Positional signalling and the development of the humerus in the chick limb bud

The positional signal model for specification of the cartilaginous elements in limb development has been tested by examining the effect on the humerus of grafting a polarizing region to different positions along the anteroposterior axis of the limb bud at stage 16. The humerus between the host and grafted polarizing region was largely normal though there were variations in width, particularly the distal epiphysis. The humerus often showed mirror-image symmetry along the anteroposterior axis. When the grafted polarizing region was in a very anterior position, there were a few cases where a second humerus developed. Anterior to the graft an additional humerus often developed. This was associated with the splitting of the bud into two domains. It is suggested that these results are not consistent with a positional signal model and that an additional mechanism involving an isomorphic prepattern may be involved in the specification of the cartilaginous elements.     

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.     

Development of avascularity during cartilage differentiation in the embryonic limb

The differentiation of cartilage and muscle in limb-bud mesenchyme has been interpreted by some investigators in terms of a vascular pre-pattern model. It has been argued that a pre-pattern of the early limb vasculature compartmentalises the mesenchyme into specific microenvironmental areas in which, depending on the oxygen tension and nutrient supply, cartilage or muscle will differentiate. However, recent analyses of the development and differentiation of blood vessels in limbs have shown that regional variations in vascularization develop co-incidentally with the earliest indication of cartilage formation or mesenchymal condensation. The simple model described in the present study suggests that the mechanical compression/tension forces generated by the condensing mesenchyme are sufficient to constrict and eventually close off the thin-walled undifferentiated vessels caught in the condensation foci, thus leading to the avascularity of cartilage rudiments. This view suggests that the vasculature has no major function in governing the pattern of cartilage differentiation.     

Normal development of the skeleton in chick limb buds devoid of dorsal ectoderm

It has been suggested that the ectoderm on the dorsal and ventral faces of the limb bud plays a part in controlling the pattern of cartilage differentiation. To test this, the dorsal wing bud ectoderm in the chick embryo was destroyed by irradiation with ultraviolet light at stage 17-19, at the very beginning of limb bud development, but the apical ectodermal ridge was spared. The irradiated ectoderm disappeared within 24 hr (by stage 23-24) and did not regenerate thereafter; thus the dorsal surface of the limb bud was kept denuded throughout most of the period of skeletal pattern formation. By 6 or 7 days after the irradiation (stage 35), when the rudiments of all the adult skeletal elements are normally present in recognizable form, the irradiated wings could be placed into two categories, those that were approximately normal in shape and those that had curled dorsally. All of these limbs were reduced in size, to varying degrees, when compared to their controls and lacked dorsal soft tissues. The limbs that were normal in shape, however, even though sometimes denuded over practically the whole extent of their dorsal surface, almost always had a complete and normally proportioned cartilage pattern, suggesting that ectoderm (other than the apical ectodermal ridge) does not exert any direct control over the development of the limb cartilage pattern. However, many of those limbs that had curled as a result of the irradiation did have major pattern deformities, suggesting that the topology of cartilage differentiation does depend on the shape of the limb bud.     

Regeneration potency of mouse limbs

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.     

Limb morphogenesis in the branchiopod crustacean, Thamnocephalus platyurus, and the evolution of proximal limb lobes within Anostraca

Crustacean limbs exhibit highly diverse morphologies. One major route of diversification is in the number and position of branches arising from the proximal part of the limb. Here I describe development of larvae of the branchiopod crustacean, Thamnocephalus platyurus and describe in detail the development of the thoracic limbs. The thoracic limbs bear proximal branches both medially and laterally. The most proximal branches on either side (gnathobase and pre-epipod) show a similar developmental history: they develop via fusion of two rudiments into a single adult branch. However, phylogenetic analysis suggests that the developmental fusions have distinct evolutionary histories. In one case (gnathobase), the developmental rudiments reflect the ancestral adult morphology of two distinct branches. In the other (pre-epipod), the rudiments are an apparent novelty within the Anostraca and develop into two adult structures in only a single derived family.     

Differential cell affinity and sorting of anterior and posterior cells during outgrowth of recombinant avian limb buds

To examine the role of position-specific differences in cell-cell affinity, recombinant limb buds composed of dissociated and reaggregated cells derived from anterior (A) and posterior (P) limb bud fragments were analyzed. Dissociated anterior and/or posterior cells were differentially labeled, and their behavior was analyzed during recombinant limb bud outgrowth. We find that anterior and posterior cells sort out from one another to form alternating anterior and posterior stripes of cells that extend distally along the proximal-distal axis. These alternating stripes are prominent across the A/P axis in whole-mount preparations of recombinant limb buds after 48 h of outgrowth when the presumptive autopod is dorsal-ventrally flattened and digit rudiments are not evident. After 96 h, when digital and interdigital regions are clearly defined, we find evidence that A/P stripes do not follow obvious anatomical boundaries. The formation of A/P stripes is not inhibited by grafts of ZPA tissue, suggesting that polarizing activity does not influence cell-cell affinity early in limb outgrowth. In vitro studies provide evidence that cell sorting is not dependent on the limb bud ectoderm or the AER; however, cells sort out without organizing into stripes. Gene expression studies using anterior-specific (Alx-4) and posterior-specific (Shh, Bmp-2, and Hoxd-13) marker genes failed to reveal expression domains that corresponded to stripe formation. Control recombinant limb buds composed of anterior, central, or posterior mesenchyme formed digits in a position-specific manner. A/P recombinant limb buds that develop to later stages form digits that are characteristic of central recombinant limbs. These data provide the first definitive evidence of A/P cell sorting during limb outgrowth in vivo and suggest that differential cell affinities play a role in modulating cell behavior during distal outgrowth.     

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

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

Postnatal ontogeny of limb proportions and functional indices in the subterranean rodent Ctenomys talarum (Rodentia: Ctenomyidae)

Burrow construction in the subterranean Ctenomys talarum (Rodentia: Ctenomyidae) primarily occurs by scratch‐digging. In this study, we compared the limbs of an ontogenetic series of C. talarum to identify variation in bony elements related to fossorial habits using a morphometrical and biomechanical approach. Diameters and functional lengths of long bones were measured and 10 functional indices were constructed. We found that limb proportions of C. talarum undergo significant changes throughout postnatal ontogeny, and no significant differences between sexes were observed. Five of six forelimb indices and two of four hindlimb indices showed differences between ages. According to discriminant analysis, the indices that contributed most to discrimination among age groups were robustness of the humerus and ulna, relative epicondylar width, crural and brachial indices, and index of fossorial ability (IFA). Particularly, pups could be differentiated from juveniles and adults by more robust humeri and ulnae, wider epicondyles, longer middle limb elements, and a proportionally shorter olecranon. Greater robustness indicated a possible compensation for lower bone stiffness while wider epicondyles may be associated to improved effective forces in those muscles that originate onto them, compensating the lower muscular development. The gradual increase in the IFA suggested a gradual enhancement in the scratch‐digging performance due to an improvement in the mechanical advantage of forearm extensors. Middle limb indices were higher in pups than in juveniles–adults, reflecting relatively more gracile limbs in their middle segments, which is in accordance with their incipient fossorial ability. In sum, our results show that in C. talarum some scratch‐digging adaptations are already present during early postnatal ontogeny, which suggests that they are prenatally shaped, and other traits develop progressively. The role of early digging behavior as a factor influencing on morphology development is discussed. J. Morphol. 275:902–913, 2014. © 2014 Wiley Periodicals, Inc.     

Biophysical stimuli induced by passive movements compensate for lack of skeletal muscle during embryonic skeletogenesis

In genetically modified mice with abnormal skeletal muscle development, bones and joints are differentially affected by the lack of skeletal muscle. We hypothesise that unequal levels of biophysical stimuli in the developing humerus and femur can explain the differential effects on these rudiments when muscle is absent. We find that the expression patterns of four mechanosensitive genes important for endochondral ossification are differentially affected in muscleless limb mutants, with more extreme changes in the expression in the humerus than in the femur. Using finite element analysis, we show that the biophysical stimuli induced by muscle forces are similar in the humerus and femur, implying that the removal of muscle contractile forces should, in theory, affect the rudiments equally. However, simulations in which a displacement was applied to the end of the limb, such as could be caused in muscleless mice by movements of the mother or normal littermates, predicted higher biophysical stimuli in the femur than in the humerus. Stimuli induced by limb movement were much higher than those induced by the direct application of muscle forces, and we propose that movements of limbs caused by muscle contractions, rather than the direct application of muscle forces, provide the main mechanical stimuli for normal skeletal development. In muscleless mice, passive movement induces unequal biophysical stimuli in the humerus and femur, providing an explanation for the differential effects seen in these mice. The significance of these results is that forces originating external to the embryo may contribute to the initiation and progression of skeletal development when muscle development is abnormal.     

Fibroblast growth factor-induced gene expression and cartilage pattern formation in chick limb bud recombinants

To clarify the roles of fibroblast growth factors (FGF) in limb cartilage pattern formation, the effects of various FGF on recombinant limbs that were composed of dissociated and reaggregated mesoderm and ectodermal jackets were examined. Fibroblast growth factor-soaked beads were inserted just under the apical ectodermal ridge (AER) of recombinant limbs and the recombinant limbs were grafted and allowed to develop. Control recombinant limbs without FGF beads formed one or two cartilage elements. Recombinants with FGF-4 beads formed up to five cartilage elements, which were aligned along the anteroposterior (AP) axis. Each cartilage element showed digit-like segmentation. In contrast, recombinants with FGF-2 beads showed formation of multiple thick and unsegmented cartilage rods, which elongated inside and outside the AP plane from the distal end of the recombinants. Recombinants with FGF-8 beads formed a truncated cartilage pattern and recombinants with FGF-10 beads formed a cartilage pattern similar to that of the control recombinants. The expression of the Fgf-8, Msx-1 and Hoxa-13 genes in the developing recombinant limbs were examined. FGF-4 induced extension of the length of the Fgf-8-positive epidermis, or AER, along the AP axis 5 days after grafting, at which time the digits are specified. FGF-2 induced expansion of the Msx-1-positive area, first in the proximal direction and then along the dorsoventral axis. The functions of these FGF in recombinant and normal limb patterning are discussed in this paper.     

Retinoids reprogramme pre-bud mesenchyme to give changes in limb pattern

Retinoic acid was locally applied to presumptive limb regions of chick embryos to find out the earliest time at which the limb pattern can be reprogrammed. When beads soaked in retinoic acid were placed in the appropriate positions in embryos at stage 10 or older, duplicated or reduced leg patterns resulted. To pin point the time at which the cells in the limb rudiment respond to the retinoid, beads were removed at various times and the lengths of exposure required to reprogramme limb development found. The early limb rudiments require longer exposures to give duplications than late rudiments. The effective treatment periods last at least until stage 17 when the limb bud and apical ectodermal ridge develop. In contrast, the length of exposure to reduce the limb is constant at early stages. Retinoids first start acting to produce duplicated structures between stages 10 and 13. Therefore, retinoids appear to begin to reprogramme the cells as soon as they are determined to give rise to a limb.     

Patterns of clavicular bilateral asymmetry in relation to the humerus: variation among humans

Studies of directional asymmetry in the human upper limb have extensively examined bones of the arm, forearm, and hand, but have rarely considered the clavicle. Physiologically, the clavicle is an integrated element of the upper limb, transmitting loads to the axial skeleton and supporting the distal bones. However, clavicles develop in a manner that is unique among the bones of the upper limb. Previous studies have indicated that the clavicle has a right-biased asymmetry in diaphyseal breadth, as in humeri, radii, ulnae, and metacarpals, but unlike these other elements, a left-biased length asymmetry. Few studies have assessed how clavicular asymmetry relates to these other bones of the upper limb. Bilateral directional asymmetry of the clavicle is examined in relation to the humerus in a large, geographically diverse human sample, comparing lengths and diaphyseal breadths. Dimensions were converted into percentage directional (%DA) and absolute (%AA) asymmetries. Results indicate that humans have same-side %DA bias in the clavicles and humeri, and contralateral length %DA between these elements. Diaphyseal breadths in both clavicles and humeri are more asymmetric-both in direction and amount-than lengths. Differences in diaphyseal asymmetry are shown to relate to variation in physical activities among groups, but a relationship between activity and length asymmetry is not supported. This further supports previous research, which suggests different degrees of sensitivity to loading between diaphyseal breadths and maximum lengths of long bones. Differences in lateralized behavior and the potential effects of different bone development are examined as possible influences on the patterns observed among human groups.     

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.     

Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function

The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern.     

Cellular contribution to symmetrical forelimbs from triploid-marked "polarizing region" in the embryo of axolotl, Ambystoma mexicanum

Grafts of posterior tissue placed anterior to the limb bud in the salamander embryo exert a polarizing influence. To explain this result, the idea that the anteroposterior axis of the developing forelimb is polarized by a diffusible morphogen has been proposed. An alternative hypothesis, and the working hypothesis of the present study, is that the polarization of the developing salamander forelimb is accomplished by short-range cellular interactions resulting in intercalation rather than by the more global influence of a diffusible morphogen. One prediction of this intercalation hypothesis is that cells will be contributed to the limb from the "polarizing tissue." To test this idea, grafts of triploid marked polarizing tissue were implanted anterior to the limb bud in 82 diploid axolotl embryos at stages 32-34 of development. A total of 27 (33%) of the limbs that resulted were symmetrical and ranged in complexity from one to seven digits. Histological analysis of a subgroup of the original symmetrical limbs revealed that mesodermally derived tissues in the anterior side of these limbs (the side which formed as a duplication in response to the influence of the graft) contained high percentages of trinucleolate cells (muscle, 12.1%; connective tissue tissue, 12.5%; and cartilage, 13.4%) when compared to similar tissues in the posterior side of the same symmetrical limbs (muscle, 1.8%; connective tissue , 0.7%; and cartilage, 0.6%). When symmetrical limbs were amputated, 73% regenerated symmetrical limbs. When these regenerated limbs were again amputated, 63% formed symmetrical secondary regenerates. Histological analysis of the first generation of regenerated limbs revealed that the pattern of distribution of trinucleolate cells in each regenerate was similar to the pattern seen in the original symmetrical limb. These results indicate that there is considerable cellular contribution to the anterior side of the symmetrical forelimb from the mesoderm of grafted "polarizing tissue." This result supports the idea that short-range cellular interaction are sufficient for formation of symmetrical forelimbs in salamander embryos.     

Subepithelial type II collagen deposition during embryonic chick limb development

Summary Type II collagen is a major component of hyaline cartilage but recent studies have demonstrated the presence of this protein in a variety of interfaces that separate epithelia from mesenchyme, particularly in early stages of embryonic chick development. In the present study an immunohistochemical analysis of the distribution of type II collagen was performed on closely staged wing buds of early chick embryo. This report describes how using two different monoclonal antibodies against type II collagen and the peroxidase or fluorescence staining technique reveals that deposition of type II collagen at the ectoderm-mesenchyme interface occurs in the proximal part of the limb coincidentally with the appearance of this protein in the proximal core region, where chondrogenesis begins (stage 25). Then the staining in the subepithelial region spreads distallly with time, following the progression of the formation of cartilage rudiments. At about 7 days of development type II collagen is present under the apical ectoderm ridge and surrounds completely the wing bud underneath the epithelium. At the same time, another antibody directed against the cartilage-specific proteoglycan core protein only stains the chondrogenic central core of the limb and not the subepithelium. Although type II collagen and cartilage-specific proteoglycan are closely associated in the cartilage, the observations presented here suggest that the deposition of these proteins can be regulated independently during limb formation. The role of type II collagen at the epithelium-mesenchyme interface during limb formation is still to be determined.     

Early limb patterning in the direct‐developing salamander Plethodon cinereus revealed by sox9 and col2a1

Direct‐developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct‐developing frogs, but little attention has been given to direct‐developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct‐developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior‐to‐posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote‐like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct‐developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct‐developing salamander species.     

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)     

Autoradiographic analyses of35S-sulfate uptake in regenerating limbs of larvalAmbystoma

Summary Autoradiographic and histochemical techniques were used to determine whether chondrocytes continue to synthesize chondroitin sulfate or closely related compounds during morphological dedifferentiation of these cells in regenerating limbs of larvalAmbystoma. Forelimbs were amputated either through the mid-diaphysis or the distal epiphysis of the humerus and each animal was subsequently injected with Na₂ ³⁵SO₄ at an appropriate stage of regeneration. Incorporation of the isotope and metachromatic staining responses were used as indices of cell specialization.In autoradiographs of unamputated limbs, epiphyseal chondrocytes exhibited moderate sulfate incorporation, whereas isotope uptake was slight in diaphyseal regions. Accordingly, in early stages of regeneration, limbs amputated through the diaphysis showed a low level of sulfate incorporation by cartilage-derived cells; since these cells dispersed during blastema formation, they were not identifiable in later stages. When limbs were amputated through the epiphysis, the matrix here underwent slow dissolution and epiphyseal-derived chondrocytes and their progeny consequently remained identifiable as they contributed to the blastema. These cells continued to exhibit isotope uptake, even during early and middle stages of regeneration —results which support the idea of tissue-specific regeneration of cartilage.Further inspection of the stained autoradiographs revealed that in addition to chondrocytes and blastema cells derived from chondrocytes, fibroblast-like cells located lateral to the limb skeleton and seemingly derived from muscle or muscle-associated cells also exhibited a moderate label during certain stages in the restoration of the limb. In several respects isotope incorporation and related metachromatic responses by these two types of cells during blastemal and early redifferentiating stages of regeneration were seen to parallel results reported in the literature of histochemical and autoradiographic studies of differentiating chick fimb buds. These observations, which may be added to previous analogies concerning developing and regenerating limbs, suggest a similar mechanism of cytodifferentiation in the two systems. The possibility is also considered that the observed isotope uptake by cells of non-cartilaginous origin may indicate the synthesis of sulfated glycosaminoglycans which alfect cell interactions during the regenerative processes.A portion of a dissertation submitted to the University of New Hampshire in partial fulfillment of the requirements for the degree of Doctor of Philosophy.NASA Predoctoral Trainee during the course of this work.The authors wish to thank Nr. Carl Paulitz for his assistance in photography of the autoradiographs.