Early limb development of Xenopus laevis

It has been established by the work of several investigators that interaction between epithelial and mesenchymal components is essential for limb morphogenesis. Experimental analysis of this problem (Saunders & Gasseling, 1968) shows that in the chick such interaction takes place mainly in the tip of the limb bud where the ectoderm is thickened to form a longitudinal ridge curving round the apex. This is referred to as the apical ectodermal ridge and most authorities (Saunders, 1948; Zwilling, 1961; Tschumi, 1957) have ascribed it to 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 of vertebrates (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 of amphibian limb development concerned with this problem present contradictory findings. Thus while Tschumi (1957) reports the presence of this structure in Xenopus laevis, Balinsky (1965) states: ‘there are no apical ridges on amphibian limb buds’, and in a more recent publication Tschumi (Dober & Tschumi, 1969) withdraws his earlier assertion and now states that a ‘well-developed crest in the sense of Saunders cannot be proven’.

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

The hind limb was preferred because of its larger size, accessibility and ease of orientation. However, we have also examined a limited series of forelimb buds for the presence of the ridge. The work presented here is a preliminary to an investigation of the ultrastructural features associated with epithelial-mesenchymal interactions in limb development.

Xenopus laevis tadpoles were reared and staged according to the instructions in Nieuwkoop & Faber (1967). These animals were regularly examined with a binocular dissecting microscope and eight individuals of each stage from 44 to 53 inclusive were removed. Subsequently it was found that stages 51 and 52 were the most interesting and ten further animals were therefore taken in this range. A representative bud of each of these stages was photographed in order to provide the outline drawings shown in Fig. 1. These tadpoles were fixed in Bouin’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 microscope and orientated in molten agar (45 °C) in order to provide transverse and ventral longitudinal sections of the limb buds (ventral longitudinal section: a section of the limb bud in a plane parallel to the ventral surface of the animal). When the agar had set, thus holding the specimen in the desired position, it was removed from the mould, routinely processed and blocked out in paraffin wax. Sections were cut at 8 μm and stained with haematoxylin and eosin. Since both buds of each of the 90 animals were examined, we have studied a total of 90 buds sectioned 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 were processed, cut and stained in the same manner, providing ten buds sectioned transversely and ten longitudinally at right angles to the expected course of the ridge.

Four 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 °/₀ alcohol in which the trunk segments containing the hind limb buds were cut out as previously. Dehydration was completed in a graded series of alcohols, after which the specimens were immersed in Fluorisol for 1 h as recommended by Nott (1969). Finally, they were removed and allowed to dry out by evaporation at room temperature. For examination in the microscope the specimens were stuck to an aluminium chuck with Durofix. The chucks were then covered with a thin film of silver in a vacuum coating unit and examined in a Cambridge Stereoscan electron microscope.

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

By stage 47 mesenchymal cells had increased in number; those immediately below the epidermis showed a tendency to align themselves at right angles to the surface (Fig. 3). In this zone the cells were more tightly packed than in deeper regions. The epidermis covering the bud had begun thickening at stage 45, the inner layer becoming cuboidal, and many cells contained prominent nucleoli. The caudal boundary of the developing limb was marked by an inward projecting spur of epidermis.

From stage 47 onwards the limb bud increased dramatically in size, and at stage 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 the epidermis and in the distal part of the bud they remained aligned perpendicular to the surface. Close to this region a few columnar cells were sometimes present in the epidermis. Mitoses were common both in the epidermis and the mesenchyme. These first became noticeable at stage 47, whereas prior to this time we saw none, although the number of mesenchymal cells was apparently increasing.

Between stages 50 and 51 we observed the structure which we consider to be the 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 longer find it. The ridge was a narrow band of thickened epidermis which ran round the tip of the bud and extended a short distance proximally on both the dorsal and ventral aspects of the bud. It consisted of three clearly defined layers of epidermal cells, the inner one of which was high columnar, the cells being at least 2−$212$ times as long as they were broad (Figs. 5, 6). Fig. 6 also shows a vacuolar space containing some eosinophilic bodies, the significance of which is unknown. 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 the course of the ridge round the tip of the bud. Its appearance coincided with the formation of the apical ectodermal ridge but it persisted after the disappearance of the latter.

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

In this phase the mesenchymal cells immediately subjacent to the apical ectodermal ridge no longer displayed the features of regular alignment and close contiguity described in earlier stages. The entry of nerves into the limb bud was first observed during this phase at stage 51.

At stage 52 the distal part of the limb bud became flattened on its medial and lateral aspects and this constituted the initial phase in the formation of the paddle. The diminishing apical ectodermal ridge was confined to the distal margin of the developing paddle, and the marginal sinus followed a similar course in the subjacent mesenchyme. Between stages 52 and 53 the flattened extremity of the limb bud expanded to form the fully developed fan-shaped paddle. This was accompanied by the regression of the ridge and by the disappearance 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 of mesenchymal cells in the core of the limb bud. By stage 53 these mesenchymal condensations had formed the cartilaginous precursors of the long bones of the leg, and the forerunners of digits 4 and 5 were recognizable in the developing paddle. During this phase of development we often noted small regions of sparsely distributed cells which had pale nuclei and scanty irregular cytoplasm. These appearances were suggestive of cell death and disruption (Fig. 8) and it was considered that they represented areas undergoing dissolution in connection with the moulding of the shape of the limb.

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

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

The significance of the eosinophilic bodies (Fig. 6) we have seen in the ridge is also unknown. Although not pyknotic in the strictly pathological sense, they might correspond to the ‘pyknotic cells’ reported to be present in the ridge by Dober (1968) and by Amprino (1965). It seems very likely that these bodies are the products of cellular degeneration, but whether their presence in the ridge indicates a high level of such activity in this structure or whether they represent cellular debris phagocytosed by the epidermal cells and carried into the ridge by their peripheral migration (Dober, 1968) is a matter of conjecture. For the present we propose to refrain from further comment until we have examined them with the electron microscope.

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

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

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

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

In conclusion, the development of the amphibian limb appears to be fundamentally similar to the formation of the limb in birds, reptiles and mammals, including man. The most noticeable difference is the modest size of the apical ridge in comparison with that in the classes of vertebrates mentioned above. It is so small in amphibians that some earlier investigators denied its existence (Balinsky, 1965). Others described ‘a simple thickening of the ectoderm over the conical apex of the bud instead of a longitudinal crest’ (Braus, 1906, cited by Saunders, 1948). The present investigation shows, however, that this epidermal thickening is undoubtedly localized to form a longitudinal crest-like ridge in Xenopus. It is also pertinent to note that a thickened ectodermal ‘cap’ is formed in the regenerating amphibian limb, and that those species of amphibia and other vertebrates which are incapable of limb regeneration, do not form one after amputation (Thornton, 1968).

If the ridge, as claimed by some workers, plays a major role in limb morphogenesis (see review by Ede, 1971), its disappearance during the paddle stages in Xenopus presumably means that it ceases to exert a direct influence on subsequent development. However, we cannot exclude the possibility that the biochemical activity of the apical ectoderm persists and influences the underlying mesenchyme after the ridge morphologically regresses.

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

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