Effects of an augmented nerve supply on forelimb regeneration in the adult mud frog, Rana rugosa.

Forelimbs of the adult mud frog Rana rugosa, when amputated midway through the zeugopodium, regenerate heteromorphically. The resulting regenerative outgrowths were mostly rod shaped and consisted of a cartilaginous core, in which the base was ossified, and muscle elongated distally along the cartilage, the whole being covered by connective tissue and skin. The tip of the regenerating muscle reached a point distally about one third of the length of the regenerative outgrowths. When the innervation of forelimb stumps was augmented by surgical diversion of the ipsilateral sciatic nerve, the amputated limbs regenerated mostly as spatula-shaped outgrowths, which were longer than those of normally innervated forelimbs. Such hyperinnervated regenerates exhibited less ossification of cartilage, or sometimes none at all. However, the regeneration of muscle was more extensive. That is, it reached more than half way along the regenerative outgrowth. Furthermore, denervation resulted in the absence of regeneration in all cases examined. These results clearly indicate that limb regeneration in Rana rugosa is dependent upon the degree of innervation, not only for the early stages of regeneration, but also for the growth and differentiation of the regenerative outgrowth.     

Forelimb regeneration in the postmetamorphic bullfrog: stimulation by dimethyl sulfoxide and retinoic acid

To examine the effects of retinoic acid and dimethyl sulfoxide on regenerative ability of anuran amphibians, the left forelimbs of 60 postmetamorphic froglets of Rana catesbeiana (bullfrogs) were amputated through the distal zeugopodium. Fifteen of the froglets had their left forelimb stumps immersed in dimethyl sulfoxide (DMSO) for 3 minutes, once immediately after amputation and once on each of 5 subsequent days. Another 15 frogs had their left forelimb stumps immersed in a 0.01 M solution of retinoic acid dissolved in DMSO for the same period of time. The remaining 30 control froglets did not regenerate structures distal to the amputation surface, while all limbs in both treated groups produced regenerates by 120 days postamputation. Regenerates of limbs treated with both DMSO alone and DMSO combined with retinoic acid, although hypomorphic, were composed of multiple cartilage elements, which in many cases (46.7%) were organized as patterns partially resembling the skeletal arrangement of a normal forelimb. All of these regenerates exhibited bundles of striated muscle. In addition, nearly half (46.7%) of the regenerates in the DMSO + retinoic acid group possessed two separate regenerate outgrowths. The results demonstrate that young bullfrogs (Rana catesbeiana) possess a latent epimorphic regenerative capability, which can be stimulated by topical application to the wound surface of DMSO alone or DMSO combined with retinoic acid.     

The failure of double-half forelimbs to undergo distal transformation following amputation in the axolotl, Ambystoma mexicanum

Although capable of initiating early regenerative responses, axolotl forelimb stumps which are composed of double-half limb tissues fail to undergo the events that normally lead to the replacement of missing parts. In the present study, the posterior halves of right forelimbs were exchanged with the anterior halves of left forelimbs, or the dorsal halves of right forelimbs were exchanged with the ventral halves of left forelimbs. Forelimbs were amputated through the graft region 30 days after grafting. Limb stumps bearing double-dorsal, double-ventral or double-posterior tissues either produced hypomorphic regenerates or failed to form any externally visible outgrowth. When the limb stump bore double-anterior tissues, no externally visible structures were formed. Normal and multiple regenerates were never formed by double-half limbs. These results are discussed in terms of the polar coordinate model and suggest that the regeneration blastema requires a complete circumference of positional values in order to complete distal transformation.     

Regenerative response of amputated forelimbs of Xenopus laevis froglets to partial denervation

Xenopus laevis froglet forelimbs normally respond to amputational injury by forming a heteromorphic cartilaginous rod-shaped outgrowth. However, partial denervation of a forelimb by ablation of the N. radialis or the N. ulnaris, followed in 2 days by amputation through the mid radius-ulna, results in a size deficiency of the regenerative outgrowth 14 and 21 days postamputation. The decreasing quantity of forelimb innervation, as a result of partial denervation by 55 or 45%, apparently has a graded effect on the cell population and on the extent of cartilage development in the outgrowth. As a consequence of amputational injury, a nerve independent response of the periosteum was also found. This response produced considerable thickening in the periosteum and was due to cell proliferation in both the control and denervated cases.     

Cellular behavior in the anteroposterior axis of the regenerating forelimb of the axolotl, Ambystoma mexicanum

Cellular behavior along the anteroposterior axis of the regenerating axolotl forelimb was studied by use of triploid (3N) tissue grafted into diploid (2N) hosts and three-dimensional computer reconstructions. Asymmetrical upper forelimbs were surgically constructed with one half (anterior or posterior) 3N and the other half 2N. Limbs were amputated immediately after grafting or were permitted to heal for 5 or 30 days prior to amputation. When regenerates had attained the stage of digital outgrowth, the limbs were harvested and sectioned in the transverse axis for histological analysis. When all limbs bearing anterior grafts were considered as a group, 77% of the 3N mesodermal cells were observed in the anterior side of the regenerates and 23% were located in the posterior side of the regenerates. When all limbs bearing posterior grafts were considered as a group, 76% of the 3N mesodermal cells were found in the posterior side of the regenerate and 24% had crossed into the anterior side. Healing times of 0, 5, or 30 days prior to amputation had no effect on the experimental outcome. Three-dimensional computer reconstructions revealed that most 3N cells of mesodermal origin underwent short-distance migration from anterior to posterior or from posterior to anterior and intermixed with diploid mesodermal cells near the midpoint of the regenerated anteroposterior axis. Some 3N cells were observed at greater distances from the graft-host interface. By contrast, labeled epidermal cells from both anterior and posterior grafts exhibited long-distance migration across all surfaces of regenerated limbs. Details of a computer-assisted reconstructive method for studying the three-dimensional distribution of labeled cells in tissues are presented.     

Changes in polyamine content during limb regeneration in adult Xenopus laevis

Polyamine contents in the regenerates were determined at various stages after amputation of the forelimbs of the adult female Xenopus laevis. Putrescine, spermidine, spermine, and sym-homospermidine were detected in all the specimens examined. Cadaverine was detected only in a limited number of samples. At 5 days after amputation of forelimbs, well before the formation of regenerates, the putrescine content in the stump tissues increased, followed by the increase in spermidine content. The putrescine level in the forelimb regenerates was highest between 30 and 50 days after amputation, and then decreased. The spermidine concentration in the regenerates was about 20 times greater than that in intact forelimbs all throughout the experiments. The concentration of spermine was initially lower than that of both putrescine and spermidine and further decreased soon after amputation. The concentration of sym-homospermidine was originally very low and increased slightly during regeneration. The significance of these results, with respect to the function of polyamines in forelimb regeneration of Xenopus laevis, is discussed.     

The effect of healing time on the proximodistal organization of double-half forelimb regenerates in the axolotl,Ambystoma mexicanum

The effect of healing on the proximodistal organization of regenerates from double-half forelimbs was studied. Double-anterior and double-posterior upper forelimbs were prepared surgically and amputated at 5, 10, 15, 20, 30, and 60 days after grafting. All experimental groups regenerated hypomorphic skeletal patterns. Double-half forelimbs amputated at Days 5 and 10 regenerated more distally complete skeletal patterns than did limbs amputated at Days 30 and 60. The mean numbers of skeletal elements regenerated were seen to decrease as a function of time after grafting, with the maximal suppression of skeletal patterns observed to occur when limbs were amputated 30 days following grafting. There was no appreciable difference between limbs amputated at Days 30 and 60. These results suggest that healing time has a profound effect on the proximodistal organization of limbs regenerated from double-half forelimb stumps.     

Nerve-dependent and -independent events in blastema formation during Xenopus froglet limb regeneration

Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during blastema formation. We also investigated the lateral wound healing in comparison to blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the blastema were affected by denervation, suggesting that these events specific for blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.     

Caspase inhibition supports proper gene expression in ex vivo mouse limb cultures

We standardized conditions for ex vivo mouse limb culture to study cartilage maturation and joint formation. We compared 12.5 d.p.c. mouse forelimbs that were cultured either mounted or freely rotating for up to 72 h. Limb outgrowth progressed ex vivo at a variable rate as compared to its development in vivo, spanning approximately 48 h. Although cartilage maturation and joint formation developed grossly normal, aberrant expression of skeletal marker genes was seen. Interestingly, no regression of the interdigital webs took place in mounted cultures, in contrast to limited webbing under freely rotating conditions. Caspase inhibition, by addition of zVAD-fmk to the culture medium of freely rotating limbs, supported proper gene expression associated with skeletal development, and prevented interdigital web regression. Taken together, a freely rotating ex vivo culture for mouse limb outgrowth that is combined with caspase inhibition provides a good model to study cartilage maturation and joint formation.     

Positional information in the forelimb of the axolotl: experiments with double-half tissues

It has been demonstrated recently that upper forelimbs of axolotls comprised of symmetrically arranged soft tissues do not regenerate (P. W. Tank, 1978,J. Exp. Zool.204, 325–336). These double-half forelimb stumps contained skin, muscle, and loose connective tissues in symmetrical arrangement. The present study explores the roles of muscle, skin, and epidermis in the regeneration of double-half forelimbs by grafting them separately to create forelimb stumps bearing symmetrical arrangements of these individual tissues. Forelimb stumps bearing symmetrically arranged flexor and extensor muscles and normally arranged skin underwent complete regeneration (96%). Forelimbs comprised of double-half skin overlying normally arranged muscles and deep tissues formed hypomorphic structures and nonregenerates (56%) with some single and multiple regenerates. Limbs with double-half deep tissues and complete epidermis either regenerated distally incomplete patterns (47%), single patterns (33%), or multiple patterns (20%). Those forelimbs comprised of double-half skin and no muscle regenerated incomplete patterns in the majority of cases (56%) but single and multiple limbs also were formed. Based on these results it can be concluded that no single type of tissue is solely responsible for the regenerative failure experienced by double-half forelimbs in the earlier study. The complete failure of forelimb regeneration occurs only when all types of soft tissues tested (skin, muscle, and deep connective tissues) are present in symmetrical arrangement.     

Ability of various injuries to promote resorption of denervated, nerve independent regenerates of adult newts

Young blastemas of the newt resorb if the limb is denervated, and are thus called "nerve dependent". Late bud and later stage regenerates are termed "nerve independent" because, while denervation inhibits their growth, they proceed through differentiation to form a normally patterned regenerate. Schotté and Liversage ('59) found that reamputation of a denervated nerve independent regenerate causes it to resorb. The present study asked whether injuries of varying severity are equally effective at promoting resorption. Newt forelimbs were amputated through the mid-radius/ulna. At nerve independent stages, the regenerates were denervated and injured in one of a variety of ways, then monitored for signs of resorption. Reamputation of the tip or incisions which created large gaps in the wound epidermis promoted resorption in 77-90% of the cases. Histology showed that the tissue removed by tip reamputation was a small proportion of the entire regenerate, suggesting that blastema resorption is not determined simply by the number of cells directly affected by the injury. Pin prick injuries, which created small disruptions of the wound epidermis, never caused resorption. Devascularization, caused by severing the brachial artery, promoted resorption in 17% of cases. These results are not consistent with the hypothesis that avascularity is a major causative factor in nerve dependence.     

Some distal limb structures develop in mice lacking Sonic hedgehog signaling

Patterning of the limb is coordinated by the complex interplay of three signaling regions: the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the non-ridge limb ectoderm. Complex feedback loops exist between Shh in the ZPA, Bmps and their antagonists in the adjacent mesenchyme, Wnt7a in the dorsal ectoderm and Fgfs in the AER. In contrast to the previously reported complete absence of digits in Shh(-/-) mice, we show that one morphologically distinct digit, with a well-delineated nail and phalanges, forms in Shh(-/-) hindlimbs, while intermediate structures are severely truncated and fused. The presence of distal autopod elements is consistent with weak expression of Hoxd13 in Shh(-/-) hindlimbs. Shh(-/-) forelimbs in contrast have one distal cartilage element, a less-well differentiated nail and fused intermediate bones. Interestingly, Ihh is expressed at the tip of Shh mutant limbs and could account for formation of distal structures. In contrast to previous studies we also demonstrate that Shh signaling is required for maintenance of normal Fgf8 expression, since expression of Fgf8, unlike some other AER marker genes, is rapidly lost from anterior to posterior after E10.5, with only a small domain of Fgf8 expression remaining posteriorly. Furthermore, loss of expanded Fgf8 expression is paralleled by a collapse of the handplate. Our data show that development of most intermediate elements of the hindlimb skeleton are Shh-dependent, and that Shh signaling is required for anterior-posterior expansion of the AER in both limbs and for the subsequent branching of zeugopod and autopod elements. Finally, we show that Shh is also required for outgrowth of the limb ectoderm and thus for the formation of a distinct limb compartment.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

Regeneration of symmetrical forelimbs in the axolotl,Ambystoma mexicanum

Surgically constructed symmetrical double-anterior and double-posterior upper forelimbs of the axolotl were amputated immediately after surgery. Double-anterior limbs either failed to regenerate or formed single digits or spikes. Double-posterior limbs formed symmetrical double-posterior regenerates in 60% of the cases, thus extending the previous finding that the amount of distal transformation in surgically constructed double-half limbs is inversely proportional to the time between grafting and amputation (Tank and Holder, 1978). When these symmetrical regenerates were amputated through the forearm region, all but one formed a symmetrical secondary regenerate. The majority of the secondary regenerates had a larger number of digits than did their corresponding primary regenerates. Reamputation of the secondary regenerates resulted in symmetrical tertiary regenerates, and the majority of these also had a larger number of digits than did their corresponding primary regenerates. The results are compared to those of Slack and Savage (1978a, b) on embryonically derived double-posterior limbs and they are discussed in terms of a formal model for distal transformation (Bryant and Baca, 1978).     

Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro: the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions. The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles. In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations. On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth. In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates. In contrast, processes of individual cells did not appear to orient themselves toward the capillary. The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth. These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal.     

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog,Xenopus laevis

Summary The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.     

Hyperinnervation improves Xenopus laevis limb regeneration

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors.     

Histological and histochemical studies on the nervous influence on minced muscle regeneration of triceps surae of the rat.

The degree of minced rat muscle regeneration in the absence of nerve fibers was compared with that of normal regenerates between one and 270 days postoperatively. Up to around 30 days, the number of muscle fibers and their morphology were comparable in both normal innervated and denervated regenerates; both showed clear cross striations and peripherally located nuclei. Histochemically, SDH and myofibrillar ATPase (pH=9.4) reactions were positive, but there were no typical signs of fiber types in either case of regeneration. The only consistent difference in the early period was the smaller fiber cross sectional areas in denervated regenerates than in innervated ones. Starting about 40 days, the muscle fibers in innervated regenerates became differentiated into different fiber types (fast-twitch-oxidative-glycolytic, FOG., fast-twitch-glycolytic, FG., slow-twitch-oxidative, SO.) but there were no such activities in denervated regenerates, although their SDH and myofibrillar ATPase reactions remained positive for a long time. Degenerating muscle fibers could no longer be identified in innervated regenerates. In the denervated regenerates, however, muscle fibers underwent atrophic or degenerative changes and were replaced by connective tissue. The complete disappearance of muscle fibers varied with individual regenerates. In some cases, it occurred about 90 days and in others, traces of muscle fibers could still be seen as late as 150 days postoperatively. Thus, nerves seem to be important primarily in the late phase of regeneration; namely, differentiation of fiber types and maintenance of the structural integrity of muscle fibers.     

Effects of retinoic acid on the muscle patterns produced during forelimb regeneration in larval salamanders (Ambystoma)

After systemic treatment with retinoic acid (RA), Ambystoma opacum and A. punctatum larvae regenerated forelimbs with a wide variety of skeletal and gross anatomical abnormalities. Yet the musculatures within the RA-treated limb regenerates were normal even in instances where the cartilages were deformed beyond recognition as components of the limb skeleton. RA is known to induce reduplication of limb structures, sometimes entire segments. When the latter condition occurred in the present study, the corresponding replicates exhibited limb musculatures which were perfect down to minute details, yet of opposite bilateral symmetry. The results attest, firstly, to the independence of myogenesis and chondrogenesis during limb regeneration. Secondly, RA treatment unmasked an otherwise hidden potential within postembryonic salamander limb tissue for the morphogenesis of the contralateral musculature.     

Unusual muscle abnormalities associated with thalidomide treatment in a rhesus monkey: a case report.

Unusual musculotendinous abnormalities were observed in the forelimbs of a rhesus monkey treated prenatally with thalidomide (10 mg/kg maternal body weight, gestation days 33, 34, and 35). Although the hindlimbs exhibited malformations typical of thalidomide dysmelia, the forelimbs appeared relatively normal externally. However, dissection revealed totally anomalous insertions of preaxial muscles of both forearms. Extensor muscles inserted on flexor surfaces even though normal sites of insertion were present and unoccupied. Radiographs revealed only minor ossification deficiencies in the carpus and distal radius. These observations suggest that thalidomide, given at the appropriate time, can disrupt condensation and splitting of pre-muscle masses without greatly disturbing condensation of corresponding preskeletal elements. These types of musculoskeletal relationships are unusual findings in congenital malformations of the limbs and have not been widely associated with thalidomide teratogenesis.