The occurrence of supernumerary limbs following blastemal transplantation in the regenerating forelimb of the axolotl, Ambystoma mexicanum

Regeneration blastemas at the stages of medium bud and palette were transplanted to contralateral limb stumps so that either their anterior and posterior positions or their dorsal and ventral positions were apposed to those of the stumps. Grafts were shifted from distal levels to proximal levels, or from proximal levels to distal levels, or remained at either a proximal or a distal level. When anterior and posterior positions of graft and stump were apposed, supernumerary limbs were produced at the graft-stump junction in anterior and posterior positions relative to the stump. All analyzable supernumerary limbs were of stump handedness. Apposition of dorsal and ventral positions of graft and stump led to the formation of supernumerary limbs at dorsal and ventral positions relative to stump tissues. All analyzable supernumerary limbs were once again of stump handedness. Shifts from distal levels to proximal levels never resulted in skeletal deletions, as potential deletions in the proximal-distal axis were always filled in. Shifts from proximal levels to distal levels resulted in a low frequency of serial duplications. The results are discussed in view of a recently presented formal model for pattern regulation in epimorphic fields.     

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.     

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.     

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.     

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 structure of 180° supernumerary limbs and a hypothesis of their formation

The results of a detailed analysis of 100 supernumerary limbs generated by 180° ipsilateral rotation (on the same limb stump) of regeneration blastemas is presented. The limbs were analyzed in terms of their position of origin, frequency, cartilage structure by Victoria blue staining, and muscle structure by serial sections. Single, double, or triple supernumeraries can be produced at no unique position of origin, although the posterodorsal quadrant was preferred. Four classes of supernumerary limbs were generated by such operations—normal; double dorsal or double ventral; part normal/part mirror imaged; part normal/part inverted in approximately equal frequencies. After amputation of these supernumeraries the same muscle patterns are faithfully regenerated. A hypothesis to explain the production of these abnormal limbs is proposed based on the observed phenomenon of fusion of supernumerary blastemata, but their regenerative behaviour presents problems for current models of pattern formation. Similar results have been obtained with developing limb buds and the relation between development and regeneration is discussed.     

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].     

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.     

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.     

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.     

Cellular contribution to supernumerary limbs resulting from the interaction between developing and regenerating tissues in the axolotl

The relationship between limb development and limb regeneration is considered with regard to the mechanisms by which pattern is established during limb outgrowth. In a previous paper (Muneoka, K. and Bryant, S. V. 1982 Nature (London) 298, 369-371) the interaction between cells from the developing limb bud and the regenerating limb blastema was found to result in the production of organized supernumerary limb structures. In this paper the relative cellular contribution from developing and regenerating cells to supernumerary limbs resulting from contralateral grafts between limb buds and blastemas has been analyzed using the triploid cell marker in the axolotl. Results show that there is substantial participation from both developing and regenerating limb cells to all supernumerary limbs analyzed. These data lend further support to the hypothesis that developing and regenerating limbs utilize the same patterning mechanisms during limb outgrowth. This conclusion is discussed in terms of patterning models for developing and regenerating limbs and it is proposed that the rules of the polar coordinate model can best explain the behavior of cells during limb development as well as limb regeneration.     

Regeneration and supernumerary limb formation under sparsely innervated conditions

Normally, urodele limb regeneration is nerve-dependent. Reduction in nerve-dependency has been reported for regenerating, transplanted newt limbs (Singer and Mutterperl, '63). Aneurogenic limbs can regenerate without nerves (Yntema, '59). Induction of supernumerary limbs may be obtained from aneurogenic limbs of larval Ambystoma after transplantation orthotopically to innervated larvae and with normal nerve ingrowth to the limb transplant prevented by repeated section of brachial nerves. Of the 13 (of 43) grafts with supernumeraries, nerve counts showed 11 with 0–5; 1 with 5–10; and 1 with 20⁺ fibers. Orthotopically grafted aneurogenic limbs allowed to become innervated showed 14 supernumeraries in 49 grafts. This supernumerary limb induction is thus not nerve-dependent. Normally, innervated larval Ambystoma limbs grafted orthotopically and heteroplastically regenerated in 17 of 37 cases after repeated section of brachial nerves. Of the 17 regenerates nerve counts showed 4 with 0–5; 5 with 5–10; 7 with 10–19; and 1 with 20⁺ fibers. Larval limbs heteroplastically transplanted may require very few or no nerves for regeneration.     

BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb.

We demonstrate that signaling via the bone morphogenetic protein receptor IA (BMPR-IA) is required to establish two of the three cardinal axes of the limb: the proximal-distal axis and the dorsal-ventral axis. We generated a conditional knockout of the gene encoding BMPR-IA (Bmpr) that disrupted BMP signaling in the limb ectoderm. In the most severely affected embryos, this conditional mutation resulted in gross malformations of the limbs with complete agenesis of the hindlimbs. The proximal-distal axis is specified by the apical ectodermal ridge (AER), which forms from limb ectoderm at the distal tip of the embryonic limb bud. Analyses of the expression of molecular markers, such as Fgf8, demonstrate that formation of the AER was disrupted in the Bmpr mutants. Along the dorsal/ventral axis, loss of engrailed 1 (En1) expression in the non-ridge ectoderm of the mutants resulted in a dorsal transformation of the ventral limb structures. The expression pattern of Bmp4 and Bmp7 suggest that these growth factors play an instructive role in specifying dorsoventral pattern in the limb. This study demonstrates that BMPR-IA signaling plays a crucial role in AER formation and in the establishment of the dorsal/ventral patterning during limb development.     

Inductive activity and enduring cellular constitution of a supernumerary apical ectodermal ridge grafted to the limb bud of the chick embryo.

Apical ectodermal ridges (AERs) isolated from 3- to 4-day chick and quail embryos were prepared by means of trypsinization and microdissection and then were grafted to the dorsal or ventral side of a host chick wing bud. They induced supernumerary limb outgrowths from the host bud showing, respectively, a bidorsal or biventral organization, as determined by the patterns of feather germs. The grafted ridge cells persisted, as revealed by histological sections of supernumerary chick limb parts growing under the influence of quail AERs, whose cells are readily distinguished after application of the Feulgen reagent.These results show that the AER induces limb outgrowth regardless of whether it is associated with dorsal or ventral limb ectoderm and that its continued existence is not dependent on contributions of ectodermal cells from the opposed ectodermal faces of the limb bud. The AER is pictured as maintaining the subjacent mesoderm in a condition of developmental plasticity without specifying its differentiation with respect to the proximodistal axis. It remains uncertain whether the positional values of cells that develop under the influence of the AER arise within these cells themselves or appear in response to influences from proximal sources.     

Alteration of the retinotectal projection map by the graft of mesencephalic floor plate or sonic hedgehog

The floor plate plays crucial roles in the specification and differentiation of neurons along the dorsal-ventral (DV) axis of the neural tube. The transplantation of the mesecephalic floor plate (mfp) into the dorsal mesencephalon in chick embryos alters the fate of the mesencephalon adjacent to the transplant from the tectum to the tegmentum, a ventral tissue of the mesencephalon. In this study, to test whether the mfp is involved in the specification of the DV polarity of the tectum and affects the projection patterns of retinal fibers to the tectum along the DV axis, we transplanted quail mfp into the dorsal mesencephalon of chick embryos, and analyzed projection patterns of dorsal and ventral retinal fibers to the tectum. In the embryos with the mfp graft, dorsal retinal fibers grew into the dorsal part of the tectum which is the original target for ventral but not dorsal retinal fibers and formed tight focuses there. In contrast, ventral retinal fibers did not terminate at any part of the tectum. Transplantation of Sonic hedgehog (Shh)-secreting quail fibroblasts into the dorsal mesencephalon also induced the ectopic tegmentum and altered the retinotectal projection along the DV axis, as the mfp graft did. These results suggest that some factors from the mesencephalic floor plate or the tegmentum, or Shh itself, play a crucial role in the establishment of the DV polarity of the tectum and the retinotectal projection map along the DV axis.     

Pattern regulation in the embryonic chick limb: Supernumerary limb formation with anterior (non-ZPA) limb bud tissue

The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host's limb bud or reversed to that of the host's. Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen.     

Position of origin of donor posterior chick wing bud tissue transplanted to an anterior host site determines the extra structures formed

The formation of supernumerary limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. Different “wedges” (ectodern and mesoderm) of posterior donor right wing bud (stage 21) were transplanted to a slit made in stage 20–23 host right wing buds. Donor posterior tissue was transplanted to an anterior position in a host wing bud or, as a control, to the same position as its position of origin. Transplanting different wedges of posterior tissue to the same anterior host position results in wings with supernumerary structures, and different extra structures form depending on the position of origin of the donor tissue. The identification of extra limb structures formed was based on the skeletal and integumentary patterns of resulting wings and the pattern of muscles as seen in serial sections of resulting limbs. The results of experiments presented here are considered in light of current models that have been used to describe the formation of supernumerary limb structures by the embryonic chick limb bud.     

A hierarchical polar coordinate model for epimorphic regeneration

In the framework of polar coordinates three rules are postulated which can describe epimorphic regeneration in amphibian limbs. The rules can be seen as an extension of the polar coordinate model. When cells with different positional values are confronted, cell proliferation at the junction restores the continuity of positional values. Reestablishment of continuity is associated with the eventual congruence of the intercalating cell sequence with the host or graft, or both. The intercalating contours can be simple or twisted. The possible contours are graded within a plausible hierarchical scheme where congruent paths are favored versus non-congruent paths and simple contours are favored versus twisted contours. The model correctly predicts the multiplicity, position and different structures of supernumerary outgrowths resulting from both contralateral graftings and 180 degrees ipsilateral limb rotations. Development and regeneration of mirror-symmetric limbs are also accounted for. Several other experimental results are in agreement with the model. Many model predictions and correlations still remain to be tested.