Newt Limb Regeneration Research Articles

Injury, nerve, and wound epidermis related electrophoretic and fluorographic protein patterns in forelimbs of adult newts.

Polyacrylamide slab gel electrophoresis and [35S]methionine fluorography were used to examine proteins in regenerating newt limbs, amputated denervated limbs, unamputated denervated limbs, and separated blastema mesodermal core and wound epidermis. A total of 27 protein electrophoretic bands were obtained from amputated limbs and 24 bands from unamputated limbs. Amputation resulted in the appearance of 4 new bands and the loss of 1 band as compared to unamputated limbs. These 5 banding differences were apparent on stained gels 3 days postamputation and were maintained through 10 weeks postamputation (complete regenerate stage). Only one band in unamputated limbs was always detectable on fluorographs, whereas virtually all of the stainable bands of amputated limbs were visible on fluorographs. Amputation clearly stimulated a marked, generalized increase in the synthesis of limb proteins. The 5 amputation induced changes were equally evident in stained gels of both innervated and denervated limbs. Amputated denervated limbs possessed a full set of fluorographic bands (including the 5 differences) through 18 days postamputation. However, denervation without amputation was not sufficient to alter the stainable banding pattern. Wound epidermis and mesodermal core both displayed the 5 banding differences and had identical banding patterns with the exception of one epidermal specific band. This band was also present in whole limb skin but was absent in unamputated mesodermal limb tissue. This was the only band of unamputated limbs that was consistently detectable by fluorography. It is concluded that amputation induces nerve independent changes in protein synthesis that are common to both mesodermal core and wound epidermis. These changes may represent preparation for cellular proliferation.

Effects of radius—ulna removal on forelimb regeneration in Xenopus laevis froglets

Regeneration of boneless amputated forearms of adult newts was found to progress at a rate and to a degree comparable to amputated control limbs in which stump bones were not removed. In contrast, regeneration of boneless amputated Xenopus froglet forearms was significantly delayed and did not occur until two to three weeks following amputation. This is in comparison with the initiation of distal cartilage formation observed one week postamputation in control forelimbs of Xenopus froglets. The regeneration of cartilage in boneless forearms of adult newts was found to occur distal to the amputation level. In contrast, distal as well as proximal (centripetal) regeneration of cartilage was observed in the amputated boneless forearms of Xenopus. In froglets and newts, unamputated forelimbs in which forearm bones were extirpated did not initiate cartilage regeneration. Our findings support the hypothesis that forelimb regeneration in Xenopus froglets is primarily a tissue response. In comparison, limb regeneration in the adult newt is predominantly an epimorphic response.

In vitro effects of insulin on macromolecular events in newt limb regeneration blastemata.

This work provides data demonstrating a stimulatory effect of insulin on macromolecular events occurring in cultured regeneration blastemata and demonstrates a synergistic interdependence between nerves and insulin in newt limb regeneration. The current experiments provide evidence for the following: (1) Insulin is paramount for expression of the mitogenic effect of nerves on cultures blastemata. (2) Insulin stimulates the incorporation of (3H)uridine into the acid-insoluble fraction of blastemal homogenates, but it does not alter the turnover rate of incorporated labeled uridine. (3) Insulin also stimulates the incorporation of 35SO4 and (3H)leucine into both chondroitinase-sensitive and chondroitinase-resistant blastemal proteoglycans. (4) Insulin increases the uptake of radiolabeled precursors by the blastemata, namely, (3H)leucine, (3H)uridine, 35SO4, (3H)alpha-aminoisobutyrate, and (3H)2-deoxy-D-glucose. The importance of insulin in the regulation of newt limb regeneration is discussed.

Neurotrophic and neuronotrophic effects in the regenerating newt limb bud after electrical stimulation of brachiospinal nerves

An earlier work demonstrated that electrical stimulation of newt brachiospinal nerves produces a 20% increase in protein synthesis in the regenerating limb bud at 6 h post-stimulation. The present study shows that if stimulation of nerve cell bodies is prevented by placing procaine between the cell bodies and the stimulating electrode, there is no increase in limb bud protein synthesis compared to the non-stimulated, contralateral control limb bud. Similarly, if colchicine is applied to the brachiospinal nerves at the site of and prior to stimulation, there is no increase in limb bud protein synthesis after stimulation. Colchicine applied to brachiospinal nerves in the absence of stimulation results in a reduction of limb bud protein synthesis that is of the same magnitude as the increase seen with stimulation. The results suggest that the neurotrophic increase in limb bud protein synthesis after stimulation is under the control of the cell body and that this control is mediated by changes in fast axonal transport. A neuronotrophic increase in axonal density in the stimulated side limb bud is seen at the same time as the increase in protein synthesis after stimulation.

Monoclonal antibodies identify blastemal cells derived from dedifferentiating limb regeneration.

Blastemal cells arise after the amputation of limbs or tails in urodele amphibians. These histologically undifferentiated mesenchymal cells divide and subsequently differentiate to regenerate a new appendage. Various studies (reviewed in ref. 1) indicate that blastemal cells arise from tissues near the site of amputation, including muscle, cartilage, nerve and dermis. The multinucleated myofibre, however, is a controversial source of blastemal cells. The suggestion that myofibres can dedifferentiate is based on their histological appearance during the early stages of limb regeneration. This is contrary to the widely accepted view of muscle regeneration in higher vertebrates which attributes it to satellite cells. One prediction of the dedifferentiation hypothesis is that a population with properties of both myofibres and blastema cells should be present during the early stages of regeneration. Here we described the isolation of two monoclonal antibodies, one that recognizes an antigen found only in myofibres and another that recognizes an antigen restricted to blastemal cells. By using these antibodies as cell markers, we can detect a small population of cells in the regenerating limbs of adult newts that bear both the myofibre and blastemal cell antigens. The time and location of these double-labelled cells supports the idea that blastemal cells originate, in part, by dedifferentiation of myofibres.

Abnormal limb regeneration in adult newts exposed to the fungicide Maneb 80. A histological study.

The effects of the fungicide Maneb 80 (manganese ethylenebisdithiocarbamate, 80% active ingredient) on the regenerating limb of the adult crested newt, Triturus cristatus carnifex, was studied. Female newts were exposed percutaneously to 5 ppm Maneb 80. One group of control newts was exposed to the inert ingredients of Maneb 80 (sodium lignin sulfonate and n-butylnaphthalene sulfonate), and another control group was kept in tap water. The limbs were examined histologically at weekly intervals throughout the regeneration period and at the end of the experiment (10-12 wk postamputation). The regenerating limbs of all the animals exposed to Maneb 80 showed growth retardation and skeletal abnormalities. Histological examination provided evidence that vascular disturbances are important for the genesis of the developmental abnormalities induced by Maneb 80. The inert ingredients had a promoting effect on limb growth and had no teratogenic effects under our experimental conditions. There were no histological differences between the two control groups.

An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt

Fibronectin and laminin are two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation describes changes in their distribution during regeneration of the newt forelimb, as determined by indirect immunofluorescence. The distribution of fibronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myofibers corresponding to its basement membrane; the perineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithelium, and dermal glands. The cytoplasm of myofibers, axons, skin epithelium, and bone matrix lacked fluorescence for both glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, blastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the myoblast fusion zone. Laminin was first observed around the regenerated myotubes; this was followed by the appearance of fibronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distribution of laminin and fibronectin was similar to that seen in normal limb. Based on the distribution pattern of these glycoproteins, it is concluded that fibronectin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fibronectin may be important in the determination of the architecture of the regenerated limb.

Limb regeneration blastema cells in the adult newt, Notophthalmus viridescens, give aberrant response to colchicine

This study was designed to investigate the effect of colchicine on the mitotic index (number of mitotic figures per 1000 cells) in the newt limb regeneration blastema. Colchicine doses from 0.1 to 1000 μg per gram body weight and treatment durations from 2 to 72 h were used to determine the dose of colchicine and treatment period resulting in optimum metaphase arrest. A preliminary experiment demonstrated that there was no circadian rhythm of mitosis in the newt limb regeneration blastema. A dose of 5 μg colchicine per gram body weight was found to give the maximum mitotic index; both higher and lower doses had less effect. Very high doses of colchicine resulted in a mitotic index the same as that of the controls. The mitotic index increased with increasing treatment time up to 36 h, after which there was no further increase. In other newt tissues such as duodenal mucosa and liver, a more typical dose–response relationship was observed in that increased colchicine dose led to increased mitotic indices up to the level of lethal doses of colchicine.

Effects of a Carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine on Blastema Cells and Blastema Formation in Newt Limb Regeneration: (blastema cells/carcinogen/cell surface properties/basement lamella/abnormal regeneration).

The effects of a potent, carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on blastema formation and blastema cells were studied in the adult newt by means of scanning electron microscopy. By administration of MNNG to the blastema of the limbs the formation of the basement lamella, which lies between the epithelium and the mesenchyme, was effectively inhibited at least for a month. Basement lamella in treated limbs is formed 30 or 40 days after carcinogen administration. MNNG altered significantly cell surface morphology and cell motility of blastema cells isolated in vitro. Membrane's specializations such as the formation of filopodia were inhibited. Motility of the treated cells was much reduced compared to the control. The role of these alterations in the carcinogen-induced abnormal regeneration is discussed.

Changes in the Distribution of Fibronectin During Limb Regeneration in Newts Using Immunocytochemistry

The distribution of fibronectin in regenerating newt limbs was studied using immunocytochemistry. At appropriate intervals after the initial amputation at the elbow (10-30 days), animals were reamputated at the shoulder and processed for light microscopy. The peroxidase-antiperoxidase technique was used to localize affinity-purified antibodies to fibronectin in limb tissues. At the amputation site, fibronectin was associated with basal laminae and connective tissues adjacent to dedifferentiating limb tissues destined to form the regeneration blastema. Accumulation and growth of the blastema was accompanied by the apparent de novo synthesis of fibronectin, where it appeared randomly in the interstitium between blastemal cells. The onset of chondrogenesis was characterized by a central condensation of prechondroblasts that formed the cartilage anlagen. Fibronectin formed an amorphous network between presumptive chondroblasts. As the mature cartilage phenotype was expressed and chondrocytes became isolated in lacunae, fibronectin was greatly reduced and then disappeared. The extracellular matrix surrounding undifferentiated blastemal cells still contained fibronectin. Fibronectin was also found in high concentrations between differentiating myoblasts. A condensation of fibronectin was also observed beneath the epidermis at the distal limb tip at the onset of digit formation. These observations are consistent with the hypothesis that fibronectin may play a key role in the morphogenetic events that result in the spatial organization and subsequent differentiation of cells during pattern formation in the regenerating limb.

Formation of prechondrogenic mesenchymal cell aggregates in the regenerating newt limb

Development of the pattern of prechondrogenic condensations was documented by quantitating regional cell densities within blastemas of regenerating forelimbs of newts. The pattern of development resembles that of embryonic limb development in that the prechondrogenic pattern develops from proximal to distal, and by the presence of an apical epithelial thickening which is most prominent postaxially during the intermediate phase of mesenchymal cell accumulation. Development differs from that described previously in that an increased central cell density is present in the smallest blastemas analyzed, and the sequence of digit formation is from anterior to posterior. Also, a blister often intervenes between the epithelium and mesenchyme during the early phase of development. Cumulatively, these observations support models which suggest mechanisms for proximodistal development and a stimulatory role for apical epithelium. However, the presence of a central cell density prior to definitive aggregation implies a mechanism other than de novo condensation which is assumed in current models. Additionally, increased mesenchymal cell density subjacent to thickened epithelium is contrary to models which imply that such areas should be less dense. Finally, digital aggregates develop from preaxial to postaxial, which is opposite that of most amniotes, and is not predicted by current models of limb morphogenesis.

Cyclophosphamide-induced leukopenia and suppression of limb regeneration in the adult newt, notophthalmus viridescens.

Cyclophosphamide (cytoxan), a clinical immunosuppressant, produced a marked, persistent leukopenia (greater than 40% reduction) in adult newts in contrast to a transient, milder leukopenia accompanying amputations without cytoxan treatments. In addition, cytoxan suppressed the formation of regeneration blastemata initially: however, blastema formation occurred if a second amputation was performed two or more weeks following the cessation of cytoxan treatments. The failure of the leukopenias of these latter cases to be corrected suggests that although a cytoxan-induced leukopenia is correlated with the absence of forelimb regeneration, this leukopenia is not, of itself, a sufficient condition to inhibit regeneration.

Forelimb regeneration in thyroidectomized adult newts following organ culture and autografting of the thyroid glands.

Thyroidectomy and organ culture of adult newt thyroid glands three days prior to forelimb amputation was followed by autografting the glands subcutaneously into the animal's lower jaw region 9, 18 or 25 days postamputation (GC9, 18, 25 day series). This was an attempt, utilizing 515 animals, to elucidate further the role of the thyroids in regeneration. Amputated limbs of the thyroidectomized (Thx) and autografted muscle explant (MC = sham) cases underwent stumping or were significantly delayed in their regeneration rate and displayed abnormal morphogenesis compared with control regenerates. In the GC9 series newts, regenerates were identical to controls 45 days postamputation. However, regenerates of the GC18 series cases exhibited delayed and abnormal development at 45 days; but they were not as delayed and had fewer abnormalities than those cases in the Thx and MC groups. Results of the GC25 series newts were similar to those of the Thx group. Within 5 days of autografting the thyroids, epidermal moulting resumed and long-term survival ensued. We conclude that normal limb regeneration in the adult newt is thyroid hormone(s) dependent, specifically the later stages of growth, differentiation and morphogenesis.

Carcinogens on Regeneration: Effects of N-Methyl- N′-Nitro- N-Nitrosoguanidine and 4-Nitroquinoline-1-Oxide on Limb Regeneration in Adult Newts

A microcrystal (ca 5 micrograms) of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 4-nitroquinoline-1-oxide (4NQO) was directly administered to the regeneration blastema on day 7 after amputation of a forelimb in the newt in order to analyze the effect of such potent carcinogenic substances on regeneration cells. Although neither MNNG nor 4NQO arrested regeneration completely, they caused great retardation of the regeneration cone formation followed by various abnormalities in the bony structures. Abnormal regenerants could be classified into the following four categories; (1) complete absence of both ulna and radius; (2) subregeneration or superregeneration of carpals and digits; (3) multiple disorganization of skeletal elements; (4) arrest of regeneration at the stage of regeneration cone. The polarity of regenerants developed after application of MNNG or 4NQO was very often shifted, during which the regeneration cone was always formed from the site where a microcrystal of the carcinogens was administered. The secondary regeneration initiated by reamputation of the regenerating limb, which had received the carcinogens at the early blastema stage, proceeded in the same way as observed in the case of a simple amputation. This suggested local and temporal effects of the carcinogens applied. Nevertheless, tumor formation has not induced in the newt limb so far. We can learn from these data that both MNNG and 4NQO only alter behavior of the newt regeneration cells without excreting their carcinogenic effects on them, and that the newt cells are highly resistant and stable against the above-mentioned carcinogens.

N-Acetyl-Glucosaminidase Activity during Limb Regeneration in the Adult Newt

N-Acetyl-glucosaminidase activity was measured during the first 25 days of limb regeneration. It was found that the enzyme is present in the normal limb. Following amputation a significant drop was obtained at day 3. A significant increase in enzyme activity was found at day 5 followed by a second drop by day 10. For days 12–15 a second peak of enzyme activity was detected, followed by a third drop; by day 25, normal levels of enzyme activity were detected. Histochemical localization of the enzyme in tissue samples showing enzyme activity as detected biochemically (days 5 and 17 of regeneration) gave negative results. However, enzyme activity was found in the incubation medium, indicating that the enzyme is released from the cells. The peaks of enzyme activity coincide with the stages of limb regeneration where a high degree of tissue demolition and cell lysis occurs. The latter are important events in the regeneration process, cell dedifferentiation, and blastema formation.

Limb regeneration following the discontinuation of growth hormone therapy in the hypophysectomized newt, Notophthalmus viridescens.

Untreated adult newts do not undergo normal limb regeneration following hypohysectomy. A fibrocellular dermal barrier (cicatrix) atypically forms between the apical epithelium and the underlying mesenchymal tissues. Historically, continuous administration of growth hormone or of prolactin in combination with thyroxine restored regenerative capacity to these newts. In a previous investigation, we demonstrated that the initial effect of these two hormone treatments, when administered on alternate days to hypophysectomized newts beginning eight days post-amputation, was to facilitate the erosion of the fibrocellular barrier and establish the epithelial mesenchymal interface that is observed in a regenerating limb. The present investigation was designed to evaluate the necessity of continuous hormone therapy to maintain limb regeneration in hypophysectomized newts. One, two, or three injections of growth hormone or of prolactin in combination with thyroxine was administered on successive alternate days to hypophysectomized newts either immediately following limb amputation (ID) or beginning eight days post-amputation (DD). The ID and DD newts receiving one, two, or three injections of growth hormone showed evidence of regeneration to the digitiform stage by day 30 post-amputation, while those receiving prolactin and thyroxine underwent wound healing. While both hormone treatments initially promoted a dermis-free apical epithelium, only hypophysectomized newts that had received growth hormone were able to continue regenerating. We have, therefore, concluded that discontinuous growth hormone therapy is sufficient to initiate and maintain the conducive environment for limb regeneration to advanced stages in the hypophysectomized newt. While initiating this process, prolactin and thyroxine therapy on a discontinuous regime does not maintain regeneration. The direct and indirect role of growth hormone in supporting limb regeneration in normal and hypophysectomized newts is discussed.

An investigation of the prolactin-thyroxine synergism in newt limb regeneration.

The use of hormone replacement to support limb regeneration in hypophysectomized newts has been the subject of many investigations. Growth hormone, as well as prolactin (PL) in combination with exogenously supplied thyroxine, have all been shown to he effective. However, the bovine growth hormone used to support limb regeneration was contaminated by prolactin and thyroidstimulating hormone (TSH). The present investigation evaluates the significance of (1) prolactin contamination and (2) endogenous thyroxine synthesis resulting from TSH contamination on limb regeneration in hypophysectomized newts. The effect of supplying exogenous thyroxine was also evaluated. Our studies showed that when hypophysectomized newts were injected with contamination levels of PL and TSH, regeneration occurred, suggesting that the newt's thyroid synthesized sufficient thyroxine to support a prolactin-thyroxine synergism. The endogenous thyroxine was synthesized by thyroid glands that were indistinguishable from those of saline-injected, hypophysectomized controls.

Denervation effects on newt limb regeneration: Collagen and collagenase

Although the inhibitory effect of denervation on newt limb regeneration results in decreased macromolecular synthesis and changes in transcription, no specific protein has been identified to be differentially affected by denervation. This study reports the effect of denervation on two specific proteins involved in collagen metabolism in the regenerate. The activity of collagenase, a marker protein for dedifferentiation, was compared in normal regenerates and in limbs denervated at the time of amputation by means of culture techniques. The time course of collagenase activity in innervated versus denervated limbs indicates that the nerve is not required for the onset of collagenase production but suggests that inhibition of collagenase production in the regenerate may be related to formation of the regeneration blastema. The effect of denervation on synthesis of collagen, a marker protein for differentiation, relative to synthesis of noncollagen proteins was studied with tracer techniques in vivo. After unilateral denervation of palette-stage regenerateś, incorporation of proline into collagen is reduced to a larger extent (2.5- to 3-fold) than incorporation into NCP (1.8- to 2-fold). At 70 hr after denervation, the kinetics of incorporation of proline into both collagen and NCP indicate that the relatively larger effect of denervation on collagen synthesis results primarily from a differential reduction in the rate of collagen synthesis compared to NCP synthesis.

Scanning electron microscopy of epidermal cell migration in wound healing during limb regeneration in the adult newt, Notophthalmus viridescens.

The epidermal cells which migrate over the wound surface of the amputated limb of the adult newt were examined using the scanning electron microscope. Specimens were prepared routinely for scanning electron microscopy or were embedded in Epon 812 for light microscopic observations. A cuff of epidermal cells was seen at the edge of the wound, from which cells appeared to migrate over the wound surface. As early as five hours after transection of the limb, the basal layers of this cuff appeared to send out pseudopodial projections. These seemed to establish a physical contact with a fibrin-like substratum, which apparently served as a means of support for the migrating cells. Subsequently, the epidermal cells became elongate and had the appearance of streaming toward the center of the wound. Between 10 and 13 hours post-amputation, the cells in the central region of the stump were rounded up and some possessed microappendages resembling microplicae and microvilli. Throughout the entire period of wound coverage, the cells seemed to maintain contact with the fibrin network, which appeared to be the first structural element of wound architecture. As a result of these observations, the mechanism by which the epidermal cells migrate has been clarified.

Cyclic AMP and noradrenaline sensitivity fluctuations in regenerating newt tissue

LIMB regeneration in the adult newt is dependent on the nervous system1,2 but the biochemical events involved are not well understood. A role for catecholamines (CA) seems likely since histofluorescence has shown that CA are more abundant in regenerates than in other newt limb tissues3. It has also been shown it hat reserpine, which promotes the release of CA from nerve terminals, accelerates regeneration4, whereas chlorpromazine5, guanethidine5 (both CA release blockers) and α-methyl-p-tyrosine6 (CA biosynthesis inhibitor) inhibit it. Studies suggest that in many biological systems the CA effects are linked to the activation of membrane adenylate cyclase with subsequent production of cyclic AMP7. It has also been suggested that nerves may transmit trophic information by the stimulation of this enzyme8–11. To test this hypothesis further, regenerates were isolated either for direct measurements of cyclic AMP or for stimulation with CA in vitro12. This method avoids the possible nonspecific effects due to systemic administration of CA. We now report that the fluctuations of cyclic AMP found at the various stages of regeneration correlate with noradrenaline (NA) sensitivity changes.