The relationship of the zone polarizing activity to supernumerary limb formation (twinning) in the chick wing bud.

The role of the polarizing zone mesoderm in development of supernumerary distal wing parts after 180° rotation of the wing tip was investigated. Postaxial mesoderm with and without polarizing tissue was repositioned preaxially in the wing bud and duplications occurred only when polarizing zone tissue was included. When the polarizing zone was removed and the distal tip of the wing reoriented, no duplication resulted. Similarly when the polarizing zone was removed, the distal tip reoriented and postaxial, nonpolarizing mesoderm introduced to restore the tissue mass of the stump, no twinning occurred. However, with the distal aspect reoriented on a stump from which postaxial, nonpolarizing mesoderm was removed, twinning occurred in 92.9% of the cases. Further, when the polarizing zone was removed, the distal aspect reoriented and a small piece of polarizing tissue returned, twinning resulted in 93.5% of the cases. These results indicate that polarizing zone tissue is required for the twinning that results after 180° rotation of the wing tip.     

Inhibitory effect on limb morphogenesis by cells of the polarizing zone coaggregated with pre- or postaxial wing bud mesoderm.

The influence of cells of the polarizing zone mesoderm on the morphogenesis of recombinant chick limbs was studied. The recombinant buds were composed of leg bud ectoderm and different regions of the wing bud mesoderm, which had been dissociated and reaggregated. In any case where the polarizing zone mesoderm was coaggregated with the wing mesoderm the morphogenetic capabilities of the recombinant were reduced. This was the case with postaxial mesoderm, preaxial mesoderm plus polarizing tissue, and postaxial mesoderm from which a piece of the nonpolarizing mesoderm (comparable in size to the polarizing zone) had been removed. All of these gave outgrowths with digits in only a very low percentage of cases. In contrast, those recombinants without polarizing mesoderm developed outgrowths with digits in a high percentage of cases, indicating good morphogenesis. Finally, if the polarizing zone were removed prior to dissociation, the recombinant limb, composed of the total remaining wing bud mesoderm plus leg bud ectoderm, exhibited a higher percentage of complete morphogenesis than if the polarizing zone had been part of the recombinant.It is clear that cells of the polarizing zone, when dissociated, and coaggregated with wing mesoderm, are inhibitory to the morphogenetic performance of that mesoderm in the recombinant limb situation.     

The control of the anteroposterior and dorsoventral axes in embryonic chick limbs constructed of dissociated and reaggregated limb-bud mesoderm

In the 3- to 4-day embryonic avian limb bud, a unique zone of mesodermal tissue is located posteriorly at the junction of bud and body wall. Appropriately grafted to a host limb bud, it induces the formation of a supernumerary limb outgrowth from preaxial tissue and determines that its posterior side will face the graft. It is called the zone of polarizing activity (ZPA).When limb-bud mesoderm is isolated, dissociated, reaggregated centrifugally, jacketed in the mesoderm-free hull of another limb bud, and grown as a graft on a host embryo, the recombinant frequently forms a limb-like structure terminating in digits that fail to show differentiation with respect to the anteroposterior axis. When, however, a bit of ZPA tissue is implanted in the recombinant subjacent to the anterior or posterior margin of the ectoderm, the resulting outgrowth shows a characteristic anteroposterior order of digits that corresponds to the placement of the implant, regardless of its relationship with the anteroposterior axis of the ectoderm or of the host embryo.Dorsoventral differentials have been recognized only in limbs formed from reaggregated leg-bud mesoderm. The direction of the dorsoventral axis always corresponds to the original axis of the ectodermal jacket regardless of the orientation of the recombinant on the host.     

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.     

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.     

Constitutive activation of sonic hedgehog signaling in the chicken mutant talpid(2): Shh-independent outgrowth and polarizing activity.

We have examined the developmental properties of the polydactylous chicken mutant, talpid(2). Ptc, Gli1, Bmp2, Hoxd13, and Fgf4 are expressed throughout the anteroposterior axis of the mutant limb bud, despite normal Shh expression. The expression of Gli3, Ihh, and Dhh appears to be normal, suggesting that the Shh signaling pathway is constitutively active in talpid(2) mutants. We show that preaxial talpid(2) limb bud mesoderm has polarizing activity in the absence of detectable Shh mRNA. When the postaxial talpid(2) limb bud (including all Shh-expressing cells) is removed, the preaxial cells reform a normal-shaped talpid(2) limb bud (regulate). However, a Shh-expressing region (zone of polarizing activity) does not reform; nevertheless Fgf4 expression in the apical ectodermal ridge is maintained. Such reformed talpid(2) limb buds develop complete talpid(2) limbs. After similar treatment, normal limb buds downregulate Fgf4, the preaxial cells do not regulate, and a truncated anteroposterior deficient limb forms. In talpid(2) limbs, distal outgrowth is independent of Shh and correlates with Fgf4, but not Fgf8, expression by the apical ectodermal ridge. We propose a model for talpid(2) in which leaky activation of the Shh signaling pathway occurs in the absence of Shh ligand.     

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.     

Intercalation and the cellular origin of supernumerary limbs in Xenopus

The hypothesis that a specialized polarizing zone controls the pattern of the anterior-posterior axis during limb development in Xenopus has been tested by analysing the cellular contribution to supernumerary limbs. Supernumerary limbs were generated by grafting hindlimb buds contralaterally between X. borealis and X. laevis to appose anterior and posterior limb tissues. Cells derived from these two species of Xenopus are readily identified by staining with quinacrine. The analysis of cellular contribution showed that supernumerary limbs consist of approximately half anterior-derived (57%) and half posterior-derived (43%) cells. These data are not consistent with the polarizing zone theory but are consistent with the hypothesis that both supernumerary limbs and normally developing limbs arise from intercalary interactions between limb bud cells with different positional values.     

The in vitro maintenance of the apical ectodermal ridge of the chick embryo wing bud: an assay for polarizing activity.

We have devised an in vitro bioassay for limb bud polarizing activity in the chick embryo. This assay has proven to be a relatively quick and effective test for a morphogenetic factor asymmetrically distributed in the limb bud which is capable of maintaining or thickening the apical ectodermal ridge.A small section of the preaxial border of the chick embryo wing bud was cultured alone, with tissue from the posterior border, mid-dorsal or anterior corner of a second donor wing, or from the flank. The tissue from the preaxial border (responding tissue) consisted of mesoderm with overlying ectoderm and apical ectodermal ridge. When the responding tissue was cultured alone, with flank, or with anterior corner limb tissue, the apical ectodermal ridge flattened in 24–36 hr and many macrophages appeared in the underlying mesoderm. When cultured with posterior border limb tissue however, the apical ridge of the responding tissue remained thickened for up to 48 hr., and no macrophages appear in the underlying mesoderm. The behavior of responding tissue was intermediate between these two extremes when cultured with mid-dorsal limb tissue. The morphogenetic activity assayed by this procedure thus seems to be present as a gradient in the wing bud, with activity decreasing from posterior to anterior. Contact with the responding tissue is not required to enable posterior border tissue to elicit ridge thickening and inhibit the cell death.     

The ability of the chick wing bud to regulate positional disparity along the anterior-posterior axis

When wedges of wing bud tissue are added to a host wing bud so there is positional disparity between graft and host, skeletal duplications result (L. E. Iten and D. J. Murphy 1980) Dev Biol. 75, 373-385. The polarity of the duplications is predictable by the polar coordinate model, leading to the interpretation that the positional disparity caused the duplications. To determine whether positional disparity alone causes duplications, without the complication of added tissue, we rotated wedges of ectoderm and mesoderm around the proximodistal axis within the wing bud. Wedges measuring 200-800 micron along the distal edge were rotated 180 degrees at stages 20-22, reversing the anteroposterior and dorsoventral axes relative to the bud. This caused positional disparity, similar to that achieved by Iten and Murphy (1980), without the addition of tissue. We found that rotations involving no polarizing zone tissue produced normal wings or wings lacking some distal parts, as did rotations of tissue lying entirely within the polarizing zone. However, when polarizing zone mesoderm was displaced, so that polarizing and nonpolarizing tissues were juxtaposed, a majority of the operations produced polarized skeletal duplications. Our data demonstrate that positional disparity alone does not cause skeletal duplications in the chick wing bud, unless polarizing zone tissue is displaced. Further, these data demonstrate that the chick wing bud can regulate to form a normal wing skeleton in the face of large positional disparity, provided that the polarizing zone is not moved. Finally, our results may be explained by the action of the proposed polarizing morphogen on the displaced cells causing repolarization.     

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.     

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].     

Retinoic acid induces a pattern of digits in anterior half wing buds that lack the zone of polarizing activity

Wing buds whose posterior half is excised, develop into wings lacking distal structures. However, such experimentally generated preaxial half wing buds can be rescued by implanting a retinoic-acid-releasing bead at their anterior margin. The polarity of the pattern that originates from preaxial half wing buds is reversed. For example, instead of a 234 digit pattern typical for normal wings, the order of digits is 432. This result implies that retinoic acid has the capacity to reprogram anterior limb bud tissue, and that the resulting change in cell fate does not depend on the presence of posterior tissue regions such as the zone of polarizing activity (ZPA).     

Normal development of the chick wing following removal of the polarizing zone.

The negative results of assays for polarizing activity along the posterior border of the chick wing 24 and 48 hours after removal of the polarizing zone demonstrate that this zone is not regenerated following removal. These results, and the fact that normal wing development can occur after polarizing zone removal from stages 15 through 24 wing buds, indicate that during these stages the polarizing zone has no direct role in normal development of the limb bud. It is speculated that the polarizing zone is effective only during limb induction and that after this time it exists in latent or residual form.     

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.     

Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation

The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6-12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.     

Disrupting the establishment of polarizing activity by teratogen exposure.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.     

Role of the chicken homeobox-containing genes GHox-4.6 and GHox-8 in the specification of positional identities during the development of normal and polydactylous chick limb buds.

During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is "posteriorized" and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative Activation of HoxD Homeobox Genes by Factors from the Polarizing Region and the Apical Ridge in Chick Limb Morphogenesis

When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted into the anterior margin of the chick limb bud, the expressions of the chick homeobox genes HoxD12 and D13 were induced prior to the formation of chick extra digits. This induction was observed in a restricted domain close to both the grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the distaloposterior region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, act cooperatively to provide positional information to induce the sequential expression of the HoxD genes.