Lens formation from the cornea following implantation into hindlimbs of larval Xenopus laevis: the influence of limb innervation and extent of differentiation

Corneal fragments of larval Xenopus laevis at stage 48 (according to Nieuwkoop and Faber, '56), were implanted into sham denervated unamputated hindlimbs, denervated unamputated hindlimbs, amputated and sham denervated hindlimbs, and amputated and denervated hindlimbs of larvae at stages 52 and 57. The results show that unamputated limbs at stage 52, either innervated or denervated, manifest a weak capacity to promote the first lens-forming transformations of the outer cornea. This capacity is absent in both limb types at stage 57. After amputation, limbs of both early and late stages form a regenerative blastema and support lens formation from the outer cornea. Denervation of early stage limbs has no appreciable effect on blastema formation and lens-forming transformation of corneal implants. However, denervation of late stage limbs inhibits both processes. These results indicate that the limb tissues of the early stage limbs contain non-neural inductive factors at a low level and that after limb amputation and blastema formation the level of these factors becomes high enough to promote lens formation from implanted cornea, even after denervation. In contrast, the limb tissues of late stage limbs do not contain a suitable level of non-neural inductive factors.     

Responses to amputation of denervated ambystoma limbs containing aneurogenic limb grafts

The developing neural tubes and associated neural crest cells were removed from stage 30 Ambystoma maculatum embryos to obtain larvae with aneurogenic forelimbs. Forelimbs were allowed to develop to late 3 digit or early 4 digit stages. Limbs amputated through the mid radius-ulna regenerated typically in the aneurogenic condition. Experiments were designed to test whether grafts of aneurogenic limb tissues would rescue denervated host limb stumps into a regeneration response. In Experiment 1, aneurogenic limbs were removed at the body wall and grafted under the dorsal skin of the distal end of amputated forelimbs of control, normally innervated limbs of locally collected Ambystoma maculatum or axolotl (Ambystoma mexicanum) larvae. In Experiment 1, at the time of grafting or 1, 2, 3, 4, 5, 7, or 8 days after grafting, aneurogenic limbs were amputated level with the original host stump. At 7 and 8 days, this amputation included removing the host blastema adjacent to the graft. The host limb was denervated either one day after grafting or on the day of graft amputation. These chimeric limbs only infrequently exhibited delayed blastema formation. Thus, not only did the graft not rescue the host, denervated limb, but the aneurogenic limb tissues themselves could not mount a regeneration response. In Experiment 2, the grafted aneurogenic limb was amputated through its mid-stylopodium at 3, 4, 5, 7, or 8 days after grafting. By 7 and 8 days after grafting, the host limb stump exhibited blastema formation even with the graft extending out from under the dorsal skin. The host limb was denervated at the time of graft amputation. When graft limbs of Experiment 2 were amputated and host limbs were denervated on days 3, 4, or 5, host regeneration did not progress and graft regeneration did not occur. But, when graft limbs were amputated on days 7 or 8 with concomitant denervation of the host limb, regeneration of the host continued and graft regeneration occurred. Thus, regeneration of the graft was correlated with acquisition of nerve-independence by the host limb blastema. In Experiment 3, aneurogenic limbs were grafted with minimal injury to the dorsal skin of neurogenic hosts. When neurogenic host limbs were denervated and the aneurogenic limbs were amputated through the radius/ulna, regeneration of the aneurogenic limb occurred if the neurogenic limb host was not amputated, but did not occur if the neurogenic limb host was amputated. Results of Experiment 3 indicate that the inhibition of aneurogenic graft limb regeneration on a denervated host limb is correlated with substantial injury to the host limb. In Experiment 4, aneurogenic forelimbs were amputated through the mid-radius ulna and pieces of either peripheral nerve, muscle, blood vessel, or cartilage were grafted into the distal limb stump or under the body skin immediately adjacent to the limb at the body wall. In most cases, peripheral nerve inhibited regeneration, blood vessel tissue sometimes inhibited, but other tissues had no effect on regeneration. Taken together, the results suggest: (1) Aneurogenic limb tissues do not produce the neurotrophic factor and do not need it for regeneration, and (2) there is a regeneration-inhibiting factor produced by the nerve-dependent limb stump/blastema after denervation that prevents regeneration of aneurogenic limbs.     

Lack of proliferative response in denervated, reamputated limb regenerates of larval Ambystoma

This paper describes the response of early four-digit regenerates of axolotls to reamputation and denervation. Reamputation of innervated regenerates led to sharp increases in 3H-thymidine labeling index (LI) and mitotic index (MI) on days 2-5 post-reamputation. This resembles the response of innervated limbs following initial amputation. Regenerates that were denervated at the time of reamputation exhibited no proliferative response through day 5. This is in marked contrast to denervated, original amputation limb stumps, in which LI and MI rise for several days (as in innervated stumps) before falling to background levels. Although myelin was scarce near the level of reamputation, the lack of proliferation cannot be explained solely on that basis. The results are consistent with the possibility that the "neurotrophic factor" that causes stump and blastema cell mitosis is not present in unamputated limbs but is made in response to amputation.     

A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts

We have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthalmus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1% of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contralateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/18-positive blastema. The appearance of the 22/18 responses is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.     

Effects on adult newt limb regeneration of partial and complete skin flaps over the amputation surface.

The influence of the wound epithelium on the cellular events preceding blastema formation was examined by comparing dedifferentiation, DNA labeling indices, and mitotic indices of the distal mesodermal tissues in control regenerating newt forelimbs and in amputated forelimbs covered with a flap of full thickness skin. Three kinds of results were seen following the skin-flap graft operations. Epidermal migration across the amputation surface was completely inhibited in 22% (8) of the cases and these limbs repaired the amputation wound but did not form regeneration blastemas. In 11% (4) of the experimental limbs, essentially normal wound epithelia displaced the skin flaps and the limb stumps formed blastemas and regenerated. The majority of the skin grafts (67%) exhibited epidermal migration restricted to the free edges of the flaps. These limbs formed eccentric blastemas on the ventral side of the limb next to the dermis-free epidermis and regenerated laterally in that direction.     

Increased prolactin binding and morphological changes in the wound epithelium of regenerating limbs of Notophthalmus viridescens

Summary Distribution of prolactin has been examined in regenerating forelimbs from the newt Notophthalmus viridescens. Specific prolactin binding was demonstrated in homogenates of unamputated tissue, and of regenerating limbs at from 3 to 21 days postamputation. Labeled prolactin that was injected intraperitoneally into animals with one regenerating limb accumulated in the most distal portion of the regenerate at 7 and 14 days postamputation. Light microscopic autoradiography demonstrated that labeled prolactin was localized most heavily in the apical, outer layer of the wound epithelium. Scanning electron microscopy demonstrated that, in addition to changes in prolactin affinity following amputation, morphological changes occurred in the apical wound epithelium as well. Cell surfaces of the stump epidermis were characterized by periodic dispersion of papillae among a network of interconnecting structures 1–2 m across. By contrast, the surfaces of cells from the area in which labeled prolactin was found to localize most intensely were characterized by lack of papillae and, depending on the stage of regeneration, a pattern of microvilli and microplicae. These morphological alterations appear to reflect functional and biochemical differences between stump epidermis and wound epithelium.     

Rescue of blocked cells by reinnervation in denervated forelimb stumps of larval Ambystoma

Cells of amputated, denervated larval Ambystoma forelimbs dedifferentiate and enter the cell cycle but do not subsequently proliferate sufficiently to form a blastema. The denervated limb stump resorbs slowly until reinnervation stimulates regeneration. We used this system to investigate the fate of cells in denervated limbs which undergo early but limited cycling in response to amputation. In Experiment 1, cells were labeled with [3H]thymidine (3H-T) on Day 4 postamputation (PA)/Day 3 postdenervation (PD). Labeled cells were still present on Day 7 PA, but were less frequently observed on Day 13 PA when the limbs were reinnervated and beginning to regenerate. In Experiment 2 we denervated 1 day preamputation to obtain earlier reinnervation and prevent loss of Day 4 PA labeled cells. Cells labeled with 3H-T on Day 4 PA/Day 5 PD were present throughout the denervation period and most were still present on Day 13 PA. Little or no mitotic activity was found among the labeled cells after the initial round of cycling. The apparent cell cycle block was released upon reinnervation on Days 12 and 13 PA when cycling resumed. Labeled mitotic figures were present on Day 13 PA, and the mitotic index of the labeled population increased as a result of reinnervation. These results demonstrate that blocked cells are rescued by nerves, re-enter the cell cycle, and thus contribute to the reinnervation blastema.     

Nerve interactions and regenerative processes occurring in newt limbs fused end-to-end

Peripheral nerve interactions and regenerative phenomena were studied in newt forelimbs fused end to end. After simple fusion, one or two spikelike structures regenerated at the plane of fusion in 88% of the cases. When one of the limbs was denervated at the time of fusion, no regeneration occurred from the plane of fusion. If the limbs were fused and one was amputated at the shoulder more than 10 days after fusion, regeneration from the amputation surface did not occur. When the limbs were reamputated 30 days later, regeneration of left limbs from the proximodistally reversed right limb stumps followed. If one of the limbs was denervated at the time of fusion, and amputation was subsequently carried out through the formerly denervated limb, regeneration always took place after the first amputation. On the basis of these results it is postulated that when regenerating nerves of opposite proximodistal polarity meet head-on, the majority of fibers, at least, do not grow into territories occupied by the other nerve. These results have also demonstrated that full limb regeneration can occur at a greater distance from the midline than the end of a normal limb. These experiments also provide a technique for artificially elongating peripheral nerves.     

Retinoic acid enhances monoclonal antibody WE3 reactivity in the regenerate epithelium of the adult newt.

Monoclonal antibody (mAb) WE3 recognizes an antigen that is developmentally expressed in the wound epithelium during adult newt limb regeneration. Experiments were designed to determine whether retinoic acid (RA), dissolved in dimethyl sulfoxide (DMSO) and administered by intraperitoneal injection, would enhance the temporal appearance of the WE3 antigen. RA given on days 1 or 4 after amputation, when the WE3 antigen is not yet detectable, resulted in moderate reactivity to mAb 2 days after injection and strong reactivity throughout the wound epithelium 4 days after injection. DMSO alone had no enhancing effect. RA also caused limb skin epidermis to exhibit reactivity to mAb WE3, initially near the amputation level, but then also more proximally. By 4 and 6 days after RA injection, epidermis of the flank, eye lid, and unamputated hind limbs also became strongly reactive to mAb WE3. Outer layers of skin epidermis were shed, resulting in an epidermis only one or two cells thick. Epidermis of newts given DMSO alone remained non-reactive to mAb WE3. When RA was given on days 7 and 10 after amputation, when a low level of mAb WE3 reactivity is already present in the wound epithelium, a considerable enhancement of mAb WE3 reactivity occurred through the next few days. No such enhancement was seen with DMSO alone. RA also greatly increased mAb WE3 reactivity in the wound epithelium of denervated limbs, in which case the wound epithelial reactivity to mAb WE3 is normally low. Retinol palmitate also increased mAb WE3 reactivity. The results raise the possibility that the WE3 antigen is a component of most if not all retinoid target tissues in newts.     

Changes in the Inflammatory Response to Injury and Its Resolution during the Loss of Regenerative Capacity in Developing Xenopus Limbs

Tissue and organ regeneration, unlike development, involves an injury that in postembryonic animals triggers inflammation followed by resolution. How inflammation affects epimorphic regeneration is largely uninvestigated. Here we examine inflammation and its resolution in Xenopus laevis hindlimb regeneration, which declines during larval development. During the first 5 days postamputation, both regeneration-competent stage 53 and regeneration-deficient stage 57 hindlimbs showed very rapid accumulation of leukocytes and cells expressing interleukin-1β and matrix metalloproteinase 9. Expression of genes for factors mediating inflammatory resolution appeared more persistent at stages 55 and 57 than at stage 53, suggesting changes in this process during development. FoxP3, a marker for regulatory T cells, was upregulated by amputation in limbs at all three stages but only persisted at stage 57, when it was also detected before amputation. Expression of genes for cellular reprogramming, such as SALL4, was upregulated in limbs at all 3 stages, but markers of limb patterning, such as Shh, were expressed later and less actively after amputation in regeneration-deficient limbs. Topical application of specific proinflammatory agents to freshly amputated limbs increased interleukin-1β expression locally. With aqueous solutions of the proinflammatory metal beryllium sulfate, this effect persisted through 7 days postamputation and was accompanied by inhibition of regeneration. In BeSO₄-treated limbs expression of markers for both inflammation and resolution, including FoxP3, was prolonged, while genes for cellular reprogramming were relatively unaffected and those for limb patterning failed to be expressed normally. These data imply that in Xenopus hindlimbs postamputation inflammation and its resolution change during development, with little effect on cellular dedifferentiation or reprogramming, but potentially interfering with the expression of genes required for blastema patterning. The results suggest that developmental changes in the larval anuran immune system may be involved in the ontogenetic loss of epimorphic regeneration in this system.     

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.     

Lens formation from cornea implanted into amputated hindlimbs of Xenopus laevis larvae requires innervation or proliferating cell populations in the stump

The capacity of amputated early and late limbs of larval Xenopus laevis to promote lens-forming transformations of corneal implants in the absence of a limb regeneration blastema has been tested by implanting outer cornea fragments from donor larvae at stage 48 (according to Nieuwkoop and Faber 1956), into limb stumps of larvae at stage 52 and 57. Blastema formation has been prevented either by covering the amputation surface with the skin or by reconnecting the amputated part to the limb stump. Results show that stage 52 non-regenerating limbs could promote lens formation from corneal implants not only when innervated but also when denervated. A similar result was observed in stage 57 limbs where blastema formation was prevented by reconnecting the amputated part to the stump. In this case, relevant tissue dedifferentiation was observed in the boundary region between the stump and the autografted part of the limb. However, stage 57 limbs, where blastema formation was prevented by covering the amputation surface with skin, could promote lens formation from the outer cornea only when innervated. In this case, no relevant dedifferentiation of the stump tissues was observed. These results indicate that blastema formation is not a prerequisite for lens-forming transformations of corneal fragments implanted into amputated hindlimbs of larval X. laevis and that lens formation can be promoted by factors delivered by the nerve fibres or produced by populations of undifferentiated or dedifferentiated limb cells.     

Nerve-independence of limb regeneration in larval Xenopus laevis is correlated to the level of fgf-2 mRNA expression in limb tissues

In both larval and adult urodele amphibians, limb blastema formation requires the presence of an adequate nerve supply. In previous research, we demonstrated that the hindlimb of early Xenopus laevis larvae formed a regeneration blastema even when denervated, while the denervated limb of late larvae did not. We hypothesized that the nerve-independence was due to the autonomous synthesis of a mitogenic neurotrophic-like factor by undifferentiated limb bud cells. In this paper, we demonstrate that fgf-2 mRNA is present in larval limb tissues and that its level is correlated to the extent of mesenchymal cells populating the limb: in early limbs, fgf-2 mRNA is present at high levels all over the limb, while, in late limbs, the fgf-2 expression is low and detectable only in the distal autopodium. After denervation, fgf-2 mRNA synthesis increases in amputated early limbs but not in amputated late limbs. The implantation of anti-FGF-2 beads into amputated early limbs hardly lowers the mitotic activity of blastema cells. However, FGF-2 beads implanted into the blastema of late limbs prevent the denervation-induced inhibition of mitosis and oppose blastema regression. Our data indicate that FGF-2 is a good candidate for the endogenous mitogenic factor responsible for blastema formation and growth in amputated and denervated early limbs. However, in amputated late limbs, the very limited fgf-2 expression is not sufficient to promote blastema formation in the absence of nerves.     

Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating limbs with regenerates.

Amputated, regenerating forelimbs have been compared with the contralateral, denervated non-regenerating limb stumps in the adult newt Notophthalmus viridescens, with respect to hyaluronidase activity and the incorporation of ³H-acetate into glycosaminoglycans (GAG). At 10 days after amputation, which is the time of maximum hyaluronate production in the early growing regenerate, incorporation of ³H-acetate into GAG (cpm/mg protein) in the denervated, nonregenerating limb stump was approximately 50% of that in the contralateral regenerating limbs. At this stage, hyaluronate was the major GAG being produced, but the ratio of incorporation into hyaluronate relative to chondroitin sulfate was reduced in the denervated limbs. In intact, nonamputated limbs, the incorporation into GAG was 5% of that in the regenerating limb 10 days after amputation, and 10% of that in the denervated stumps.At 25 days, cartilage is forming and chondroitin sulfate synthesis predominates in the normal regenerate whilst the contralateral, denervated limb stumps are forming scars. GAG synthesis in the latter was less than one-quarter the level seen in the regenerating limbs, mostly due to low incorporation into chondroitin sulfate.Hyaluronidase activity, which appears in the regenerating limb during differentiation of skeletal elements (20–45 days), was not detectable in limbs denervated early enough to prevent regeneration. However, limbs denervated after formation of the blastema will regenerate without nerve, and hyaluronidase activity in such limbs was normal. Thus, hyaluronidase activity appears when regeneration reaches the cartilage deposition stage, with or without nerve.     

Regenerative ability of double-half and half upper arms in the newt, Notophthalmus viridescens

The upper arms of adult newts (Notophthalmus viridescens) were surgically manipulated to create double-half dorsal, double-half ventral, double-half anterior, and double-half posterior upper arms, and longitudinal half-dorsal, half-ventral, half-anterior, and half-posterior upper arms. Amputation through the double-half upper arms usually failed to elicit normal distal regeneration, despite the fact that an apparently normal regeneration blastema was initially formed. Instead, regeneration in these cases was limited to the formation of a variable number of small cartilage elements. On the basis of these results it is concluded that a complete limb circumference is required for distal transformation in newts, in addition to the well-established requirements for a wound epidermis, adequate innervation and dedifferentiation leading to blastema formation. A model for the sequential generation of new parts of the limb pattern during distal transformation from a complete circumference is presented. This model can also account for the occurrence of normal early stages of regeneration in double-half upper arms. Half upper arms which were amputated immediately were shown to develop single, complete regenerates. If amputation of half upper arms was delayed three or more weeks to permit complete wound healing, a supernumerary limb from the lateral wound surface sometimes developed in addition to a complete, single limb from the distal amputation surface.     

Effect of membrane splitting on transmembrane polypeptides

We investigated the effect of membrane splitting on the primary structure of human erythrocyte membrane polypeptides. Monolayers of intact, chemically unmodified cells were freeze-fractured and examined by one-dimensional SDS PAGE. Silver-stained gels revealed all major polypeptides that stain with Coomassie Blue as well as all bands that stain with periodic acid Schiff's reagent. Both nonglycosylated and glycosylated membrane polypeptides could be detected at concentrations of only a few nanograms per band. Membrane splitting had no effect on the position or number of bands. Monolayers of intact erythrocytes that had been enzymatically radioiodinated with lactoperoxidase were examined by electrophoresis, fluorography, and liquid scintillation counting. Radioactivity was quantified before and after monolayer formation and splitting, and at several stages of gel staining, drying, and fluorography. Although overexposed fluorographs revealed several minor radioiodinated bands in addition to band 3 and the glycophorins, no new bands were detected in split membrane samples derived from intact cells. These observations support the conclusion that neither the band 3 anion channel nor the glycophorin sialoglycoproteins are fragmented during freeze-fracturing. Although both band 3 and glycophorin partition to the cytoplasmic side of the membrane, preliminary quantitative observations suggest an enrichment of glycophorin in the split extracellular "half" membrane. We conclude that the process of membrane splitting by planar monolayer freeze-fracture does not cleave the covalent polypeptide backbone of any erythrocyte membrane protein, peripheral or integral.     

Inhibition and axial deviation of limb regeneration in the newt by means of a digit implanted into the amputated limb

This research was designed to follow up the observation of Thornton and Kraemer ('51) that regressed, denervated limbs of Ambystoma larvae will not regenerate upon reinnervation if all digits on the limbs were not completely resorbed. The object of this experiment was to determine whether the presence of an apical structure, protruding past the amputation surface, would affect the regenerative process. Both forearms of adult newts were amputated midway between the elbow and the wrist. One limb served as a normal regeneration control, and in the other limb the third digit from the removed hand was implanted in place of the removed radius, so that the three distal phalangeal segments protruded past the plane of amputation. Blastema formation in the experimental limbs was delayed by several weeks as compared with control limbs. Approximately one third of the experimental limbs did not regenerate. The regenerates that did form were strongly deviated (45–90°) radially from the longitudinal axis of the limb. Experimental analysis showed that the delay in regeneration is due largely to the projecting part of the digit. The radial deviation of the regenerates is not due to the digital implant, but rather to the removal of the radius. Trauma alone does not account for this phenomenon.     

Nerve-independence of limb regeneration in larval Xenopus laevis is related to the presence of mitogenic factors in early limb tissues.

Early limbs of larval Xenopus laevis can form a regeneration blastema in the absence of nerves. The nerve-independence could be due to the synthesis of neurotrophic-like factors by the limb bud cells. To test this hypothesis, two series of experiments were performed. Series A: the right hindlimbs of stage 57 larvae (acc. to Nieuwkoop and Faber. 1956. Normal table of Xenopus laevis [Daudin]. Amsterdam: North-Holland Pub. Co.), which are nerve-dependent for regeneration, were amputated through the tarsalia. The regenerating limbs were submitted to: sham denervation; denervation; denervation and implantation of a fragment of an early limb, or a late limb, or a spinal cord. Series B: froglets were subjected to amputation of both forelimbs. The cone blastemas were transplanted into denervated hindlimbs of stage 57 larvae, together with a fragment of an early or a late limb. The results in series A showed that the implantation of early limb tissue into the denervated blastema maintained cell proliferation at levels similar to those observed after the implantation of a spinal cord fragment or in sham denervated blastemas. However, the implantation of late limb tissues were ineffective. The results of series B showed that the implantation of early limb tissue, but not of late limb tissue prevented the inhibition of cell proliferation and the regression of denervated limb blastemas of juveniles. These results indicate that the nerve-independence is related to the synthesis of diffusible mitogenic neurotrophic-like factors in early limb tissues, and that nerve-dependence is established when differentiated cells of late limb tissues stop producing these factors.     

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

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

Cell proliferation in the amputated limb of lizard leading to scarring is reduced compared to the regenerating tail

After amputation, the tail of lizards regenerates while the limb forms a short scarring outgrowth. Using phospho‐histone‐H3 immunohistochemistry the mitotic activity of limb tissues at 12–25 days after amputation has been studied, when a limb outgrowth of 0.5–2 mm in length is covered by wound epidermis and the underlying connective is turning into a dense scar. In comparison with a regenerating tail of 3–5 mm in length, the number of dividing cells is reduced of 40–70% in different tissues of the scarring limb 1–2 mm in length at 18 days postamputation. Dividing cells are still present at 12–25 days postamputation in the cartilaginous epiphyses of the transected tibia and fibula and of the untransected femur. Also, the injured muscles present at the base of the scarring outgrowth still contain sparse dividing cells after 25 days postamputation of the limb. Together previous studies, the present observations suggest that after the initial proliferation of fibroblasts deriving from the injured tissues, especially from the dermis and intermuscle connectives during the initial 7–15 days postinjury, these cells cover the injured tissues underneath the wound epidermis, but rapidly produce high levels of collagen turning the initial blastema into a scar.