Proximodistal Duplication Research Articles

Retinoic acid receptor regulation of epimorphic and homeostatic regeneration in the axolotl.

Salamanders are capable of regenerating amputated limbs by generating a mass of lineage-restricted cells called a blastema. Blastemas only generate structures distal to their origin unless treated with retinoic acid (RA), which results in proximodistal (PD) limb duplications. Little is known about the transcriptional network that regulates PD duplication. In this study, we target specific retinoic acid receptors (RARs) to either PD duplicate (RA treatment or RARγ agonist) or truncate (RARβ antagonist) regenerating limbs. RARE-EGFP reporter axolotls showed divergent reporter activity in limbs undergoing PD duplication versus truncation, suggesting differences in patterning and skeletal regeneration. Transcriptomics identified expression patterns that explain PD duplication, including upregulation of proximal homeobox gene expression and silencing of distal-associated genes, whereas limb truncation was associated with disrupted skeletal differentiation. RARβ antagonism in uninjured limbs induced a loss of skeletal integrity leading to long bone regression and loss of skeletal turnover. Overall, mechanisms were identified that regulate the multifaceted roles of RARs in the salamander limb including regulation of skeletal patterning during epimorphic regeneration, skeletal tissue differentiation during regeneration, and homeostatic regeneration of intact limbs.

Hedgehog family member is expressed throughout regenerating and developing limbs.

Members of the hedgehog family have been shown to play a key role in many developmental processes, including limb patterning and chondrogenesis. We have therefore investigated whether members of this family are also expressed during regeneration of the adult urodele limb and are regulated by retinoic acid (RA), since this derivative induces proximodistal duplications in regenerating limbs, and has been shown to regulate sonic hedgehog (shh) in the developing limbs of birds and mammals. We report here that a newt homologue of Xenopus banded hedgehog, called N-bhh, is uniformly expressed by mesenchymal blastemal cells from the initial stages of regeneration and is up-regulated by RA. In addition, we show that N-bhh is uniformly expressed in the early limb bud of the newt embryo. Since bhh has not been detected in developing limbs of higher vertebrates, its expression in developing and regenerating newt limbs may be related to the regenerative capability of urodeles.

Retinoic Acid Changes the Proximodistal Developmental Competence and Affinity of Distal Cells in the Developing Chick Limb Bud

In the developing chick limb bud, retinoic acid (RA) has a striking effect on anteroposterior axis formation, resulting in a duplicated pattern of digits. There is no evidence, however, that RA affects proximodistal axis formation in the developing chick limb bud, although RA induces proximodistal duplication in regenerating amphibian limbs. We describe a series of investigations on the effect of RA on the proximodistal axis in the chick limb bud. A RA-containing bead applied to the anterior margin of the chick limb bud at stage 20 induces the anteroposterior duplication of autopodial structures at the wrist level. We found that the RA-treated tissue has the ability to form more proximal structures. When a tissue graft from the RA-treated anterior region was implanted into a stage 17 wing bud (in which the stylopod is developing in the progress zone), the graft produced a humerus, radius–ulna, and digits. When the graft was implanted into a stage 19 wing bud (in which the zeugopod is developing in the progress zone), a zeugopod and digits were formed. These results were associated with changes in the expression of Hox-A genes in the RA-treated grafts, whose domains were reorganized to be similar to those in host tissues 24 h after grafting. When a small graft of RA-treated tissue was implanted into the apex of a stage 19 wing bud, the cells were found in the zeugopod and autopod, whereas cells of control fragments were found only in the autopod region.In vitro,distal cells from different stage limb buds are known to segregate from each other. However, RA-treated stage 24 distal cells did not sort out from stage 20 distal cells and mixed homogeneously. These results suggest that RA induces distal cells to adopt “younger” properties which render them susceptible to forming more proximal patterns under the direction of host signals. The effects of RA on proximodistal patterns in developing chick limb buds appear to differ from its effects on proximodistal patterns in regenerating urodele limbs because RA can induce the proximodistal duplicationin situin the regenerating limbs.

Expression of Regeneration-Associated Antigens in Normal and Retinoid-Treated Regenerating Limbs of Ambystoma mexicanum: (limb regeneration/blastema/retinoid/monoclonal antibody).

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.

The duration of the effectiveness of vitamin A at causing proximodistal duplication in regenerating limbs of the axolotl,Ambystoma mexicanum, in relation to whole body retinoid levels

Urodele amphibians regenerate their limbs after amputation. Normally, only those parts of the limb distal to the plane of amputation are regenerated. However, retinoids are capable of inducing proximodistal (PD) duplication such that structures already present proximal to the plane of amputation are duplicated in the regenerating limb. PD duplication can be induced by retinoids even if amputation occurs some time after the retinoid treatment. Thus, the purpose of this investigation was to determine for how long after administration, retinoids were effective at inducing PD duplication, and to determine whether this persistent effectiveness was due to some change in the cells of the limb or to residual levels of retinoids in the body. Thus, forelimbs of larval axolotls, Ambystoma mexicanum, were amputated at various times in relation to retinol palmitate (RP) treatment to investigate the relationship between the induction of proximodistal duplications and the timing of the retinoid treatment. RP induced PD duplications when amputation was delayed for up to 2 weeks after cessation of the retinoid treatment. To measure the levels of retinoids actually present within the body, axolotls were treated with RP, and whole body retinoid levels were colorimetrically assayed during and after the RP treatment. Since high levels of retinoids were present for several weeks after the cessation of the treatment, we conclude that the induction of PD duplications, when amputation is delayed until after the RP treatment, is due to the persistence of retinoids in the axolotl body. © 1992 Wiley-Liss, Inc.

Effects of tunicamycin on retinoic acid induced respecification of positional values in regenerating limbs of the larval axolotl, Ambystoma mexicanum.

Urodele amphibians possess a remarkable ability to regenerate limbs following experimental or accidental amputation. Since only those parts of the limb distal to the plane of amputation usually regenerate, this suggests the existence of level-specific positional values within the cells of the limb. Vitamin A and other retinoids respecify the positional values of regenerating limbs such that structures proximal to the actual plane of amputation are formed in the regenerating limb producing proximodistal duplications. Regenerating limbs of larval axolotls (Ambystoma mexicanum) treated with sufficient retinoic acid to induce proximodistal duplication were also treated via implantation with tunicamycin, a drug which blocks the synthesis of glycoproteins by blocking N-glycosylation of proteins. Tunicamycin was shown to inhibit the proximalizing effects of retinoic acid. This indicates that asparagine-linked glycoproteins may be essential to the process through which retinoic acid induces these effects in the regenerating limb and that glycoproteins may be responsible for specifying positional values in regeneration blastema cells.

The effect of retinoic acid on positional memory in the dorsoventral axis of regenerating axolotl limbs

We investigated the effect of retinoic acid (RA) on pattern regulation in the dorsoventral (DV) axis of regenerating axolotl limbs. Half and double half dorsal and ventral zeugopodia (lower arms or legs) were amputated through their distal ends, and 4 days later the animals were injected intraperitoneally with 50 (large animals) or 100 (small animals) micrograms RA/g body wt. Half and double half dorsal and ventral zeugopodia of uninjected axolotls, and sham-operated zeugopodia of untreated and RA-treated limbs served as controls. Skeletal patterns and the DV muscle patterns of control and experimental regenerates were then analyzed. Sham-operated zeugopodia of uninjected animals regenerated normally. Sham-operated, RA-treated zeugopodia regenerated normally with proximodistal duplications. Sixty percent of uninjected control dorsal half zeugopodia, 80% of control ventral half zeugopodia, and 100% of control double dorsal and double ventral zeugopodia regenerated distally, but the regenerates did not reconstitute the muscle pattern of the missing half. Thirty-eight percent of RA-treated ventral half zeugopodia and 78% of RA-treated double ventral zeugopodia failed to regenerate distally. Of those cases that did regenerate distally, none regenerated the muscle pattern of the missing half. By contrast, 100% of RA-treated dorsal half zeugopodia regenerated distally and all completed the normal DV muscle pattern. Forty-one percent of RA-treated double dorsal zeugopodia failed to regenerate, but of the remainder that did regenerate, 50% completed the normal DV muscle pattern. These represented eight cases, six of which regenerated single limbs, and two of which regenerated twin limbs, each with a normal DV muscle pattern. We interpret these data to mean that RA ventralizes the positional memory of blastema cells in the DV axis.

Serially duplicated regenerates from the anterior half of the axolotl limb after retinoic acid treatment.

Axolotl (Ambystoma mexicanum) forearms were divided, by an incision between the radius and ulna, to produce anterior and posterior halves. These were prevented from fusing together again by a graft of head skin and amputated through the wrist. This procedure enabled independent regeneration from both halves of the stump. Anterior half stumps produced a single digit while the posterior halves mainly regenerated three digits, the two halves together making a single hand. Treatment with retinoic acid, injected intraperitoneally four days after amputation, abolished regeneration from the posterior half stump and produced proximo-distally duplicated regenerates from the anterior half. The duplicated regenerates had in most cases a complete four digit hand and were therefore more than proximalised regenerates from the anterior side of the limb. Replacement of anterior limb skin with head skin had no effect on the response of the regenerating limb to retinoic acid. In species where application of retinoic acid induces anterior-posterior duplications, these are always derived from the anterior side of the limb. The results presented here show that the morphogenic effects of retinoic acid in inducing proximo-distal duplications are also due to its effects on the anterior tissues of the limb.

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).

Vitamin A inhibits amphibian tail regeneration

The objective of this investigation was to determine what effect vitamin A had on tail regeneration in Notophthalmus viridescens adults, in Ambystoma mexicanum larvae, and in Xenopus laevis tadpoles. Notophthalmus viridescens and Ambystoma mexicanum had their tails amputated and then were treated with retinol palmitate by immersion in concentrations known to cause proximodistal duplications in regenerating limbs. Xenopus laevis tadpoles had their tails amputated and then were treated with either retinol palmitate by immersion, or with retinoic acid administered by implantation of silastin blocks containing retinoic acid. The results ranged from no effect at all at the lower dose levels used, to complete inhibition of tail regeneration at higher dose levels. The degree of inhibition of tail regeneration appeared to be dose dependent. In no case were any duplicated or accessory structures formed analogous to those observed in regenerating limbs. This result suggests that the morphogenetic processes involved in tail regeneration are at least in some ways different from those occurring in limbs, where a similar vitamin A treatment would cause proximodistal duplication or production of accessory limb structures.

Effects of retinoids on regenerating limbs: comparison of retinoic acid and arotinoid at different amputation levels.

Retinoic acid and the synthetic retinoid, arotinoid, were compared for their efficacy in inducing proximodistal (PD) pattern duplication in regenerating axolotl limbs, after amputation through either the distal zeugopodium (lower arm or leg) or distal stylopodium (upper arm or leg). At each level of amputation, the morphology of the duplications produced was the same for both retinoids, and the mean level of proximalization was dose-dependent. Blastema formation was delayed by both retinoids and the delay was associated with regression of the limb stump. Blastemas which produced PD duplication to the stylopodial or girdle level grew out from the stump in a posterior direction. In several zeugopodial regenerates, a partially duplicated, PD-reversed zeugopodium regenerated between the stump cartilages and a completely duplicated zeugopodium distally. Arotinoid was 50 times more effective than retinoic acid in evoking duplication. The dose of arotinoid required to duplicate a stylopodium in a stylopodial regenerate was several times higher than the dose required to duplicate a zeugopodium in a zeugopodial regenerate, suggesting differences either in the sensitivity of zeugopodial and stylopodial cells to retinoid, or in the numbers of positional value specifying these segments.

The effects of two retinoids on limb regeneration in Pleurodeles waltl and Triturus vulgaris

The effects of two vitamin A analogues, retinol palmitate and retinoic acid, on pattern formation during limb regeneration in larvae of two European newts, P. waltl and T. vulgaris are described. The response of the regenerating limb to retinoid treatment differed according to the larval stage of development for P. waltl. Young larval limbs, which were anterior limb buds at the time of amputation, duplicated transversely while limbs of older larvae duplicated proximodistally. Proximodistal duplications were usually limited to the production of supernumerary carpals or a second zeugopod. Complete limbs regenerating from a distal amputation plane were rarely seen. T. vulgaris larvae regenerated limbs with either one or the other type of duplication, but never both on the same limb, at all larval stages tested. When larval P. waltl were kept in normal laboratory light during the treatment with retinol palmitate suspended in the rearing water the percentage of limbs which duplicated was very small for young larvae and increased with the age of the larvae used. Keeping the animals in the dark during the treatment period greatly increased the percentage of duplicate limbs obtained on the young larvae but not on the older larvae. This result is discussed in terms of the photodegradation of the retinoid and the length of the sensitive period for the regenerating limb. A dose-response relationship between the dose of retinol palmitate and either the percentage of limbs duplicated or the degree of duplication was not found. Such a relationship, however, was observed when retinoic acid was injected intraperitoneally into stage-54+ P. waltl larvae. Additionally, this technique revealed a peak of sensitivity to retinoic acid on the 6th day after amputation. Limb regeneration in older larvae was temporarily blocked by retinoid treatment. The limbs showed little or no regression and began blastemal development shortly after the treatment ended. Limbs of young larvae, however, often regressed. Such regressions were followed by blastemal formation and middle- to late-bud blastemas were found at the end of 11 or 14 days treatments with retinol palmitate.

The effects of local application of retinoic acid on limb development and regeneration in tadpoles of Xenopus laevis

Vitamin A can have different effects on developing and regenerating limbs depending on the mode of administration. Previous work has demonstrated the differential effect of retinol palmitate on limb development and regeneration in Xenopus laevis. The purpose of the present investigation was to determine the effects of vitamin A on limb development and regeneration in Xenopus when administered by a local implantation method. Xenopus tadpoles had both hindlimbs implanted with either a block of silastin carrying retinoic acid or an anion exchange resin bead carrying retinoic acid and then the right hindlimb was amputated and the effect of the retinoic acid on limb development and regeneration was studied. The results showed that in developing hindlimbs the effects of silastin implants carrying retinoic acid was to cause skeletal reductions or deletions similar to those induced by immersion of the tadpole in retinol palmitate. On the other hand, in regenerating hindlimbs, the silastin implants caused a range of skeletal reductions and deletions as well as occasional accessory structures but notably induced no proximodistal (PD) duplications, unlike the effect of immersion in retinol palmitate where PD duplications were a common response. Implantation of anion exchange resin beads carrying retinoic acid had no significant effect on either development or regeneration beyond stage 50, presumably because the dose of the retinoic acid was so low. Thus the results suggest that the mode of administration of vitamin A has a very significant influence on its effects. The significance of this observation for vitamin A experiments on limbs is discussed.

Comparison of the effects of vitamin A on limb development and regeneration in the axolotl, Ambystoma mexicanum

The objective of this investigation was to compare the effect of vitamin A on limb development and limb regeneration in the same animal, at the same time, thus eliminating the possibility that species differences or different rates of uptake between animals would influence the results. Axolotl larvae had both right limbs amputated and then were treated with retinol palmitate by immersion at 60 or 300 mg l-1 for 4 or 10 days. Intact left developing limbs at the cone, two-digit, or four-digit stages responded to the treatment by deletion of skeletal elements producing hypomorphic limbs. Severity of the deletions was correlated with higher dose, longer times, and earlier stages of limb development. In contralateral right regenerating limbs, the effect of the same treatment was to cause various degrees of proximodistal duplication as well as occasional hypomorphic regenerates. Thus, there is a marked difference in response to vitamin A between developing and regenerating limbs. The implications of this observation are discussed especially with respect to the underlying morphogenetic mechanisms.

The characteristics of local application of retinoic acid to the regenerating axolotl limb

The work described here is concerned with the effects of local application of retinoic acid to the regenerating limb, administered in silastin blocks. Using 14 C RA we have shown that its rate of diffusion into medium was the same as into regenerating limbs and that the curve of percent loss shows a fast rise tailing off to a plateau. RA caused specific alterations in the proximodistal axis of the regenerate such that complete limbs could be produced from distal amputation planes. Increasing concentrations of RA caused regeneration to commence from increasingly more proximal levels. The effect of time of administration after amputation on proximodistal duplication has been investigated as well as position effects with silastin blocks placed either at the anterior, posterior or dorsal poles of the blastema. These data permitted an estimate of the absolute amount of RA needed to cause alterations in the proximodistal axis −2 to 16 μg per limb or 1 ng per cell. Supernumerary limbs were also induced by these local implants and here there was a distinct position effect with the dorsal side causing the highest frequency. This reveals a possible effect of RA on the anteroposterior axis of regenerating limbs, but some of these supernumeraries were thought to arise from the irritant action of RA rather than a specific effect on pattern formation. The relevance of these results to those obtained on other systems is discussed.

Stage-dependent effects of retinoic acid on regenerating urodele limbs

Following amputation through the distal zeugopodium, regenerating limbs of larvalAmbystoma mexicanum and pre and post-metamorphicPleurodeles waltlii were treated with 150 μg of retinoic acid (RA) per gram of body weight, at the dedifferentiation, early bud, medium bud, late bud or early redifferentiation stages of regeneration. The effect of RA on regenerate morphogenesis differed as a function of the stage at which it was administered. When given during dedifferentiation or at early bud stages, RA evoked proximodistal duplications of stump segments in the regenerates. The maximum duplication index (DI) inAbystoma was achieved when RA was injected at 4 days post-amputation, which corresponds to the stage of dedifferentiation; and inPleurodeles at 10 days post-amputation, which corresponds to a stage midway between early bud and medium bud. When RA was administered at later stages, the DI declined progressively to zero or nearly zero by the stage of early redifferentiation in both species. The decline in DI was due to a decreased frequency of duplication, not to a decrease in the magnitude of duplication in individual regenerates. At the same time, there was an increase in hypomorphism and aberrant morphogenesis of both duplicating and non-duplicating regenerates. These results indicate that regenerative cells are differentially sensitive to RA in a stage-dependent way.

Retinoic acid-induced pattern duplication in regenerating urodele limbs

The effects of varying doses of retinoic acid on forelimb regeneration in larval Ambystoma mexicanum amputated through the wrist joint and in adult Notophthalmus viridescens amputated through the basal carpals were compared. In both species, the major effect of retinoic acid was to cause the proximodistal duplication, in the regenerate, of stump segments proximal to the amputation plane. Transverse axial duplications (anteroposterior and dorsoventral) occurred in a smaller percentage of cases; these consisted of cartilage spurs in axolotls, and extra digits in newts. The frequency and magnitude of the proximodistal and (in the newt) transverse duplications were dose dependent, and the regenerating limbs were maximally sensitive to the retinoid during the period of dedifferentiation and accumulation of blastema cells. The effect of retinoic acid is exerted on cells local to the amputation surface, as shown by the fact that retinoic acid caused the proximodistal duplication of stump segments in regenerates derived from amputated distal lower arm segments grafted to the eyesocket.

Does vitamin A act on pattern formation via the epidermis or the mesoderm?

AbstractTo determine whether the effect of vitamin A on pattern formation in the regenerating axolotl limb was due to a change in the blastemal mesoderm or the epidermis, tissue grafts were exchanged between normal and retinoic acid‐treated animals. The results were conclusive. The combination of treated epidermis and untreated mesoderm resulted in normal regenerates. The combination of treated mesoderm and untreated epidermis produced regenerates that were serially duplicated in the proximodistal axis just as the controls were. We may, therefore, conclude that vitamin A acts on the mesodermal cells of the blastema in causing proximodistal duplications.