/ . Embryol. exp. Morph. Vol. 26, 2, pp. 169-179, 1971 \ 69

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Early limb development of Xenopus laevis

By D. TARIN1 AND A. P. STURDEE1

From the Department of Anatomy, School of Medicine,University of Leeds

SUMMARYThis investigation has used histological techniques and the scanning electron microscope

to establish the presence of an apical ectodermal ridge in the developing limbs of Xenopuslaevis.

The ridge appeared at stage 50, reached its maximal size at stage 51, and subsequentlydisappeared by stage 53.

The course of the ridge was consistently related to a marginal sinus in the underlyingmesenchyme.

The other features of limb morphogenesis, such as the formation of a paddle and thesequence of condensation of skeletal rudiments in the mesenchyme, corresponded closely tothose seen in other vertebrates.

It remains to be seen whether the ridge we have demonstrated in Xenopus exercises asimilar function to that claimed for its counterpart in the chick.

INTRODUCTION

It has been established by the work of several investigators that interactionbetween epithelial and mesenchymal components is essential for limb morpho-genesis. Experimental analysis of this problem (Saunders & Gasseling, 1968)shows that in the chick such interaction takes place mainly in the tip of thelimb bud where the ectoderm is thickened to form a longitudinal ridge curvinground the apex. This is referred to as the apical ectodermal ridge and mostauthorities (Saunders, 1948; Zwilling, 1961; Tschumi, 1957) have ascribed itto an important role in control of the growth, shape and orientation of the limb.

An apical ectodermal ridge has also been described in other classes ofvertebrates (mammals, Milaire, 1956; reptiles, Milaire, 1957; and man, O'Rahilly,Gardner & Gray, 1956), where it is presumed to exercise the same function.However, it is not certain that a ridge exists in amphibia, for the few studies ofamphibian limb development concerned with this problem present contradictoryfindings. Thus while Tschumi (1957) reports the presence of this structure inXenopus laevis, Balinsky (1965) states: 'there are no apical ridges on amphibianlimb buds', and in a more recent publication Tschumi (Dober & Tschumi, 1969)withdraws his earlier assertion and now states that a ' well-developed crest in thesense of Saunders cannot be proven'.

1 Authors' address: Department of Anatomy, School of Medicine, Leeds LS2 9NL, U.K.

170 D. TARIN AND A. P. STURDEE

We were therefore especially interested in ascertaining whether such a ridgedoes exist in amphibia, and if so, in establishing its size and the duration of itspresence. To accomplish this we have conducted a histological study of limbdevelopment from its first appearance to the formation of the paddle.

The hind limb was preferred because of its larger size, accessibility and ease oforientation. However, we have also examined a limited series of forelimb budsfor the presence of the ridge. The work presented here is a preliminary to aninvestigation of the ultrastructural features associated with epithelial-mesen-chymal interactions in limb development.

MATERIALS AND METHODS

Light microscopy

Xenopus laevis tadpoles were reared and staged according to the instructionsin Nieuwkoop & Faber (1967). These animals were regularly examined with abinocular dissecting microscope and eight individuals of each stage from 44 to

48 49 50 51 52

Fig. 1. Outline drawings (x 40) to show the relative size and shape of the bud atvarious stages. These are tracings of photographs of the bud seen from the lateralside. Note the position of the ankle constriction (£>), the paddle (P) and digits IVandV.

53 inclusive were removed. Subsequently it was found that stages 51 and 52were the most interesting and ten further animals were therefore taken in thisrange. A representative bud of each of these stages was photographed in orderto provide the outline drawings shown in Fig. 1. These tadpoles were fixed inBouin's fluid for 16 h and then transferred to 70 % alcohol. The trunk segments,containing the two hind limb buds, were cut out under the dissecting microscopeand orientated in molten agar (45 °C) in order to provide transverse and ventrallongitudinal sections of the limb buds (ventral longitudinal section: a section ofthe limb bud in a plane parallel to the ventral surface of the animal). When theagar had set, thus holding the specimen in the desired position, it was removedfrom the mould, routinely processed and blocked out in paraffin wax. Sectionswere cut at 8 /«n and stained with haematoxylin and eosin. Since both buds ofeach of the 90 animals were examined, we have studied a total of 90 budssectioned transversely and 90 sectioned in the ventral longitudinal plane.

For the brief study of the forelimb, two animals from each of the stages 49-53

Early limb development o /Xenopus laevis 171

were processed, cut and stained in the same manner, providing ten buds sectionedtransversely and ten longitudinally at right angles to the expected course of theridge.

Scanning electron microscopyFour tadpoles from each of stages 49-53 were fixed for 1 h in cacodylate

buffered glutaraldehyde at pH 7-35. Fixed specimens were transferred to 30 %alcohol in which the trunk segments containing the hind limb buds were cut outas previously. Dehydration was completed in a graded series of alcohols, afterwhich the specimens were immersed in Fluorisol for 1 h as recommended byNott (1969). Finally, they were removed and allowed to dry out by evaporationat room temperature. For examination in the microscope the specimens werestuck to an aluminium chuck with Durofix. The chucks were then covered witha thin film of silver in a vacuum coating unit and examined in a CambridgeStereoscan electron microscope.

RESULTS

First appearance of the limb bud

The first recognizable feature of limb-bud development occurred at stage 44.This consisted of a small condensation of mesenchymal cells with prominentnucleoli, located beneath the flank epidermis (Fig. 2). The mesenchymal cellswere in close proximity to the anal canal, the caudal part of the coelomic cavityand the muscle of the body wall. The two latter are derived from the somaticlayer of lateral mesoderm, which according to Balinsky (1965) and Milaire (1965)is also the source of limb mesenchyme. Our own evidence, being purely morpho-logical, can neither confirm nor deny this claim, although the proximity of thebud mesenchyme to the somatopleure would be consistent with an origin fromthis source.

Early enlargement of the budBy stage 47 mesenchymal cells had increased in number; those immediately

below the epidermis showed a tendency to align themselves at right angles tothe surface (Fig. 3). In this zone the cells were more tightly packed than indeeper regions. The epidermis covering the bud had begun thickening at stage45, the inner layer becoming cuboidal, and many cells contained prominentnucleoli. The caudal boundary of the developing limb was marked by aninward projecting spur of epidermis.

From stage 47 onwards the limb bud increased dramatically in size, and atstage 48 we first noticed the presence of a vascular supply. At stage 49 (Fig. 4),the mesenchymal cells were, as before, densely packed immediately below theepidermis and in the distal part of the bud they remained aligned perpendicularto the surface. Close to this region a few columnar cells were sometimes presentin the epidermis. Mitoses were common both in the epidermis and the mesen-chyme. These first became noticeable at stage 47, whereas prior to this time wesaw none, although the number of mesenchymal cells was apparently increasing.

172 D. TARIN AND A. P. STURDEE

Early limb development o/Xenopus laevis 173

Apical ectodermal ridge phase

Between stages 50 and 51 we observed the structure which we consider to bethe counterpart of the apical ectodermal ridge (Saunders' ridge) of the chick.This was first recognizable at stage 50, attained its maximum size at stage 51(Fig. 5), and subsequently diminished, so that by stage 53 we could no longerfind it. The ridge was a narrow band of thickened epidermis which ran round thetip of the bud and extended a short distance proximally on both the dorsal andventral aspects of the bud. It consisted of three clearly defined layers of epi-dermal cells, the inner one of which was high columnar, the cells being atleast 2-2} times as long as they were broad (Figs. 5, 6). Fig. 6 also shows avacuolar space containing some eosinophilic bodies, the significance of which isunknown. Such appearances are common in the ridge.

In the mesenchyme, immediately subjacent to the apical ectodermal ridge,there lay a large blood vessel. This was the marginal sinus and it followed thecourse of the ridge round the tip of the bud. Its appearance coincided with theformation of the apical ectodermal ridge but it persisted after the disappearanceof the latter.

The presence of the ridge was confirmed in surface appearances seen with thescanning electron microscope. This showed a consistent single elevation to bepresent, in a number of different limb buds, indicating that the ridge, as seenwith this instrument, was a genuine structure and not an artifactual creaseproduced by drying (Fig. 7).

In this phase the mesenchymal cells immediately subjacent to the apicalectodermal ridge no longer displayed the features of regular alignment andclose contiguity described in earlier stages. The entry of nerves into the limb budwas first observed during this phase at stage 51.

Formation of the paddle

At stage 52 the distal part of the limb bud became flattened on its medial andlateral aspects and this constituted the initial phase in the formation of thepaddle. The diminishing apical ectodermal ridge was confined to the distalmargin of the developing paddle, and the marginal sinus followed a similar

Fig. 2. Ventral longitudinal section of stage 44 tadpole to show the earliest visiblefeatures of the hind limb (x 600). M indicates the mesenchymal condensation lyingclose to the anal canal (£/), the coelomic cavity (C) and the somatopleure (S).Fig. 3. Ventral longitudinal section, stage 47 tadpole (x 630) showing earlyenlargement of the bud. The alignment of mesenchymal cells perpendicular to thesurface is clearly seen in the bud on the right. Thickening of the epidermis and itsinward projection at the caudal boundary are also obvious on the same side.Fig. 4. Stage 49 tadpole; ventral longitudinal section (x 630) illustrating furtherenlargement of the bud, the presence of a blood vessel (B) and mitotic figures(arrows) in the mesenchyme.

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174 D. TARIN AND A. P. STURDEE

Fig. 5. Stage 51 tadpole; ventral longitudinal section (x 900) demonstrating theapical ectodermal ridge (A). Notice that the epidermis has three layers at this pointand also that the basal cells are clearly columnar. The marginal sinus (V) is visible inthe subjacent mesenchyme.

Early limb development 0/Xenopus laevis 175course in the subjacent mesenchyme. Between stages 52 and 53 the flattenedextremity of the limb bud expanded to form the fully developed fan-shapedpaddle. This was accompanied by the regression of the ridge and by the dis-appearance of its characteristic columnar cells. The marginal sinus, however,persisted in its position at the periphery of the paddle.

Development of cartilage was first seen at stage 52 as condensations ofmesenchymal cells in the core of the limb bud. By stage 53 these mesenchymalcondensations had formed the cartilaginous precursors of the long bones of theleg, and the forerunners of digits 4 and 5 were recognizable in the developingpaddle. During this phase of development we often noted small regions ofsparsely distributed cells which had pale nuclei and scanty irregular cytoplasm.These appearances were suggestive of cell death and disruption (Fig. 8) and itwas considered that they represented areas undergoing dissolution in connectionwith the moulding of the shape of the limb.

Fore limb development

The general features of forelimb development corresponded closely with thosedescribed above, although they occurred at somewhat later stages. Thus, theforelimb also possessed an apical ectodermal ridge, present between stages 52and 53. The proximo-distal sequence in laying down of cartilage, and thepresence of a marginal sinus closely related to the ridge, were also noted.

DISCUSSION

On the basis of this investigation we can state with confidence that a ridge ofthickened apical ectoderm with specific features such as basal columnar cellsand a three-layered arrangement is consistently present at certain stages inXenopus limb development. We therefore strongly contest Tschumi's revisedopinion (Dober & Tschumi, 1969) that a ridge does not exist in this species, andBalinsky's (1965) similar assertion. Consequently, the morphological features ofXenopus limb development are closely comparable to those of other vertebratesincluding the chick. In the latter, experiments performed by Saunders (1948) andalso by Zwilling (for review see Zwilling, 1961) have resulted in their belief thatthe ectodermal ridge is indispensable for the proximo-distal outgrowth of thelimb and influences the orientation of the paddle (Zwilling, 1956). It has beenclaimed by Tschumi (1957) to perform the same function in amphibia but thishas not yet been confirmed. However, even in the chick where the role of theridge has been far more extensively investigated, research workers disagree withthis interpretation of the results (Amprino, 1965; Bell, Kaighn & Fessenden,1959). Therefore, the role of the ridge in amphibian limb development cannot beassumed until there is a much larger body of evidence available.

The significance of the eosinophilic bodies (Fig. 6) we have seen in the ridge isalso unknown. Although not pyknotic in the strictly pathological sense, they

176 D. TARIN AND A. P. STURDEE

10 fi 10 fim

Early limb development o/Xenopus laevis 177might correspond to the 'pyknotic cells' reported to be present in the ridge byDober (1968) and by Amprino (1965). It seems very likely that these bodies arethe products of cellular degeneration, but whether their presence in the ridgeindicates a high level of such activity in this structure or whether they representcellular debris phagocytosed by the epidermal cells and carried into the ridge bytheir peripheral migration (Dober, 1968) is a matter of conjecture. For thepresent we propose to refrain from further comment until we have examinedthem with the electron microscope.

We noted that mitoses were frequently seen in the limb mesenchyme fromstage 47 onwards. Prior to this time, however, we saw none although the numberof mesenchymal cells was increasing. This suggests that the increase in size ofthe limb bud between stages 44 and 47 is produced by continuing influx of cellsfrom elsewhere (see above).

The alignment and packing of mesenchymal cells described above is compar-able with observations on other developing systems where induction is occurring,such as the kidney (Saxen & Wartiovaara, 1966); the tooth (Koch, 1967, Fig. 18)and the central nervous system (Tarin, 1971, in the Press). This supports theview that there is some developmental interrelationship between the mesenchymeand the epidermis in the tip of the bud.

The areas of cellular degeneration first seen in stage 52, in the region of theankle constriction, probably play a role in the modelling of the gross morphologyof the limb. This interpretation is supported by observations on mammals(Milaire, 1965) where necrosis and absorption of mesenchymal cells in theinterdigital parts of the paddle provide the mechanism for its division into fiveseparate digits.

We noted that skeletal development in the proximal portion of the limb atstage 53 is clearly more advanced than that further distally. This conforms to theproximo-distal sequence of development in the limb first demonstrated bySaunders (1948) for the chick and by Tschumi (1957) for Xenopus.

In conclusion, the development of the amphibian limb appears to be funda-mentally similar to the formation of the limb in birds, reptiles and mammals,

Fig. 6. Ventral longitudinal section of late stage 50 limb bud (x 1950) showing avacuolar space in the apical epidermis. This shows a slightly less well-developedridge than Fig. 4. The space contains three eosinophilic bodies (E) of unknownnature. These appearances are typical only of the apical epidermis where they arecommon.Fig. 7. Scanning electron microscopic view of a stage 51 limb bud ( x 275). The budis viewed here looking almost vertically downwards on its caudal tip. The apicalectodermal ridge (A) runs diagonally from left to right and extends for a shortdistance proximally on the dorsal and ventral surfaces which slope steeply away inthis picture. Disregard the contaminating particles marked with the asterisks.Fig. 8. Ventral longitudinal section through the ankle constriction. Stage 52 tadpole(x 550). This shows one of the small regions (R) where the cells are considered tobe undergoing dissolution in connection with the moulding of the limb.

178 D. TARIN AND A. P. STURDEE

including man. The most noticeable difference is the modest size of the apicalridge in comparison with that in the classes of vertebrates mentioned above. Itis so small in amphibians that some earlier investigators denied its existence(Balinsky, 1965). Others described 'a simple thickening of the ectoderm over theconical apex of the bud instead of a longitudinal crest' (Braus, 1906, cited bySaunders, 1948). The present investigation shows, however, that this epidermalthickening is undoubtedly localized to form a longitudinal crest-like ridge inXenopus. It is also pertinent to note that a thickened ectodermal' cap' is formedin the regenerating amphibian limb, and that those species of amphibia andother vertebrates which are incapable of limb regeneration, do not form oneafter amputation (Thornton, 1968).

If the ridge, as claimed by some workers, plays a major role in limb morpho-genesis (see review by Ede, 1971), its disappearance during the paddle stages inXenopus presumably means that it ceases to exert a direct influence on subsequentdevelopment. However, we cannot exclude the possibility that the biochemicalactivity of the apical ectoderm persists and influences the underlying mesen-chyme after the ridge morphologically regresses.

Some of the features revealed by this investigation will be further investigatedby histochemical techniques and by transplantation and electron microscopy.

This work was financed by a research grant from the Tenovus Organization, Cardiff, whosesupport is gratefully acknowledged. We also wish to thank Professor R. L. Holmes forreading and criticizing the manuscript, Dr J. Sikorski for permission to use the scanningelectron microscope in the department of Textile Physics, and Mr T. Buckley for assisting usin operating this instrument.

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(Manuscript received 26 November 1970)