Régulation des excédents dans le développement du bourgeon de membre de l'embryon d'oiseau. Analyse expérimentale de combinaisons xénoplastiques caille/poulet

Summary In order to support the demonstration of the regulative capacity of the chick limb bud, already stressed by one of us (Kieny, 1964, 1967), heterospecific combinations were made between chick and quail tissues, the cells of the latter bearing a distinctive nuclear marker. A Japanese quail whole limb bud (stage-18 to 21 of H. H., wing or leg) was grafted distally onto the prospective zeugopod of a chick (stage-22) wing bud sectioned at the prospective wrist level. Thus, from a heterospecific surplus recombinant containing five prospective limb segments (stylopod and zeugopod from the chick host; stylopod, zeugopod and autopod from the quail graft), it was possible to obtain a normally shaped appendage that comprised either upper arm, lower arm and hand in the case of a wing bud graft, or heteromorphic upper arm, lower leg and foot in the case of a hind-limb bud graft. In these cases, regulation for excess appeared to take place mainly within the host tissues. The three proximal segments of the recombinant, namely the chick stylopod and zeugopod of the host's stump and the quail stylopod of the graft, became reorganized and gave rise to a single stylopodial segment, which usually contained a double stylopodial bone element, one of chick, the other of quail origin.The absence of development of the squeezed prospective zeugopod can be interpreted as follows: owing to an interaction with the stylopodial graft tissues, the zeugopodial cells of the juxtaposed stump boundary have shifted proximally their originally more distal positional values, so that they changed their prospective pattern of differentiation to that of stylopod. These reset zeugopodial cells combine with the stylopodial cells of host and graft and form a huge composite stylopod, in which, due to an asynchronous determination in the two species, chick and quail tissues do not cooperate fully for the development of a single bone.

Ce travail a été effectué avec l'aide de la D.G.R.S.T. (Action complémentaire coordonnée: Biologie de la reproduction et du développement, convention n^o 73-7-1661)     

Intercalary regeneration of symmetrical thighs in the axolotl,Ambystoma mexicanum

Intercalary regeneration of stylopodial and zeugopodial skeletal elements takes place in axolotl limbs composed of normal wrist blastemas autografted or homografted to double half-anterior or half-posterior thighs. Analysis of the morphological pattern of the skeleton and, in homografts, of pigmentation pattern, shows that the intercalated elements are derived from the host double half-thigh. Intercalary regeneration from double half-posterior thighs is expected since they normally can undergo complete proximal-distal regeneration, but is not necessarily expected from double half-anterior thighs, since they normally do not regenerate more distal segments. These results demonstrate that (1) cells of double half-anterior thighs are not inherently incapable of undergoing distal transformation, (2) cells of a distal blastema grafted to a more proximal level do not form patterns proximal to their level of origin, and (3) there is an inhibitory interaction between blastema cells derived from double half-anterior thighs that is expressed after simple amputation, but not when these cells are in contact with a more distal, normal blastema. Using these and other data, a three-dimensional boundary model of limb regeneration is proposed.     

Retinoic acid-induced pattern duplication in regenerating urodele limbs

The effects of varying doses of retinoic acid on forelimb regeneration in larval Ambystoma mexicanum amputated through the wrist joint and in adult Notophthalmus viridescens amputated through the basal carpals were compared. In both species, the major effect of retinoic acid was to cause the proximodistal duplication, in the regenerate, of stump segments proximal to the amputation plane. Transverse axial duplications (anteroposterior and dorsoventral) occurred in a smaller percentage of cases; these consisted of cartilage spurs in axolotls, and extra digits in newts. The frequency and magnitude of the proximodistal and (in the newt) transverse duplications were dose dependent, and the regenerating limbs were maximally sensitive to the retinoid during the period of dedifferentiation and accumulation of blastema cells. The effect of retinoic acid is exerted on cells local to the amputation surface, as shown by the fact that retinoic acid caused the proximodistal duplication of stump segments in regenerates derived from amputated distal lower arm segments grafted to the eyesocket.     

Stage-dependent effects of retinoic acid on regenerating urodele limbs

Summary Following amputation through the distal zeugopodium, regenerating limbs of larvalAmbystoma mexicanum and pre and post-metamorphicPleurodeles waltlii were treated with 150 g of retinoic acid (RA) per gram of body weight, at the dedifferentiation, early bud, medium bud, late bud or early redifferentiation stages of regeneration. The effect of RA on regenerate morphogenesis differed as a function of the stage at which it was administered. When given during dedifferentiation or at early bud stages, RA evoked proximodistal duplications of stump segments in the regenerates. The maximum duplication index (DI) inAbystoma was achieved when RA was injected at 4 days post-amputation, which corresponds to the stage of dedifferentiation; and inPleurodeles at 10 days post-amputation, which corresponds to a stage midway between early bud and medium bud. When RA was administered at later stages, the DI declined progressively to zero or nearly zero by the stage of early redifferentiation in both species. The decline in DI was due to a decreased frequency of duplication, not to a decrease in the magnitude of duplication in individual regenerates. At the same time, there was an increase in hypomorphism and aberrant morphogenesis of both duplicating and non-duplicating regenerates. These results indicate that regenerative cells are differentially sensitive to RA in a stage-dependent way.     

Intercalary leg regeneration in the large milkweed bug Oncopeltus fasciatus.

Grafting operations were performed on the metathoracic legs of fourth instar Oncopeltus fasciatus within 24 hr after ecdysis. Different levels along the tibia were combined so that a lengthened tibia, approximately 1.5 times the normal length, or a shortened tibia, about half the normal length, were created. Intercalary regeneration occurred between the graft and stump in both combinations and the extra tissue was visible on the adult leg. The intercalary regenerate produced by the lengthened tibia showed reversed bristle polarity, while that produced in the shortened segment showed normal polarity. It is suggested that a regenerate with reversed polarity represents a mirror image duplication of the graft and might originate from the graft, whereas a regenerate with normal polarity may originate, as in normal regeneration, from the stump. It appears that each level in the appendage has the developmental capacity to produce only more distal structures. This conclusion is supported by the results of a grafting operation in which a portion of the tibia was grafted back on to the stump with its proximo-distal axis reversed. Regeneration of appropriate distal structures proceeded from the free proximal surface of the grafted tibia.     

Avian long bones, flight and bipedalism.

Morphological, geometrical, chemical and mechanical characteristics of avian long bones are reviewed. Important differences exist between long bones of birds and mammals. Differences are also present in appendicular bones of birds, either between wing bones and leg bones or proximal (stylopodial) long bones and distal (zeugopodial) long bones. Special emphasis is put on pneumatization, in terms of both phylogenetic origin and geometrical and mechanical characteristics linked to it. Cortical thickness, bending strength and flexural Young's modulus were significantly lower in pneumatized bones than in marrow-filled bones. Possible adaptive reasons for the differences shown are discussed.     

Leg Regeneration in Insects: Cell Interactions and Lineage

SYNOPSIS. Developing insect legs have positional informationspecified down the length and around the leg circumference.After grafting or amputation of larval cockroach or cricketlegs healing confronts epidermal cells with different positionalvalues. This stimulates growth, the intercalary regenerationof intervening tissue, the regeneration of all more distal tissuefrom a complete leg circumference and often the formation ofan incomplete distal regenerate from a symmetrical part-circumference.These processes will lead to regeneration of missing structures,duplication of structures, or the formation of branched supernumerarylegs, depending on the situation. During regeneration, cellscannot cross lineage restrictions which divide the leg intoanterior and posterior compartments.     

Regeneration of appendages in the large milkweed bug, Oncopeltus fasciatus

An analysis was made of the regeneration of legs and antennae of Oncopeltus. Amputations were performed on first instar larvae within 24 hr after hatching, and on later instars within 24 hr after ecdysis. The resulting regenerates were then measured at each instar. When amputations were performed soon after hatching, there was no significant effect on the duration of any instar. The regenerate was usually visible after the second post-operative ecdysis, and was smaller than a normal appendage (hypomorphic). Removal of the three distal segments of the antenna usually resulted in regeneration of only one segment which was abnormally long and showed a combination of the bristle patterns characteristic of the two most distal segments of the control. In a few such cases a partial intersegmental membrane was present in the regenerated segment. Removal of the tarsus resulted in a structurally complete regenerate which was smaller than the control tarsus. The largest leg regenerates were obtained when amputation was performed through the tibia. With amputation through the femur, a decrease in length of the remainder of this segment was observed after the first ecdysis. This type of amputation and amputation through the trochanter in some cases resulted in the formation of a globular stump containing tarsal claws. The results indicate that amputation of part of an appendage in Oncopeltus does not stimulate an increased growth rate in the stump, but merely causes reorganization of the stump material which subsequently grows at the normal rate. Since even the most hypomorphic regenerates contained well-formed claws, even though proximal parts were missing, it appears that the reorganization process must begin at the most distal point and proceed proximally.     

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 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.     

Dosage Compensation in Drosophila: Nadp-Enzyme Activities and Cross-Reacting Material

The relationships between gene dosage, enzyme activities and CRM levels have been determined for G6PD and 6PGD. Enzyme activities and CRM levels were directly proportional and increased in genotypes carrying duplications of the respective structural genes. When a duplication consisting of the distal 45% of the X chromosome was used to duplicate Pgd+, 6PGD activity and CRM increased and G6PD activity decreased. When the proximal 55% of the X chromosome was duplicated, G6PD activity and CRM increased whereas 6PGD activity and CRM levels decreased. These observations support the model of dosage compensation of X-linked genes that invokes an autosomal activator in limited concentrations for which X-linked loci compete. The distal 45% of the X chromosome, when duplicated, caused a significant increase in NADP-malic enzyme activity and CRM levels, as if a structural gene for NADP-ME is sex-linked.     

Evaluation of the sensitive step of inhibition of chondrogenesis by retinoids in limb mesenchymal cells in vitro

The sensitive step of inhibition of chondrogenesis in vitro by retinoids was investigated in modified micromass cultures of limb bud mesenchymal cells from mouse embryos of day 11 and 12. Evaluation of chondrogenesis was performed after alcian blue staining, using a simple random hit counting of cartilage nodules. All-trans-retinoic acid, 13-cis-retinoic acid, and a newly developed arotinoid, RO 13-6298, were tested for their ability to inhibit chondrogenesis. We found that inhibition of chondrogenesis depended on the dosage and the duration of treatment with the different retinoids. Further analysis showed that chondrogenesis in limb bud mesenchymal cells from the proximal part was irreversibly inhibited after one hour of treatment, whereas distal cells showed a reduction of cartilage development only after a treatment period of 12 and more hours. In respect to the doses of the retinoids, proximal cells were about one magnitude more vulnerable than distal cells. These proximo-distal differences were obtained with 13-cis-retinoic acid at 10 micrograms/ml, with all-trans-retinoic acid at 1 microgram/ml and with arotinoid RO 13-6298 with 10 ng/ml. It is supposed that the late blastemal stage of chondrogenic differentiation before the onset of matrix synthesis is the step which is most vulnerable to retinoid treatment.     

The effects of local application of retinoids to different positions along the proximo-distal axis of embryonic chick wings

Summary Two retinoids, all-trans-retinoic acid and a synthetic analog, TTNPB, were locally applied to different positions along the proximo-distal axis of embryonic chick wing buds using controlled release carriers. Truncations or limbs with duplicated structures across the antero-posterior axis develop after retinoid application to distal positions in buds from stage 20–24 embryos. Phocomelic limbs develop when the retinoids are applied more proximally to buds of stage 23–24 embryos. Duplications of the pattern of structures along the proximo-distal axis never occur.Using TTNPB that is relatively stable, the amount of retinoid in the wing tissue when phocomelia is induced was measured. There is twice as much retinoid per cell in the proximal half of the bud as in the distal half of the bud. The concentration of TTNPB in proximal tissue is estimated to be three times higher than in distal tissue in which pattern formation and cartilage morphogenesis are relatively normal.At early stages in the development of phocomelia, the shape of the bud changes and the indentation that marks the elbow does not arise. Neither retinoid-induced cell killing nor effects on the pattern of programmed cell death were detected.The induction of phocomelia by retinoids appears to be based on effects on proximal cells, whereas retinoids produce pattern changes by acting on distal cells. Furthermore, compared with pattern changes, higher concentrations of retinoid in the bud tissue are required to produce phocomelia.     

Pattern formation in imaginal discs of Drosophila melanogaster after irradiation of embryos and young larvae

Embryos and first instar larvae of Drosophila melanogaster were X-irradiated in order to study pattern formation in discs damaged at early stages. After treatment, appendages were found in which some pattern elements were duplicated and others were absent. In some strains legs were preferentially duplicated, and in others, antennae were preferentially duplicated. Duplicated appendages were mirror images and resulted most frequently when animals were irradiated during late embryonic or early larval stages. Appendages varied from those which showed complete duplication of only the distal parts (claws or aristae), to those which showed duplication of almost the entire appendage. Examination of the cuticular patterns in duplicated mesothoracic legs showed that in legs with complete duplication solely in distal regions, only extreme lateral leg parts were duplicated, and medial leg parts were absent. In legs with duplication extending into proximal regions, much of the lateral side was duplicated, and only extreme medial parts were missing. The situation for partially duplicated antennae was similar. Prothoracic legs were found fused in some X-rayed flies. The cuticular patterns were almost perfect mirror images, although the amount of fusion varied widely between different individuals. Apparently the pattern forming processes in the right and left first leg discs are coordinated in X-rayed animals. The results were consistent with a model embracing a gradient of developmental capacity in the early disc similar to that postulated to exist in the late third instar leg disc. This model is also consistent with results of various surgical experiments reported in the literature. Several predictions of the model are explained including the possible mode of action of a specific class of mutants which may affect pattern formation by altering a morphogenetic gradient.     

Retinoic-acid-induced pattern completion in regenerating double anterior limbs of urodeles

The effects of retinoic acid on the regeneration of double anterior lower arms in the adult newt, Notophthalmus viridescens, were investigated. Normally, double anterior lower arms regenerate a hypomorphic, symmetrical pattern of structures, which are distally complete; and double anterior upper arms regenerate a hypomorphic, symmetrical but distally incomplete pattern of structures. In limbs with a normal anteroposterior axis, the major effect of retinoic acid is to alter the proximodistal (PD) positional value of cells local at the amputation level to a much more proximal value, thereby creating duplications in the regenerate of structures proximal to the amputation plane (Thoms and Stocum, '84). Therefore, we predicted that double anterior lower arms treated with retinoic acid would regenerate like double anterior upper arms. However, in a substantial number of cases, each half of these double anterior lower arms regenerated a limb that was complete in the anteroposterior (AP) axis, with asymmetry corresponding to the half of origin. In addition, these regenerates were serially duplicated in the PD axis. These results indicate that retinoic acid can posteriorize the positional value of midline cells, leading to restoration of normal AP pattern, when the set of posterior-half positional values is removed from the cross section of the limb.     

Forelimb regeneration in the postmetamorphic bullfrog: stimulation by dimethyl sulfoxide and retinoic acid

To examine the effects of retinoic acid and dimethyl sulfoxide on regenerative ability of anuran amphibians, the left forelimbs of 60 postmetamorphic froglets of Rana catesbeiana (bullfrogs) were amputated through the distal zeugopodium. Fifteen of the froglets had their left forelimb stumps immersed in dimethyl sulfoxide (DMSO) for 3 minutes, once immediately after amputation and once on each of 5 subsequent days. Another 15 frogs had their left forelimb stumps immersed in a 0.01 M solution of retinoic acid dissolved in DMSO for the same period of time. The remaining 30 control froglets did not regenerate structures distal to the amputation surface, while all limbs in both treated groups produced regenerates by 120 days postamputation. Regenerates of limbs treated with both DMSO alone and DMSO combined with retinoic acid, although hypomorphic, were composed of multiple cartilage elements, which in many cases (46.7%) were organized as patterns partially resembling the skeletal arrangement of a normal forelimb. All of these regenerates exhibited bundles of striated muscle. In addition, nearly half (46.7%) of the regenerates in the DMSO + retinoic acid group possessed two separate regenerate outgrowths. The results demonstrate that young bullfrogs (Rana catesbeiana) possess a latent epimorphic regenerative capability, which can be stimulated by topical application to the wound surface of DMSO alone or DMSO combined with retinoic acid.     

Morphogenetic interactions between rotated skin cuffs and underlying stump tissues in regenerating axolotl forelimbs

Rotation of skin cuffs 180° around the longitudinal axis of the underlying tissues in the axolotl forelimb results in a high percentage of multiple regenerates after amputation through the rotated skin. Similar results occur after rotation of only the anteroposterior (A-P) axis of the skin. Rotation of only the proximodistal (Pr-Ds) axis of the skin results in normal regenerates whereas dorsoventral (D-V) axial skin rotation results in single regenerates with some disturbances in symmetry. Rotation of anterior or posterior half cuffs of skin produces results similar to those obtained after A-P rotation of full skin cuffs, and rotation of dorsal or ventral skin halves duplicates the results obtained by rotating full skin cuffs about the D-V axis. Skin cuffs rotated for periods from 6 months to over 2 years before amputation are also capable of causing multiple regenerates to form. No significant difference in the percentage of multiple regenerates was seen after skin rotation and limb amputation through shoulder, upper arm, and forearm levels. X-Radiation (4000 r) of either the skin or underlying tissues before skin rotation resulted in single regenerates after amputation. If a strip of normal skin was turned perpendicularly to the long axis of the irradiated underlying stump tissues, the regenerative response was blocked. In some of the above experiments, regenerates with longitudinally duplicated upper arm and forearm segments appeared. It is postulated that normally both the skin and the underlying limb tissues can influence morphogenesis during regeneration and that they work in harmony. In contrast, rotated skin and the underlying tissues each exert a morphogenetic influence upon the regenerating limb, and the regenerate is not able to integrate these disharmonious influences. This is reflected in the highly abnormal morphology of the regenerates. The nature of the morphogenetic influence disrupted by skin rotation is not yet known.     

The relationship of innervation and differentiation to regenerative capacity in the reamputated hindlimb of larval Xenopus laevis

Regenerated hindlimbs of larval Xenopus laevis were reamputated at critical larval stages and levels, viz when amputation of the control limb at the same larval stage and level is followed by reduced regeneration. Reamputations were performed at the level of (1) the original plane of amputation, (2) the early regenerate (cone/palette stage), (3) the late regenerate (digit stage). Reamputation increased both the percentage rate of regeneration and the morphological complexity of the regenerates in all experimental series. Cell counts in lateral motor columns and spinal ganglia innervating the hindlimb, together with histological observations and mitotic index and labelling index determinations in reamputated and control limbs showed that improved regeneration in the reamputated limb was related to an increase in undifferentiated and proliferating cells in the stump. We did not find any evidence suggesting that renewed regeneration in reamputated anuran limbs results from an increase in innervation, as has previously been hypothesized. We support our conclusions by demonstrating an improvement in regenerationen in the reamputated and denervated hindlimbs.     

Heterochronic differences of Hoxa-11 expression in Xenopus fore- and hind limb development: Evidence for lower limb identity of the anuran ankle bones

 The wrist (carpus) and ankle (tarsus) of most tetrapods, as well as the wrist of anurans, contains relatively small nodular skeletal elements. The anuran tarsus, however, comprises a pair of long bones, the proximal tarsals tibiale and fibulare, which resemble the lower leg bones, tibia and fibula (zeugopodium). In this paper we investigate whether the proximal tarsals of Xenopus are of zeugopodial character identity, i.e. whether they develop under the influence of the same genes that pattern the lower limb. We compare Hoxa-11 expression in the forelimb bud with that in the hind limb bud by whole-mount in situ hybridization. Hoxa-11 has been implicated in the development of the lower limb. In Xenopus we note three differences between Hoxa-11 expression in fore- and hind limb buds: (1) Hoxa-11 expression is maintained until the hind limb bud reaches a larger size (2 mm) than that of the forelimb bud (1.5 mm); (2) Hoxa-11 expression is maintained over larger spatial domains than in the forelimb; and (3) Hoxa-11 expression has a pronounced posterior polarity in the hind limb, but not in the forelimb. Hind limb expression of Hoxa-11 can be understood as a heterochronic prolonging of the expression dynamic in the forelimb. Finally we found that the proximal tarsals start to develop within the expression domain of Hoxa-11, while in the forelimb the lower arm elements reach the distal expression limit of Hoxa-11. The gene expression data presented here support the notion of a zeugopodial identity of the proximal tarsal elements in Xenopus. Received: 20 January 1998 / Accepted: 27 March 1998     

Retinoid stimulation of epidermal differentiation in vivo

Retinoids are potent inducers of epidermal hyperplasia in vivo. Epidermal transglutaminase activity and stratum corneum turnover were examined in all-trans-retinoic acid and arotinoid ethyl ester treated hairless mice, to evaluate the possible contribution of decreased epidermal cell loss to the induction of hyperplasia by retinoids. Stratum corneum turnover was enhanced, and the absolute amounts (activity/cm2), but not the specific activities, of soluble and particulate transglutaminase increased following retinoid treatment. Since epidermal differentiation was enhanced after retinoid treatment, the hyperplastic response is due to increased cell formation and not decreased cell loss.