the graft and stump contributed cells to the supernumerary structures. These results are discussed in the light of two current models describing the developing chick limb and analysed diagrammatically within the framework of one of these models, the polar coordinate model.

Neotenic blastemal morphogenesis

Regeneration in arthropods and amphibians follows an analogous principle making comparisons between the two phyla possible.

Putative Immunological Influence upon Amphibian Forelimb Regeneration

The effect on amphibian forelimb regeneration of several substances known to be immunoactive in mammals was examined. Only cobra venom factor and antilymphocyte serum adversely affected the rate of regeneration. Interferon, thymic ‘helper’ (TE-H) and thymic ‘suppressor’ (TE-S) fractions (from calf thymus extracts) increased the frequency of morphologically abnormal regeneration. In addition, TE-S increased while TE-H decreased regenerate volume relative to normal and experimental control regenerates. These data are consistent with the notion that the immune system might be influencing the progress of limb regeneration and that the later stages and events of regeneration might be most sensitive. However, proof that the aberrations produced were the result of actual alterations in immunological activity is not yet available.

Amphibian regeneration and cellular heterochrony

It is posited that the initiating event of amphibian regeneration of a limb, is ‘retrodifferentiation’* of what are to become the developing cells of the blastema. These cells reiterate a larval or premetamorphic ontogenic repertoire, induced by elevated levels of prolactin with adequate innervation. Subsequent redifferentiation of the blastema cells occurs, controlled by thyroxine and innervation.

Cyclic nucleotide metabolism during amphibian forelimb regeneration : II. The protein kinases.

The hypothesis that cAMP mediates neural and endocrine influences on limb regeneration was examined by studying the protein kinases in regenerating limb tissues. Since these enzymes are the vehicles through which cAMP acts intracellularly, an understanding of changes in their concentrations and behaviors during regeneration can be instrumental in elucidating the role of cAMP in this process. Mean activities oscillated throughout regeneration with maximal activities being observed during the mid-late bud stage. The phosphorylation of histone, added to the assay, varied with the stage of regeneration-greatest activity occurring during the early bud stage and very weak activity during the palette and early digital stages. Histone actually appeared to inhibit endogenous phosphorylation during dedifferentiation. In addition, cAMP demonstrated different degrees of enhancement of histone phosphorylation during regeneration-producing its greatest effect at the palette stage and having the least effect at the early bud stage. The results of this study suggest that changes in the absolute amounts of protein kinase are probably not significant in the regulation of regeneration. In addition, the variable acceptability of histone as an exogenous substrate and the variations in the cAMP effects on phosphorylation suggest that physiological changes are occurring in which cAMP might play a significant role. In particular, these data suggest that cAMP might be instrumental in influencing events associated with differentiation and morphogenesis.

Simpler rules for epimorphic regeneration: The polar-coordinate model without polar coordinates

The polar-coordinate model of French, Bryant & Bryant (1976) describes epimorphic regeneration in insects and amphibians, and correctly predicts some surprising phenomena. Their model rests on two main rules. It is shown here that the first of these, the Rule of Intercalation, expresses a requirement that the pattern of positional values of the cells shall be continuous, and that the other, the Rule of the Complete Circle, is not an independent hypothesis, but simply a consequence of the continuity requirement. The formulation of the rules in terms of continuity is coordinate-free and applicable in any number of dimensions: polar coordinates do not have the fundamental significance attached to them by French, Bryant & Bryant (1976), nor are there any grounds to think that the system of positional values in an amphibian limb is two-dimensional. Some simple topology helps to clarify the concepts and prove these points. The phenomena codified by French et al. are to be expected in any epimorphically regenerative organ whose pattern of growth and pattern of positional values are defined by a maintained system of diffusible chemical signals.

Cyclic nucleotide metabolism during amphibian forelimb regeneration : I. The cyclic AMP phosphodiesterases.

Concentrations of the cyclic nucleotides in regenerating limb tissues change in a manner which suggests that they might mediate neural or endocrine influences upon specific developmental events. Since modulation of the role of cAMP within this process can be achieved through cAMP phosphodiesterase, enzymatic activity, relative intracellular distribution, and the kinetic parameters of this enzyme were examined at several stages of limb regeneration in adultNotophthalmus viridescens. Both forms of the phosphodiesterase displayed decreased activity about the time of bud formation. Total phosphodiesterase activity was reduced between 66% and 85% (as compared to intact limbs) between wound healing and palette stages. Relative intracellular distribution (soluble vs. particulate), however, remained essentially constant, 93%-98% soluble for the highK m form and 61%-71% soluble for the lowK m form of the enzyme, throughout this process. The apparentK m of the highK m form increased more than 2-fold during wound healing then fell to approximately 10% (0.7-1.1 μM) of the value of intact limbs (8.3 μM) during dedifferentiation and bud formation. A return to pre-amputational levels was subsequently achieved. In contrast, the apparentK m of the lowK m form increased (from 0.064 to 0.86 μM) during dedifferentiation and began decreasing thereafter. These results are consistent with the hypothesis that one or more mechanisms are operating to modify either the quantity, activity, or physical characteristics of the cAMP phosphodiesterases and that such changes are instrumental in regulating endogenous concentrations of cAMP in limb tissues during regeneration.

Regeneration of ankylosed teeth in the adult frog premaxilla

The relationship between the ankylosed amphibian tooth and regeneration of a jaw segment was studied. A section of the premaxilla was removed in 95 young leopard frogs. Subsequent would healing was observed at intervals of 0-180 days. The dental lamina formed new teeth and by 90 days, teeth in varying stages of development could be seen extending across the wound segment. Teeth within the wound grew to normal size and shape and were replaced by their successors without support of underlying jaw bone which grew in later and often was incomplete.

Rigenerazione e totipotenza

Abstract Regeneration and totipotency.—Regeneration and totipotency of cells and nuclei in plants and animals are discussed. The paper consists of the following sections: regeneration in coelenterates and planarians; limb regeneration in amphibians; regeneration of mammalian liver; Wolffian lens regeneration in urodeles; nuclear transplantation in animal eggs; regeneration of plants from single cells (in vivo; in vitro; from somatic cell hybrids); general remarks.

Mise en évidence du rôle, dans la régénération des Amphibiens, d’une glycoprotéine sécrétée par la cape apicale: étude cytochimique et autoradiographique en microscopie électronique

ABSTRACT Evidence for the role of an apical cap glycoprotein in amphibian regeneration : cytochemical and autoradiographic electron-microscopic studies Early during limb regeneration in the newt, an ectodermal apical cap covering a mesodermal blastema is formed. High-resolution autoradiography of these tissues has been carried out after incorporation of [3H]fucose, which is a precursor of glycoproteins. Autoradiography shows that silver particles are located at first on epithelial cells, then on mesenchymatous cells. This observation is consistent with a hypothesis in which the apical cap would elaborate a glycoprotein acting on the blastema. Substructural autoradiography and cytochemistry also show the importance of cellular surfaces for both cells producing glycoprotein and those which are target cells.

General and selective inhibition of amphibian regneration by vinblastine and dactinomycin.

General and selective inhibition of amphibian regneration by vinblastine and dactinomycin.

The influence of aminopterin on limb regeneration in Ambystoma mexicanum

ABSTRACT During the last twenty-five years a number of authors have studied the influence of chemical substances on limb regeneration in amphibians. Examples of compounds which have been tested so far are: (1) the antimitotic agent, colchicine (Thornton, 1943); (2) the salt, beryllium nitrate (Thornton, 1949, 1950, 1951); (3) the carcinogens, dibenzanthracene and methylcholanthrene (Karczmar & Berg, 1952; Ruben & Balls, 1964); (4) the lathyrus factor, β-aminopropionitrile (Chang, Witschi & Ponseti, 1955); (5) the hormone, thyroxine (Hay, 1956); (6) atropine and other neuropharmacological drugs (Singer, Davis & Scheuing, 1960); (7) the metachromatic dye, toluidine blue (Csaba, Bierbauer & Tôrô, 1961); and (8) semicarbazide, an inhibitor of histamine formation (Deck & Shapiro, 1963). Most of these substances caused growth retardation as well as malformations of the limb regenerates.

Influence of head skin on limb regeneration in urodele amphibians.

Journal of Experimental ZoologyVolume 150, Issue 1 p. 5-15 Article Influence of head skin on limb regeneration in urodele amphibians† Charles Stead Thornton, Charles Stead Thornton Kenyon College, Gambier, OhioSearch for more papers by this author Charles Stead Thornton, Charles Stead Thornton Kenyon College, Gambier, OhioSearch for more papers by this author First published: June 1962 https://doi.org/10.1002/jez.1401500103Citations: 22 † Supported by a grant (B-3193) from the United States Public Health Service. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume150, Issue1June 1962Pages 5-15 RelatedInformation