Supernumerary limgs in amphibians: experimental production in Notophthalmus viridescens and a new interpretation of their formation.

The formation of supernumerary limbs was studied in the adult newt, Notophthalmus viridescens. Forelimb blastemas at the stages of medium bud and early digits were either transplanted to the contralateral forelimb with their dorsal-ventral axis opposed to that of the limb stump, or removed, rotated through 180°, and replaced on the same limb stump with both dorsal-ventral and anterior-posterior axes opposed to those of the stump, or as a control, removed, and replaced in normal orientation. Supernumerary limbs were produced in both experimental series, but not in the controls.Following contralateral transplantation, supernumerary limbs arose close to the graft junction at the two positions where dorsal limb tissue was in contact with ventral limb tissue. Both dorsal and ventral supernumerary limbs were of the same handedness as the limb stump and they were mirror-images of the regenerate developing directly from the transplanted blastema. Following 180° rotation, supernumerary limbs arose close to the graft junction at those positions where anterior-ventral and posterior-dorsal limb tissues were in contact. The supernumerary limb which arose in the posterior-dorsal position with respect to the limb stump was a mirror-image of the transplant, and was therefore of opposite handedness to both transplant and stump. The supernumerary limb which arose in the anterior-ventral position was of the same handedness as both transplant and stump. A new model of pattern regulation in epimorphic fields which can account for these results and which has retrospective value in the interpretation of earlier experiments on developing limbs is discussed.     

Interactions between irradiated and unirradiated tissues during supernumerary limb formation in the newt.

The interactions between irradiated and unirradiated blastemas and stumps in the newt forelimb were studied. Irradiated right blastemas at the stage of early digits were grafted to unirradiated left stumps and unirradiated left blastemas were grafted to irradiated right stumps. Grafts were oriented with their anterior-posterior axes opposed to that of the stumps. Supernumerary limbs ranging in completeness from one to four digits were found to arise predominantly on the anterior or posterior sides of the host limb. The graft developed well when the blastema was unirradiated and had reversed handedness with respect to the stump. Irradiated grafts developed poorly. On occasions, limbs with two supernumerary structures were found. The results are discussed in terms of the origin of the cells which comprise the supernumerary limbs and their bearing on a recently presented model concerned with pattern specification and regulation in epimorphic fields.     

Regeneration in the tail fan of crayfish

Summary The tail fan of a crayfish consists of the caudal end of the body, the telson, and the most caudal limbs, the uropods. We investigated the positional information in these structures with grafting operations. The uropods are biramous; they bifurcate to a lateral exopodite and a medial endopodite. After the distal part of a uropod ramus was grafted to the stump of a ramus, medio-lateral or dorso-ventral mismatch of surfaces provoked the production of supernumerary distal parts. Proximo-distal intercalation between exopodite and endopodite yielded a mosaic ramus. The results show that the two rami contain equivalent ramus fields in congruent orientation. The exopodite consists of basal and distal segments; each of these segments seems to have an equivalent segmental field.The telson regenerated an ablated distal portion poorly, unlike the limbs of crayfish. After the posterior lobe of the telson was inverted dorso-ventrally and grafted into the telson stump, supernumerary posterior lobes regenerated dorsal and ventral to the graft. Thus the dorsal and ventral surfaces of the telson embody different positional information. A grafted uropod endopodite or exopodite healed to the telson, but dorsoventral inversion of the graft did not provoke the formation of supernumerary structures at the graft-host boundary. Because supernumeraries did not form, the relations between positional information in the telson (a body axis structure) and the uropod (a limb) remain unclear.     

The interaction between the blastema and stump in the establishment of the anterior-posterior and proximal-distal organization of the limb regenerate

Interactions between the limb stump and the developing regenerate were studied in the limbs of adult newts, Notophthalmus viridescens. Forelimb blastemas at various stages were transplanted to the contralateral forelimb such that the anterior-posterior axes of stump and blastema were opposed. The blastemas were transplanted either from a proximal to distal, distal to proximal, proximal to proximal, or distal to distal level limb stump. The results indicate that at the earliest stage studied the anterior-posterior axis of the blastema is established but is not stable. An interection between the stump and blastema at this early stage results in the production of a variety of limbs intermediate in polarity between the graft and the stump. At all later stages, the original anterior-posterior axis of the blastema can be retained, although under certain grafting conditions the stump can still exert considerable influence over the anterior-posterior organization of the final regenerate. In those circumstances in which the blastema retains its original handedness, the interaction between stump and blastema results in the production of separate anterior and posterior supernumerary regenerates.The results of transplanting proximal blastemas to a distal limb level indicate that the proximal boundary of the blastema has been established by the earliest stage studied, leading to the production of limbs with serially duplicated segments. However, irrespective of the stage of a blastema transplanted from a distal to proximal level, there are no deleted structures in the proximal-distal axis of the resulting limb. From both histological examination of transplanted regenerates and the arrangement of skeletal elements of the resulting limbs, it is postulated that the stump plays an important role in the production of the intercalary regenerate.     

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.     

Types of supernumerary outgrowths produced after inverting the dorsoventral limb axis of the anuranBufo bufo

Summary Contralateral grafts were performed on the larval limb buds of the anuranBufo bufo. The dorsoventral axis of 80 buds at stages IV or V was inverted. Ten tadpoles were used as controls. Fifty-two supernumerary structures developed, all of them in dorsal or ventral locations on the host stump. The majority (32 out of the 44 outgrowths with more than 3 toes) were normal limbs of stump handedness. However, the following abnormal structures were also observed: 2 double-posterior, 3 mixed-symmetric, and 7 undetermined cases. These results are in agreement with the predictions of a hierarchical polar coordinate model for epimorphic regeneration.     

Organization of positional information in the axolotl limb

We have used the phenomenon of position-dependent growth stimulation, brought about by the confrontation of cells with dissimilar positional values, to reveal the organization of positional information in the center of the upper and lower arms of axolotls. When either humerus or radius was transplanted into either dorsal or posterior positions, extra growth leading to the formation of supernumerary digits occurred following amputation through the graft. However, transplants of humerus or radius into anterior or ventral positions did not lead to the formation of any additional digits. The ulna by contrast was capable of stimulating supernumerary digit formation when transplanted into anterior, posterior, dorsal, or ventral positions. We interpret these results to indicate that the humerus and radius are surrounded by symmetrically arranged anterior and ventral positional values, whereas the ulna is surrounded by a complete asymmetrical set of angular positional values. We use our proposed arrangement for the positional information in the limb center to explain a number of previous experimental findings. In addition, we provide an explanation, in terms of the underlying positional information, for the structural and developmental relationships between the different skeletal elements of the vertebrate limb, and in particular for the anatomical pattern known as Gregory's pyramid.     

Formation of supernumerary structures by the embryonic chick wing depends on the position and orientation of a graft in a host limb bud

The relationship between the position transplanted in a host limb bud, the orientation of a graft in a host limb bud, and the extra limb structures formed was studied by juxtaposing normally nonadjacent embryonic chick wing bud tissue. In one series of transplantation operations, two different wedges (ectoderm and mesoderm) of stage 21 right donor posterior wing bud tissue were transplanted to the middle of a host stage 20 to 22 right wing bud such that the dorsal-ventral polarity of the graft and host were the same or reversed. The results of these transplantation operations show that the formation of supernumerary limb structures depends on the position of origin of the donor tissue, the anterior-posterior position transplanted in a host limb bud, and the orientation of the graft in the host limb bud. In a second series of transplantation operations, the relationship between the proximodistal position where posterior donor tissue is transplanted in an anterior host site and the extra structures formed was studied. A wedge of posterior stage 21 right wing bud tissue was transplanted to an anterior proximal or anterior distal site of a stage 22 to 24 host right wing bud. The results of these transplantation operations show that when the donor tissue is transplanted to an anterior proximal position in a host wing bud, then limbs with only a duplicated humerus result, whereas, when transplanted to an anterior distal position, then limbs with a duplicated forearm element and extra digits result.     

Supernumerary regeneration in the large milkweed bug Oncopeltus fasciatus.

As an approach to the problem of pattern formation in the insect appendage, various graft combinations were studied in the legs of the large milkweed bug Oncopeltus fasciatus. Metathoracic legs of fourth instar larvae were amputated through the tibia within 24 hr after ecdysis and grafted back onto the stumps. The orientation of the graft was altered by rotation through 90 or 180° and/or by exchanging right and left stumps and grafts, yielding seven possible orientations in addition to the control. Many of these grafts resulted in the production of one or two supernumerary regenerates of the distal segments, which appeared at the graft junction after the second postoperative ecdysis. When two supernumerary regenerates resulted, one appeared to be produced from the stump and the other from the graft. When one regenerate was present, it appeared to be a composite of material produced from both the stump and the graft. In contrast to the results obtained in cockroaches, the external face of the leg appeared to be the only one capable of giving rise to a supernumerary regenerate.     

Diverse supernumerary structures develop after inverting the anteroposterior limb axis of the anuran

Contralateral limb bud graftings were performed on tadpoles of the anuran Bufo bufo. The anteroposterior axis was inverted while the larvae were at stage IV or V (e.g., between 22-30 days after fertilization). Eighty-four tadpoles were operated on, 10 of which were used as controls. At anterior or posterior location 104 supernumerary structures developed in toto. They were collected and whole-mount examined after being stained with Alcian blue. They were further prepared for serial sectioning, mounting, and staining with hematoxylin and eosin. The majority of these supernumerary structures were found to be normal limbs of the stump handedness in agreement with all models and experiments on the urodeles axolotl and newt. However, some of the structures were clearly abnormal: double symmetric or of mixed handedness. This result is consistent with a prediction of a hierarchical polar coordinate model. The fact that no such structures have been found in the experiments on the urodeles may be due to the expected low probability for their appearance and the fact that only few such limbs have been sectioned and analyzed as yet.     

Compatible limb patterning mechanisms in urodeles and anurans

We have experimentally tested the similarity of limb pattern-forming mechanisms in urodeles and anurans. To determine whether the mechanisms of limb outgrowth are equivalent, we compared the results of two kinds of reciprocal limb bud grafts between Xenopus and axolotls: contralateral grafts to confront anterior and posterior positions of graft and host, and ipsilateral grafts to align equivalent circumferential positions. Axolotl limb buds grafted to Xenopus hosts are immunologically rejected at a relatively early stage. Prior to rejection, however, experimental (but not control) grafts form supernumerary digits. Xenopus limb buds grafted to axolotl hosts are not rejected within the time frame of the experiment and therefore can be used to test the ability of frog cells to elicit responses from axolotl tissue that are similar to those that are elicited by axolotl tissue itself. When Xenopus buds were grafted to axolotl limb stumps so as to align circumferential positions, the majority of limbs did not form any supernumerary digits. However, in experimental grafts, where anterior and posterior of host and graft were misaligned, supernumerary digits formed at positional discontinuities. These results suggest that Xenopus/axolotl cell interactions result in responses that are similar to axolotl/axolotl cell interactions. Furthermore, axolotl and Xenopus cells can cooperate to build recognizable skeletal elements, despite large differences in cell size and growth rate between the two species. We infer from these results that urodeles and anurans share the same limb pattern-forming mechanisms, including compatible positional signals that allow appropriate localized cellular interactions between the two species. Our results suggest an approach for understanding homology of the tetrapod limb based on experimental cellular interactions.     

Retinoic acid modifies positional memory in the anteroposterior axis of regenerating axolotl limbs

The effects of retinoic acid (RA) on anteroposterior (AP) positional memory of regenerating axolotl limbs were tested after removing the anterior or posterior half from the zeugopodium (lower arm or leg). RA (150 micrograms/g body wt) was injected into groups of animals bearing the following types of limbs: (1) anterior and posterior half zeugopodia grafted to the eyesocket and amputated distally 7 days later; (2) unamputated anterior and posterior half zeugopodia in situ; (3) double anterior and double posterior half zeugopodia amputated distally 7 days after their construction; (4) sham-operated zeugopodia amputated distally 7 days after operation. Controls consisted of these four groups injected with the retinoid solvent, dimethyl sulfoxide, or not injected. Control half zeugopodia grafted to the eyesocket regenerated no more than one or two digits. Control unamputated half zeugopodia in situ underwent partial or complete regeneration of the missing half from the proximal and midline wound surfaces exposed during construction of the half zeugopodia. Control double anterior and posterior zeugopodia both regenerated symmetrical, hypomorphic regenerates with 1-3 digits in the double anteriors and 1-6 digits in the double posteriors. Sham-operated controls regenerated normally. Regenerating anterior and posterior halves responded differently to RA. RA-treated anterior half zeugopodia in the eyesocket, and anterior half stumps adjacent to the unamputated posterior half zeugopodia in situ both produced regenerates that duplicated stump structures in the proximodistal axis and formed a complete and normal AP pattern. RA-treated double anterior zeugopodia regenerated proximodistal-duplicated pairs of mirror-imaged limbs, each with a complete and normal AP pattern. In contrast, half posterior zeugopodia in the eyesocket, the posterior half stumps of unamputated half anterior zeugopodia in situ, and double posterior zeugopodia all failed to regenerate. These results suggest that RA modifies positional memory in only one direction in the AP axis, posterior.     

THE DYNAMICS OF WOUND CLOSURE AND ITS ROLE IN THE PROGRAMMING OF PLANARIAN REGENERATION. II — DISTALIZATION

When the dorsal and ventral epidermal layers join by first intention during the closure of the wound, the cells of their borders (M-cells) do not meet in the same manner in all sections. In anterior sections the dorsal M-cells attach themselves to the ventral basement membrane, so that only the dorsal epidermis is stretched. In posterior sections the dorsal and the ventral M-cells join by their apical edges without being closely apposed to the wound surface. Only the ventral cells are stretched because of their specific motility. In longitudinal sections the dorsal and the ventral M-cells also join by their apical edges, but since they are closely apposed to the wound surface both epidermal layers are stretched. The stretching is a process equivalent to distalization. The junction between the dorsal and the ventral epidermis is shifted ventrally in the anterior wounds (as in the intact heads) and dorsally in the posterior wounds (as in the intact tails). Some abnormalities of wound closure have been observed at levels where heteromorphic regeneration frequently occurs. These findings are consistent with the hypothesis previously advanced (3) that the modalities of wound closure establish the programme for regeneration.     

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.     

周围神经再生过程中靶器官的诱向性作用机制

以细胞培养技术与自然凝胶电泳系统方法证明,在周围神经再生过程中,损伤的坐骨神经远侧端,即起衍生的靶器官诱导神经突起的定向生长。分析探讨与神经诱向性再生相关的活性因子,其结果提示在远侧端神经组织中出现的90kDa蛋白组分具有很强的诱向性作用,诱导神经突起在神经再生过程中能够准确地到达靶器官。由此说明雪旺细胞在神经再生过程中扮演着重要角色。     

Evidence for polarizing zone in the limb buds of Xenopus laevis

To test for the presence of polarizing mesoderm in an amphibian, Xenopus laevis hindlimb bud tips were rotated 180° on the proximodistal axis and returned to the stump. Supernumerary outgrowths were induced in the preaxial stump and preaxial tip tissues, and the most postaxial digit always formed next to the grafted postaxial tissue. The occurrence of polarized supernumerary outgrowths indicated that the posterior limb border contained a polarizing zone. When the limb tip was cut at varying known lengths from the body wall, rotated, and grafted to the limb stump, the incidence of twinning along the proximodistal axis permitted insight into the distribution of the polarizing zone along the posterior border. The location of polarizing tissues was found to be similar to that in the chick wing bud at comparable stages. To confirm the posterior border stump influence on the rotated preaxial limb tip tissues, 180° tip rotations were made at the proximodistal level with the highest incidence of twinning. In these cases, the adjacent stump posterior border tissues (polarizing zone) were removed, leaving a substantial amount of the deeper postaxial stump tissue, however. The frequency of twinning from tip tissues was greatly reduced in these larvae compared to those with rotated limb tips on intact stumps. Cytological examination of supernumerary outgrowths resulting from grafts of two-nucleolate tips onto one-nucleolate stumps confirmed the preaxial source of the supernumerary outgrowths.     

两栖肢场的插值再生和图式调节

French-Bryant-Bryant的增生场图式调节机制被合理地作了修正,从而自洽地测决了同侧嫁接和异侧嫁接致生正常重肢的形成问题:(1)指出细胞插值生长和远向变换约束之间的平衡是一种自发对称性破损机制,会导致全区捷径插值被分区捷径插值局部地(在异侧嫁接)或整体地(在同侧嫁接)取代,分别在全区和分区极大周值差位置致生重肢.(2)关于重肢的手性和细胞来源的理论预测与实验结果吻合.(3)对于异常重肢的结构复杂性也提出了一种可行的解释.     

Morphogenetic interactions occurring between blastemas and stumps after exchanging blastemas between normal and double-half forelimbs in the axolotl, Ambystoma mexicanum.

Regeneration blastemas were exchanged between surgically constructed forelimbs comprised of symmetrical tissues (double-anterior and double-posterior) and normal, unoperated forelimbs. Normal blastemas grafted at the stage of medium bud (MB) onto double-half forelimb stumps regenerated normal skeletal patterns in nearly all cases. Double-half blastemas transplanted at the stage of MB onto normal forelimb stumps did not regenerate complete limb patterns. These results indicate that a double-half blastema cannot be “rescued” by transplantation to a normal stump and that a double-half limb stump does not interfere with the ability of a normal blastema to distally transform. The regeneration blastema possesses sufficient positional information at the stage of MB to permit it to develop autonomously. Supernumerary forelimbs resulted from several types of graft-stump combinations. The location and handedness of these supernumerary limbs are predicted by the rules of a recently presented model for pattern regulation in epimorphic fields [French, V., Bryant, P. J., and Bryant, S. V. (1976). Science193, 969–981].     

The free thoracoumbilical flap for resurfacing large soft-tissue defects of the lower extremity

Both cadavers and living patients were studied regarding a method to resolve large skin defects with bone exposure in the leg, with long-distance thrombosis of the anterior tibial vessels or posterior tibial vessels resulting from traumatic lesions. Forty-six casting mold specimens of cadaveric legs were investigated. There were rich communication branches among the anterior tibial artery, posterior tibial artery, and fibular artery in the foot and ankle, which complemented each other well. Twenty-six patients with large skin defects with bone exposure in the proximal or middle segment of the leg were admitted to the authors' hospital. Among those patients, 19 demonstrated long-distance thrombosis of the anterior tibial vessels or posterior tibial vessels resulting from traumatic lesions. During treatment, a thoracoumbilical flap based on the inferior epigastric vessels was anastomosed to the distal stump of the anterior tibial vessels or the posterior tibial vessels, with reversed flow. All defects were successfully repaired, with good color and texture matches of the flaps. This method can be used for patients with normal anterior tibial vessels or posterior tibial vessels, normal distal stumps of the injured blood vessels, and good reversed flow. The method has the advantages of dissecting blood vessels in the recipient area during the débridement, not affecting the blood circulation of the injured leg, not sacrificing blood vessels of the opposite leg, and not fixing the patient in a forced posture. The muscles are less bulky in the distal one-third of the leg, and the blood vessels are shallow and can be dissected and anastomosed easily. When the flap is used for reconstruction in the proximal two-thirds of the leg, the blood vessel pedicle of the free flap is at a straight angle, without kinking.     

Regulation after proximal or distal transposition of limb regeneration blastemas and determination of the proximal boundary of the regenerate.

When blastemas of several stages of differentiation were grafted in normal orientation to stump levels proximal or distal to their level of origin, normal limbs regenerated. Histological and autoradiographic studies of the development of these regulated limbs showed that the grafted blastemas formed only structures normally distal to their level of origin. In the case of a blastema transplanted proximally, regulation occurred by intercalary regeneration from the stump, whereas, when blastemas were transplanted distally, regulation appeared to take place within the blastema itself by a distal shift in its pattern of organization. The results suggest that the proximal limit of the limb regenerate is determined by level-specific properties of the limb cells but that these properties allow for interactions leading to regulation when different levels of stump and blastema are brought together.