/. Embryol. exp. Morph. 94, 231-244 (1986)
231
Printed in Great Britain © The Company of Biologists Limited 1986
Interdigital tissue chondrogenesis induced by surgical
removal of the ectoderm in the embryonic chick leg bud
J. M. HURLE AND Y. GANAN
Departamento de Anatomia, Facultad de Medicina, Universidad de Extremadura,
Badajoz, 06071, Spain
SUMMARY
In the present work, we have analysed the possible involvement of ectodermal tissue in the
control of interdigital mesenchymal cell death. Two types of experiments were performed in the
stages previous to the onset of interdigital cell death: (i) removal of the AER of the interdigit;
(ii) removal of the dorsal ectoderm of the interdigit. After the operation embryos were
sacrificed at 10-12h intervals and the leg buds were studied by whole-mount cartilage staining,
vital staining with neutral red and scanning electron microscopy.
Between stages 27 and 30, ridge removal caused a local inhibition of the growth of the
interdigit. In a high percentage of the cases, ridge removal at these stages was followed 30-40 h
later by the formation of ectopic nodules of cartilage in the interdigit. The incidence of ectopic
cartilage formation was maximum at stage 29 (60%). In all cases, cell death took place on
schedule although the intensity and extent of necrosis appeared diminished in relation to the
intensity of inhibition of interdigital growth and to the presence of interdigital cartilages. Ridge
removal at stage 31 did not cause inhibition of the growth of the interdigit and ectopic
chondrogenesis was only detected in 3 out of 35 operated embryos.
Dorsal ectoderm removal from the proximal zone of the interdigit at stage 29 caused the
chondrogenesis of the proximal interdigital mesenchyme in 6 out of 18 operated embryos. The
pattern of neutral red vital staining was consistent with these results revealing a partial inhibition
of interdigital cell death in the proximal zone of the interdigit.
It is proposed that under the present experimental conditions the mesenchymal cells are
diverted from the death programme by a primary transformation into cartilage.
INTRODUCTION
The development of the digits of most vertebrates takes place by their detachment from an initial hand or foot plate. Interdigital cell death appears to be a
major phenomenon during this process responsible for sculpturing of the digits
(Fallon & Cameron, 1977; Hinchliffe, 1982). Little is known about the causes of
these interdigital necrotic processes at the cellular level (Fallon, Brucker & Harris,
1974; Hurle, Lafarga & Hinchliffe, 1981) or at the tissue level (Hurle, FernandezTeran & Colvee, 1985; Hurle, Colvee & Fernandez-Teran, 1985). The question of
whether the cells have an intrinsic death programme, as appears to be the case for
other areas of cell death of the developing limb bud (Saunders, Gasseling &
Saunders, 1962; Fallon & Saunders, 1968), or whether they die as a consequence of
changes in the local microenvironment, remains to be elucidated. The possible
linkage between interdigital cell death and the hypothetical factors for positional
information of the developing limb are also unknown (Newman & Leonard, 1983).
Key words: limb development, cell death, chondrogenesis, chick, interdigital tissue, leg bud.
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J. M. HURLE AND Y. GANAN
The causes of interdigital cell death are of special interest in studies of limb
developmental biology due to the fact that the structure of the interdigits in the
stages immediately preceding regression is very similar to that of the growing digits
and to that of the limb bud in the early growing stages (Pautou, 1978; Hurle &
Fernandez-Teran, 1983,1984). In the chick leg 12 to 24 h prior to the onset of cell
death, the interdigit consists of a core of mesenchymal cells and blood vessels
covered by ectoderm with a distinctive apical ectodermal ridge (AER) (Hurle
& Fernandez-Teran, 1983). Furthermore, the AER of the limb bud retains its
functional properties until the stages just before cell death, as can be deduced
from experiments of heterochronic recombinations of ectoderm and mesoderm
(Rubin & Saunders, 1972; Pautou, 1972). In the same way, fragments of subridge
interdigital mesoderm in organ culture conditions are diverted from the death
programme and differentiate into cartilage (Newman, 1977).
In a recent paper, we have surveyed the structural changes of the chick embryo
leg bud after the administration of Janus green B (Fernandez-Teran & Hurle,
1984). This procedure constitutes a typical model for the study of the morphogenetic role of interdigital cell death, since administration of Janus green appears
to inhibit cell death, leading to syndactylous limbs (Menkes & Deleanu, 1964;
Saunders & Fallon, 1967; Pautou, 1976). However, the results of our study showed
that Janus green causes early damage to the interdigital ectoderm, which might be
responsible for the inhibition of interdigital tissue regression.
In the present work, we have attempted to analyse such a possible role of the
ectoderm in the control of interdigital tissue regression. The interdigital AER or
small areas of the dorsal ectoderm of the interdigit were removed from the chick
embryo leg buds in stages previous to the onset of cell death. Our results show that
these experimental procedures cause, in a high percentage of the cases, the formation of ectopic nodules of cartilage in the interdigital space without disturbing
the structure of the neighbouring digits.
MATERIALS AND METHODS
Fertile White Leghorn eggs incubated at 38 °C were fenestrated between days 5 and 7 of
incubation and staged according to Hamburger & Hamilton (1951). In a group of embryos, at
stages 27, 28, 29, 30 and 31, the AER was removed from the third interdigital space of the right
leg with a fine tungsten needle (Fig. 1), paying special attention to avoid damaging the AER of
the neighbouring digits. To elucidate whether the effects of ridge removal were specific to this
zone of the ectoderm, in a second group of embryos a small area (0-25x0-15 mm) of the dorsal
ectoderm of the proximal zone of the third interdigit was removed at stage 29 with the same
procedure (Fig. 1). The eggs of the operated embryos were sealed with cellophane tape and
returned to the incubator. The embryos were then sacrificed at 10-12 h intervals and the leg buds
were studied by neutral-red vital staining, whole-mount cartilage staining and scanning electron
microscopy. A total of 295 surviving embryos ranging from the stage of the operation up to
stages 34-35 have been studied. In all cases the left leg was employed as control.
Neutral-red vital staining
Modifications in the shape and extension of the interdigital necrotic area were evaluated by in
ovo staining with neutral red following the method of Hinchliffe & Ede (1973).
Interdigital tissue chondrogenesis
233
Fig. 1. Schematic drawings showing the experiments performed to analyse the role of
the ectoderm on interdigital tissue regression. (A) Apical ridge removal; (B) dorsal
ectoderm removal.
Whole-mount cartilage staining
For this purpose the leg buds werefixedin Bouin's fluid, stained with alcian blue 8G-X (Gurr)
following the technique of Ojeda, Barbosa & Bosque (1970) and cleared in xylene or methyl
salicylate. After the analysis of the skeletal elements of the foot, all the specimens were rinsed in
acetone and processed for SEM.
Scanning electron microscopy (SEM)
The surface morphology of the experimental and control legs was studied in specimens fixed
in 2-5 % glutaraldehyde in cacodylate buffer and also in the specimensfixedin Bouin's fluid and
stained with alcian blue as indicated above. In all cases, after dehydration in acetone, the
specimens were dried by the critical-point method, gold sputter-coated and viewed in a Philips
SEM 501 or in a Jeol T-100 electron microscopes.
RESULTS
AER removal at stage 27
At this stage, the digital arrays of the foot plate are hardly recognizable at the
time of the operation and on some occasions the ridge of the digit was damaged in
the course of the operation. When ridge removal is limited to the prospective
interdigital space, the ablation of the ridge results in an arrest of the growth of the
interdigit. As can be seen in Fig. 2 at stage 29 (30 h after the operation) digits III
and IV appear mostly free, due to the absence of formation of most of the
interdigital tissue. The pattern of chondrogenesis of the digits was normal although slight deviations of digit III are occasionally observed. As can be seen in
Fig. 3, neutral-red vital staining revealed that the small amount of interdigital
tissue present in the experimental legs undergoes cell death at the same stages as
the control legs. As in the control legs, the process of interdigital necrosis
accounted for the disappearance of the interdigital tissue in the experimental legs.
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J. M. HURLE AND Y. GANAN
AER removal at stages 28, 29 and 30
Ridge removal at these stages resulted in a variable intensity of defective
development of the interdigital tissue accompanied in a number of cases by the
formation of ectopic interdigital cartilages (Table 1).
Fig. 2. Stage-29 experimental leg bud after ridge removal at stage 27 stained with
alcian blue. The pattern of chondrogenesis is normal. Note the presence of a
prominent cleft in the third interdigit. Scale bar, 0-3 mm.
Fig. 3. Light micrograph showing the pattern of cell death at stage 31 in the third
interdigital space of control (A) and experimental (B) legs after ridge removal at stage
27. Scale bar, 0-25 mm.
Interdigital tissue chondrogenesis
235
Table 1. Frequency of interdigital ectopic chondrogenesis 30 h or more after ridge
removal
Stage at operation
27
28
29
30
31
Number of embryos
With cartilage Without cartilage
0
28
14
20
20
13
14
31
3
32
Fig. 4. SEM micrograph showing the third interdigital space of an experimental leg
10 h after ridge removal. Arrows show the area in which the AER was removed. Scale
bar, 0-1 mm.
In all experimental legs, 10-12 h after the operation the shape of the foot was
normal except for a small indentation corresponding to the area of AER removal.
Examined under the SEM, this area appeared devoid of ectoderm (Fig. 4).
Neutral-red staining revealed a small necrotic focus associated with the area of
ridge removal in only 3 out of 15 embryos studied by this procedure.
30 h or more after ridge removal, alcian-blue staining frequently revealed the
presence of a prominent nodule of cartilage in the interdigit. The frequency of
cartilage formation was maximum when the operation was performed at stage 29
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J. M. HURLE AND Y. GANAN
Fig. 5. Alcian-blue-cartilage staining showing the presence of an ectopic cartilage in
the interdigit of a leg bud 70 h after ridge removal at stage 29. Scale bar, 0-5 mm.
and decreased at stages 28 and 30 (Table 1). The nodule was rounded or elongated
(Fig. 5). In the course of development, the nodules showed little increase in size
and remained free in the interdigit without forming an identifiable joint with the
neighbouring phalanges. In five cases, the ectopic cartilages appeared as two
pieces separated by a developing joint. The ectopic cartilages were never accompanied by reduction in the number, or deformations of the phalanges of the
neighbouring digits. Under the SEM the surface of the interdigital cartilage was
prominent, limited by a lateral sulcus (Fig. 6). Ectodermal healing of the excised
ridge appeared incomplete up to 30 h after the operation (Fig. 6). In advanced
stages of development, the ectopic cartilages were joined to the neighbouring
digits by a variable amount of interdigital tissue (Fig. 7). Neutral-red staining
showed that by stage 33 interdigital cell death was only absent in the zone of
ectopic chondrogenesis (Fig. 8). In the remaining interdigital tissue cell death
showed in most cases an intensity similar to that of the control legs.
The only morphological change observed in the experimental legs in which
AER removal resulted in failure to form a cartilage was the appearance of an
indentation located in the area of ridge removal. In these cases, vital staining with
Interdigital tissue chondrogenesis
237
neutral red revealed a diminution in the intensity of the normal interdigital cell
death process (Figs 9,10).
AER removal at stage 31
Ridge removal at this stage did not cause any modification in the shape of the leg
buds. Alcian-blue staining revealed ectopic cartilages in only 3 out of 35 embryos
operated at this stage.
Fig. 6. SEM micrograph showing a detailed view of the third interdigital space of an
experimental leg 30 h after ridge removal. The presence of an ectopic interdigital
cartilage in this area was demonstrated by a previous alcian blue staining of the
specimen. Prominent sulcus is located in the boundaries of the area of chondrogenesis.
Note the presence of a small area still devoid of ectoderm (arrow). Scale bar, 0-1 mm.
Fig. 7. Low-magnification SEM micrograph showing an experimental leg 70 h after
ridge removal at stage 30. A prominent interdigital cartilage appears in the medial
surface of the fourth digit. Note that the regression of the interdigital tissue is in
progress. Scale bar, 0-25 mm.
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J. M. HURLE AND Y. GANAN
Fig. 8. Light micrographs showing the pattern of interdigital cell death at stage 33 in
control (A) and experimental (B) legs after ridge removal at stage 30. Arrow shows the
presence of an interdigital cartilage in the experimental leg. Scale bar, 1 mm.
Fig. 9. Light micrograph showing the pattern of cell death at stage 33 in an experimental leg without interdigital cartilage after ridge removal at stage 30. Note the
diminution of cell death in the area underlying the zone of ridge removal (arrow)
(compare with the control leg showed in Fig. 7A). Scale bar, 1 mm.
Fig. 10. Light micrographs showing the pattern of cell death at stage 34 in the third
interdigital space of control (A) and experimental (B) legs after ridge removal at stage
30. In the experimental leg the interdigital cleft is more prominent and cell death is less
intense. Scale bar, 0-25 mm.
Interdigital tissue chondrogenesis
239
Dorsal ectoderm removal
Dorsal ectoderm removal at stage 29 did not cause significant changes in the
shape of the limb bud. As can be seen in Fig. 11, at stage 31 (24 h after the
operation) a prominent inhibition of interdigital cell death was detected in 5 out of
7 embryos studied by this procedure. However, the inhibition of cell death was
only present in the proximal zone of the interdigit. At more advanced stages, the
interdigital necrotic area displayed a normal appearance in the distal zone of the
interdigit and the digits became detached from each other by stage 34-35. At stage
33, alcian-blue staining revealed the presence of interdigital ectopic cartilages in
6 out of 18 embryos studied by this procedure. As can be seen in Fig. 12, in all
cases the cartilage occupied the proximal zone of the interdigit and showed no
Fig. 11. Light micrograph showing the pattern of cell death at stage 31 in the third
interdigital space of control (A) and experimental (B) legs after dorsal ectoderm
removal at stage 29. Note the absence of cell death in the experimental leg. Scale bar,
0-25 mm.
Fig. 12. Alcian-blue staining showing the presence of an ectopic cartilage in the
proximal zone of the third interdigit of an experimental leg 70 h after dorsal ectoderm
removal at stage 29. Scale bar, 0-3 mm.
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J. M. HURLE AND Y. GANAN
Fig. 13. SEM micrograph showing the appearance of an experimental leg 10 h after
dorsal ectoderm removal at stage 29. Scale bar, 0-2mm.
Fig. 14. SEM micrograph showing the third interdigital space of an experimental leg
with ectopic chondrogenesis 80 h after dorsal ectoderm removal. Note the bulging
appearance of the interdigital cartilage at this stage. Scale bar, 0-4 mm.
joints with the neighbouring phalanges. In no case was the structure of the digits
altered.
As in the case of AER removal, the efficiency of the surgical procedure was
controlled by scanning electron microscopy (Fig. 13). The area devoid of ectoderm
Interdigital tissue chondrogenesis
241
was detectable up to stage 30. At more advanced stages surface alteration of the
interdigit was not present, with the exception of the presence of a rounded
prominence in the area of ectopic chondrogenesis which in some cases appeared to
be in course of detachment into the amniotic sac (Fig. 14).
DISCUSSION
Our results provide evidence suggesting that the ectoderm might be directly or
indirectly involved in the control of interdigital cell death. Although the two types
of experimental procedures performed in this study resulted in alteration of the
pattern of interdigital cell death and eventual formation of interdigital cartilages,
there are several facts which make it necessary to discuss the two experiments
separately.
As could be expected from previous studies (Saunders, 1948; Summerbell, 1974;
Rowe & Fallon, 1982) removal of the interdigital ridge causes a local inhibition of
growth of the interdigit which leads to limbs with an abnormally precocious
interdigital cleft as compared with the control contralateral limbs. As will be
discussed below, cell death does not appear to be involved in the formation of this
abnormal interdigital cleft. This fact is in agreement with previous studies of
Rowe, Cairns & Fallon (1982) showing that after stage 23, ridge removal does not
evoke cell death in the underlying mesenchyme. The earlier the operation was
performed the more prominent the cleft which appeared. When ridge removal was
performed at stage 27 the interdigital cleft achieved a maximum intensity, whereas
the cleft was minimal or non-existent when it was done at stage 30-31. This
observation is in agreement with experiments of recombinations of AER and
mesenchyme of different stages (Rubin & Saunders, 1972; Pautou, 1972) and indicates that the AER actively induces distal growth of the interdigit up to stage 30.
On the basis of the sequence of structural alterations observed in the course
of normal regression of the interdigital tissue, it has been suggested that the
interdigital cell death might be triggered by a sudden interruption of the inductive
effect produced by the AER due to a rapid disintegration of this structure (Hurle
& Fernandez-Teran, 1983,1984). Our present observations show that the onset of
interdigital cell death takes place in the schedule independently of the stage at
which we remove the AER, making unlikely a linkage between the disappearance
of the AER and the initiation of interdigital cell death. Only a minor number of
experimental limbs displayed an abnormal necrotic focus in the area of ridge
removal but the inconstancy of this result suggest that it was due to differences in
depth of cut rather than to the absence of the inductive effect of the ridge.
Otherwise, this necrotic focus would be present in most of the experimental limbs
and presumably its extension would also be related to the age at which the
operation was performed. Contrary to the hypothesis of a causal relationship
between the disappearance of the AER and the onset of interdigital cell death,
we have observed at more advanced stages an apparent diminution in the intensity
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J. M. HURLE AND Y. GANAN
of cell death, often accompanied by the differentiation into cartilage of the
interdigital mesoderm. Although it is tempting to suggest that AER removal may
cause the arrest of the degenerative programme thus allowing the formation of
interdigital cartilages, there are other interpretations which fit better with the
results obtained here. It must be taken into account that, in spite of the apparent
diminution of interdigital cell death, in many cases the digits detach from each
other even when interdigital cartilages are formed. In fact, the inhibition of cell
death in the cases in which there is no cartilage is always associated with zones in
which the interdigital cleft is more prominent than in the contralateral control
limb. In these cases the diminution of cell death might be due to a diminution in
the interdigital tissue caused by ridge removal. It has recently been observed that
the anterior necrotic zone of developing wing buds exhibits regulatory properties;
its intensity increases or diminishes in experimental wings, depending on whether
there is an excess or deficiency of mesenchymal tissue (Yallup, 1984).
The formation of ectopic cartilages in the interdigital space was not an unexpected feature after ridge removal. We have previously reported its formation
after administration of Janus green to 6- to 6-5-day chick embryos (FernandezTeran & Hurle, 1984). In that experimental model, the formation of ectopic
cartilages was also preceded by the disintegration of the AER. Without further
experimental analysis, it is difficult to ascertain the nature of the signal which
changes the prospective fate of the interdigital mesenchyme from death to
chondrogenesis. However, since the AER appears to exhibit an important inhibitory effect on chondrogenesis in vitro (Solursh, Singley & Reiter, 1981; Kosher,
Savage & Chan, 1979), a feasible explanation is that AER removal acts by
abolishing this inhibitory factor produced by the ridge. Based on these facts, it can
be proposed that under our experimental conditions the cells are primarily
stimulated to form cartilage, thus being diverted from the death programme, as
appears to occur in tissue culture conditions (Newman, 1977).
The ability of ridge removal to induce interdigital chondrogenesis is reduced
drastically after stage 31. This suggests that either the ridge loses its potentiality
with age or that after stage 31 the mesenchymal cells are already irreversibly
programmed to die. Similarly, the absence of interdigital cartilages after ridge
removal at stage 27 suggests that insufficient interdigital mesenchymal cells are
available for transformation into cartilage.
The formation of ectopic cartilages after dorsal ectoderm removal indicates that
this property is not restricted to the ridge ectoderm. The fact that the non-ridge
ectoderm of the limb also exhibits a significant inhibitory effect on chondrogenesis
in vitro (Solursh etal. 1981) supports the idea that under our experimental
conditions the inhibition of cell death is secondary to the differentiation into
cartilage of the interdigital mesenchymal cells. The lesser incidence of chondrogenesis after dorsal ectoderm removal as compared with ridge removal at the same
stages might be indicative of a lesser inhibitory effect on chondrogenesis of the
non-ridge ectoderm. However, it must be taken into account that the surgical
procedure for dorsal ectoderm removal cannot be fully standardized. Unavoidable
Interdigital tissue chondrogenesis
243
small variations in the extension of the area of ectoderm removal may be here a
major determinant of the expression of cartilage induction.
Based on our results, it is tempting to suggest that the causes of interdigital cell
death will not be clarified until we can explain why the mesenchymal cells of the
interdigit, in contrast with those of the digital arrays, remain undifferentiated.
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