/. Embryol. exp. Morph. Vol. 26, 2, pp. 169-179, 1971
Printed in Great Britain
\ 69
Early limb development of Xenopus laevis
By D. TARIN 1 AND A. P. STURDEE 1
From the Department of Anatomy, School of Medicine,
University of Leeds
SUMMARY
This investigation has used histological techniques and the scanning electron microscope
to establish the presence of an apical ectodermal ridge in the developing limbs of Xenopus
laevis.
The ridge appeared at stage 50, reached its maximal size at stage 51, and subsequently
disappeared by stage 53.
The course of the ridge was consistently related to a marginal sinus in the underlying
mesenchyme.
The other features of limb morphogenesis, such as the formation of a paddle and the
sequence of condensation of skeletal rudiments in the mesenchyme, corresponded closely to
those seen in other vertebrates.
It remains to be seen whether the ridge we have demonstrated in Xenopus exercises a
similar function to that claimed for its counterpart in the chick.
INTRODUCTION
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'.
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 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.
MATERIALS AND METHODS
Light microscopy
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
48
49
50
51
52
Fig. 1. Outline drawings (x 40) to show the relative size and shape of the bud at
various stages. These are tracings of photographs of the bud seen from the lateral
side. Note the position of the ankle constriction (£>), the paddle (P) and digits IV
andV.
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 /«n 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
Early limb development o / X e n o p u s laevis
171
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.
Scanning electron microscopy
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 %
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.
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 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.
Early enlargement of the bud
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.
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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 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 2-2} 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.
Formation of the paddle
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
Fig. 2. Ventral longitudinal section of stage 44 tadpole to show the earliest visible
features of the hind limb (x 600). M indicates the mesenchymal condensation lying
close to the anal canal (£/), the coelomic cavity (C) and the somatopleure (S).
Fig. 3. Ventral longitudinal section, stage 47 tadpole (x 630) showing early
enlargement of the bud. The alignment of mesenchymal cells perpendicular to the
surface is clearly seen in the bud on the right. Thickening of the epidermis and its
inward projection at the caudal boundary are also obvious on the same side.
Fig. 4. Stage 49 tadpole; ventral longitudinal section (x 630) illustrating further
enlargement of the bud, the presence of a blood vessel (B) and mitotic figures
(arrows) in the mesenchyme.
!2
EMB
26
174
D. TARIN AND A. P. STURDEE
Fig. 5. Stage 51 tadpole; ventral longitudinal section (x 900) demonstrating the
apical ectodermal ridge (A). Notice that the epidermis has three layers at this point
and also that the basal cells are clearly columnar. The marginal sinus (V) is visible in
the subjacent mesenchyme.
Early limb development 0/Xenopus laevis
175
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.
Fore limb development
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.
DISCUSSION
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
176
D. TARIN AND A. P. STURDEE
10 fi
10 fim
Early limb development o/Xenopus laevis
177
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 (Saxen & 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,
Fig. 6. Ventral longitudinal section of late stage 50 limb bud (x 1950) showing a
vacuolar space in the apical epidermis. This shows a slightly less well-developed
ridge than Fig. 4. The space contains three eosinophilic bodies (E) of unknown
nature. These appearances are typical only of the apical epidermis where they are
common.
Fig. 7. Scanning electron microscopic view of a stage 51 limb bud ( x 275). The bud
is viewed here looking almost vertically downwards on its caudal tip. The apical
ectodermal ridge (A) runs diagonally from left to right and extends for a short
distance proximally on the dorsal and ventral surfaces which slope steeply away in
this 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 to
be 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 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 wasfinancedby 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.
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(Manuscript received 26 November 1970)