/. Embryol. exp. Morph. 78, 195-209 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
Fine structure of the regressing interdigital
membranes during the formation of the digits of the
chick embryo leg bud
By J. M. HURLE 1 AND M. A. FERNANDEZ-TERAN
From Departamento de Anatomia, Facultad de Medicina, Universidad de
Santander
SUMMARY
There is recent evidence showing that in addition to the well-known mesenchymal necrotic
mechanism involved in the disappearance of the interdigital membranes, the ectodermal tissue
may also play an active role in the formation of the free digits of most vertebrates. Ultrastructural study of the regressing interdigital membrane of the chick leg revealed significant changes
at the epitheliomesenchymal interface. Disruptions of the ectodermal basal lamina and an
intense deposition of collagenous material were the most conspicuous changes observed in the
extracellular matrix. In addition the basal ectodermal cells showed prominent cell processes
projected into the mesenchymal core of the membrane, and mesenchymal macrophages appeared to migrate through the epithelial tissue to be detached into the amniotic sac. It is
concluded from our results that the elimination of the interdigital membranes is a complex
process requiring the interaction of all the tissue components of the membrane.
INTRODUCTION
The development of the digits of most vertebrates takes place by their detachment from an initial hand or foot plate. On the basis of many descriptive and
experimental studies this process is usually considered as a classic case of
controlled cell death shaping morphogenesis (Saunders & Fallon, 1967; Ballard
& Holt, 1968; Pautou, 1974, 1975, 1976; Fallon & Cameron, 1977; Hinchliffe,
1981). There are however studies showing that the interdigital necrosis (interdigital necrotic zones, INZ) is accompanied by conspicuous changes in the shape
and structure of the ectodermal cells. On the basis of the ultrastructural features
of the interdigital ectoderm of human embryos, Kelley (1973) suggested that the
ectoderm may actively invaginate into the necrotic zones contributing to the
detachment of the free digits. In a recent SEM study we have observed that the
interdigital ectoderm of the chick differs significantly from that of the duck
(Hurle & Colvee, 1982), supporting the hypothesis of such an ectodermal involvement in the detachment of the digits. Furthermore a significant rupture of
^Author's address: Departamento de Anatomia, Facultad de Medicina (Poligono de
Cazofia), Santander, Spain.
196
J. M. HURLE AND M. A. FERNANDEZ-TERAN
the interdigital membrane, the ectodermal tissue being detached into the amniotic sac, was also observed in that study. These facts suggest that in the same
way that distal outgrowth of the limb is carried out by a permissive interaction
between apical ectodermal ridge (AER) and the marginal mesenchyme, the
reabsorption of the interdigital tissue may be due to a change or a cessation in
the interaction between the interdigital mesenchyme and the ectoderm, resulting
in the cessation of growth and disruption of the interdigital membranes.
Epitheliomesenchymal interaction is a basic embryological mechanism accounting for the morphogenesis of many organs (Grobstein, 1967). The two
tissues are separated by an interface of extracellular material which is a critical
element in the interaction process (Hay, 1978,1981). To summarize briefly, the
characteristics of these extracellular matrix components can modify the differentiation of the epithelium and the behaviour of the underlying mesenchymal
tissue.
The importance of the epitheliomesenchymal interface in relation to the
growth of the limb buds has motivated several studies of the ectoderm-basal
lamina-mesenchyme complex in different vertebrates (Jurand, 1965; Kelley,
1973, 1975; Kelley & Bluemink, 1974; Ede, Bellairs & Bancroft, 1974; Smith,
Searls & Hilfer, 1975; Kaprio, 1977). However, most of these studies have
focused on the early growing stages of limb organogenesis, while the structure
of the epitheliomesenchymal interface of the regressing interdigital spaces has
received little attention (Kelley, 1973).
In view of the reported information and on the basis of recent studies showing
that modifications of the basal lamina of regressing epithelial structures might
play a role in the disappearance of the tissue (Trelstad, Hayashi, Hayasi &
Donahoe, 1982), we have undertaken a detailed ultrastructural study of the
regressing interdigital membranes of the chick leg bud. Our results reveal significant changes in the epitheliomesenchymal interface during degeneration of
the interdigital membranes. Evidence of an active involvement of the ectoderm
in the detachment of the digits and the existence of a transepithelial migration
process of mesenchymal macrophages were also observed.
MATERIALS AND METHODS
The third interdigital membrane (IM) of the leg bud of white Leghorn chick
embryos was chosen for this study due to the high intensity and long duration of
the degenerative process present during the regression of this membrane.
Thirty-six embryos ranging from 7 to 11 days of incubation were sacrificed at
12 h intervals. The leg buds were fixed in 2-5% glutaraldehyde in 0-1 Mcacodylate buffer (pH7-2) for 4h, washed in buffer solution in which the third
IM was carefully microdissected and postfixed in 1 % osmium tetroxide. In some
specimens 0-1 % ruthenium red was added to the fixatives to stain the extracellular material according to the Luft (1971) procedure. The selected fragments
Structure of the regressing interdigital membranes
197
were then dehydrated in a graded series of acetones and propylene oxide and
embedded in Araldite. To ensure that the IM would be sectioned longitudinally,
the fragments were embedded in flat capsules and carefully oriented under the
binocular dissecting microscope. Serial semithin section were cut with a LKB
ultratome III and stained with 1 % toluidine blue. Ultrathin sections of selected
areas were then made, mounted on uncoated copper grids, stained with uranyl
acetate and lead citrate and examined with a Zeiss EM 109 electron microscope.
RESULTS
During the whole period studied, the interdigital membranes (IM) are
relatively simple structures consisting of a core of mesenchymal cells covered by
the ectoderm.
Day 7-7-5
The mesenchymal core of the IM shows a central loose zone rich in blood
vessels and a more condensed region located peripherally under the ectodermal
jacket. Abundant dying cells and macrophages are observed in the proximal
region of the IM (Fig. 1). No dying cells are present in the distal zone of the IM.
The extracellular space appears very poor in electron-dense components. Only
small patches of amorphous material and occasional unbanded fibrils which are
positive for ruthenium red are observed (Fig. 2). The epithelial layer of the
ventral and dorsal surfaces shows a basal layer of cuboidal cells covered by
flattened peridermal cells rich in microfilaments. At the margin of the IM the
AER is observed (Fig. 1). The presence of gap junctions is a characteristic
feature of the AER at this stage.
In the epitheliomesenchymal interspace a basal lamina, amorphous material
and collagen fibrils are observed (Figs 3, 4). Ruthenium red revealed a conspicuous periodic staining of the externa and interna lamina rara of the basal
lamina (Fig. 4). Under the basal lamina, amorphous material and fibriUar
ruthenium red-positive material are abundant (Fig. 4). Crossbanded collagen
fibrils arranged along the major axis of the IM are also observed. Cytoplasmic
cell processes of the mesenchymal cells often traverse the sublaminar space
establishing contacts with the basal lamina.
Day 8
The mesenchymal core of the IM shows a very prominent necrotic focus
located in the marginal zone of the membrane (Fig. 5). Abundant isolated dying
cells and some macrophages are present within this focus. In contrast with this
marginal necrotic zone the proximal zone of the IM also shows a necrotic area
but large macrophages are the most prominent features while isolated dead cells
are scarce. As in previous stages, electron-dense extracellular materials are
198
J. M. HURLE AND M. A. FERNANDEZ-TERAN
Structure of the regressing interdigital membranes
199
scarce within the mesenchymal core of the IM, although they can be enhanced
by ruthenium red staining (Fig. 6).
The epithelial layer of the IM shows a structural pattern similar to that observed at day 7-7-5. The only changes worth mentioning are present in the AER.
At this zone the ectodermal cells lose the elongated shape of the preceding
stages, taking on a rather rounded appearance. Gap junctions are now unusual
and numerous dead epithelial cells are observed.
The epitheliomesenchymal interface is also similar to that observed at day
7-7-5 but discontinuities of the basal lamina are occasionally observed in the
AER.
Days 8-5-10
In this stage the IM shows a very different structure from that observed in
previous stages. The changes are present both in the mesenchymal and in the
epithelial tissues and especially in the epithelial-mesenchymal interface. The
mesenchymal tissue appears very condensed and consists mainly of healthy
mesenchymal cells and large macrophages (Fig. 7). However, isolated dying cells
and cell debris are also present. The morphology of the healthy mesenchymal
cells is similar to that of the earlier stages but they now can show cytoplasmic
invaginations filled with collagen fibrils (Fig. 8). All the degenerating cells,
including macrophages, tend to be located in the proximity of the ectoderm,
establishing in many instances close contacts with the ectodermal cells. We shall
describe these features later. The extracellular matrix is now extraordinarily rich
in crossbanded or faint-banded collagen fibrils which are preferentially located
Fig. 1. Longitudinal semithin section of the IM at day 7-5. The ectodermal tissue still
displays the AER (arrows). The necrotic area (n) is only present in the proximal zone
of the membrane (n). Scale bar = 0-1 mm.
Fig. 2. 7-5-day IM stained with ruthenium red showing a dense staining of the basal
lamina and few ruthenium red-positive particles in the extracellular space. Note the
stellate shape of the mesenchymal cells with cell processes establishing contact with
the ectodermal basal lamina. Scale bar = 5 [xm.
Fig. 3. Electron micrograph of the epitheliomesenchymal interface of the dorsal
face of a 7-5-day IM illustrating the continuous basement membrane along the basal
surface of the ectoderm. Scale bar = 1 /im.
Fig. 4. Detailed view of the basal membrane of a 7-day IM after ruthenium red
staining. Ruthenium red granules are attached to the externa and interna lamina rara
of the basal lamina. Note ruthenium red granules attached to the sublaminar fibrillar
material (arrows). Scale bar = 0-5 jum.
Fig. 5. Longitudinal semithin section of a 8-day IM. A prominent necrotic focus is
present in the mesenchyme of the marginal zone of the membrane. The AER appears flattened displaying degenerating cells. Scale bar = 75 jum.
Fig. 6. Mesenchymal tissue of a 8-day IM after ruthenium red staining. All the
cellular components, including dead cells, display a prominent ruthenium redpositive (coat). The extracellular matrix only shows occasional scattered ruthenium
red-positive components. Scale bar =
200
J. M. HURLE AND M. A.
FERNANDEZ-TERAN
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Structure of the regressing interdigital membranes
201
in the most marginal zone of the IM (Fig. 9). They are grouped in large clumps
located between the mesenchymal cells and in the epitheliomesenchymal interface.
The epithelial tissue of the IM in this stage shows clear regional differences in
structure. In the ventral and dorsal faces of the IM the peridermal cells take on
a rounded appearance and display abundant microvilli. They are joined to each
other and to the underlying basal cells by desmosomes. Bundles of microfilaments are abundant within the cytoplasm. The ectodermal basal cells are low
cuboidal or rather flattened and show large bundles of microfilaments parallel to
the basal surface of the cells. In some instances dark condensations located at
various intervals are present along a bundle of microfilaments. Solitary cilia
projecting into the intercellular space are often observed. Cell junctions between
these basal cells are only present at the basal zone of the lateral cell profiles and
consist of maculae adherentes. Large macrophages are often observed within the
epithelial cells. In some cases they are located between the basal ectodermal
cells. In these cases the macrophages are separated from the underlying mesenchymal tissue by zones of junction between basal cell processes of the ectodermal
cells (Fig. 10). Clumps of extracellular material including collagen fibrils are
often located in the space between the macrophages and the ectodermal cells
(Fig. 10). In other cases the macrophages are located between the peridermal
cells and the basal ectodermal cells. In these cases the peridermal cells display
cell processes which surround the surface of the macrophage (Fig. 11). We have
never observed cell junctions between macrophages and ectodermal or peridermal cells.
In the marginal zone of the IM the AER is no longer observed. As can be seen
in Fig. 7, the epithelium of this zone is a multilayered structure. It has a layer of
basal ectodermal cells surrounded by a prominent ridge of peridermal cells. The
basal cells differ from those of the dorsal and ventral faces of the IM in that they
are more irregularly shaped. Flattened, rounded and cylindrical cells poor in cell
junctions are present. Within the cytoplasm, the most prominent feature is the
Fig. 7. Longitudinal semithin section of a 9-5-day IM. In the margin of the membrane the peridermal cells form prominent cords protruding towards the amniotic
sac. Macrophages are observed in clefts on the ectodermal tissue (black arrows) and
in the core of the peridermal cell cords (m). Note a large deposit of extracellular
material under the marginal epithelium (white arrows). Scale bar = 20|Um.
Fig. 8. Mesenchymal cell of a 9-day IM showing a vesiclefilledwith a collagen fibril.
Scale bar = ljum.
Fig. 9. Electron micrograph of a subepithelial deposit of collagen fibrils of a 9-day
IM. Scale bar = 1/im.
Fig. 10. Electron micrograph of a macrophage (m) located between the basal cells
(e) of the ectoderm of the dorsal face of a 8-5-day IM. Note the presence of collagen
fibrils in the intercellular space (arrow). Scale bar = 1 [xm.
Fig. 11. Large macrophage located under the peridermal cells of the epithelium of
a 9-5-day IM. Scale bar =
202
J. M. HURLE AND M. A. FERNANDEZ-TERAN
presence of cell invaginations or vesicles filled with collagen fibrils. These vesicles are more often observed at the basal pole of the cells (Fig. 12), but in some
instances cisternae showing a collagen fibril are observed at the apical pole of the
cells. The presence of ruthenium red staining in some of these vesicles suggests
that, at least in part, they are invaginations of the cell membrane. The peridermal cells form multilayered cords protruding towards the amniotic sac. They are
joined by desmosomes. In some instances the presence of constrictions within
the cords suggests that clumps of peridermal cells are detached into the amniotic
sac. Large macrophages are often observed in the core of the peridermal cords
(Fig. 7). The peridermal cells form a continuous sheet around these macrophages
but cell junctions between the macrophages and the peridermal cells were never
observed. Most of the peridermal cells show a healthy appearance but in some
cases they show prominent vacuolization and degenerated organelles, although
the cell nucleus retains a normal morphology. Gap junctions which are
prominent features of the AER were never observed in the marginal epithelium
at this stage.
The most characteristic feature of the epitheliomesenchymal interface is the
presence of discontinuities in the basal lamina. This feature is especially
prominent in the marginal zone of the epithelium. In this zone the ectodermal
cells are underlain by a wide space rich in collagen fibrils, clumps of amorphous
basal lamina-like material and cell detritus (Fig. 7). Small extracellular-matrix
vesicles 0-01-0-03 [im in diameter are also abundant in this space (Fig. 12). Both
in the marginal and dorsoventral zones of the IM, the ectodermal cells show
abundant cell processes projecting into the mesenchymal tissue (Figs 13, 14).
These processes originated most often at the edges of the basal surface of the
ectodermal cells but they can be present in any zone of the basal surface. Contacts between these processes and the underlying mesenchymal cells and necrotic
cell fragments (Fig. 13) are often observed. The pattern of ruthenium red staining showed conspicuous changes at these zones. The basal lamina appears irregularly stained. Patches of ruthenium red-positive material alternate with
zones lacking stain (Fig. 15). Macrophages are frequently found in association
with the basal surface of the ectodermal cells. Very often small cell processes of
the ectodermal cells establish contacts with macrophages in zones in which the
basal lamina shows discontinuities. In some instances a large segment of a
macrophage appears in clefts of the ectodermal layer (Figs 16,17). In these cases
the ectodermal cells usually show cell processes rich in microfilaments apposed
to the segment of the macrophage located under the epithelial tissue (Fig. 17).
Day 10-5-11
In this stage a progressive transformation of the regressing IM into the
definitive interdigital tissue is observed. Fig. 18 shows the appearance of the IM
at day 10-5. The necrotic zone of the mesenchymal tissue is restricted to the tip
of the remaining IM. Some very large macrophages are observed while isolated
Structure of the regressing interdigital membranes
203
Fig. 12. Basal ectodermal cell of a 8-5-day IM showing two vesicles filled with
collagen fibrils. Arrows show small extracellular matrix vesicles. Scale bar = 1 [im.
Fig. 13. Basal ectodermal cell showing a cell process establishing contact with a
degenerated cell fragment (arrow). 8-5-day IM. Scale bar = 0-5 jum.
Fig. 14. Basal ectodermal cell of a 8-5-day IM showing a prominent cell process
passing across a discontinuity of the basal lamina. Scale bar = 1 ^an.
Fig. 15. Basal ectodermal cell showing a cell process in a zone lacking basal lamina
after staining with ruthenium red. Note the presence of irregular patches of
ruthenium red granules (arrows). 9-day IM. Scale bar = 0-5 jum.
dead cells are exceptional. A prominent clump of collagen fibrils can still be seen
under the marginal epithelium. The number of macrophages located within the
epithelial tissue is significantly reduced from that of previous stages. Detaching
peridermal cell cords are still prominent at the margin of the membrane. However, macrophages located within the cords are scarce or absent. The basal lamina
tends to be continuous. At day 11 the IM reaches a rather stable appearance. In
204
J. M. HURLE AND M. A.
FERNANDEZ-TERAN
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Structure of the regressing interdigital membranes
205
the mesenchymal tissue degenerating cells are no longer present. Between the
cells, scattered crossbanded collagen fibrils are observed. A prominent clump of
collagenous material is still present near the free margin of the membrane.
However, the size of the collagenous clump is significantly reduced with respect
to that of previous stages. The epithelial tissue shows a basal layer of columnar
cells covered by flattened peridermal cells. Occasional gap junctions are again
observed between the basal cells of the free margin of the IM. As can be seen in
Fig. 19, peridermal cell cords which now show a striking ballooning appearance
are still observed at the marginal zone of the membrane. No macrophages are
present within the peridermal cords at this stage. The epithelial-mesenchymal
interface consists of continuous basal lamina underlain by occasional crossbanded collagen fibrils.
DISCUSSION
Our results show that the regression of the chick IM involves a wide range of
structural changes affecting both the ectodermal and the mesenchymal tissue.
As reported in many previous studies, the most remarkable feature of the
regressing mesenchymal tissue is the establishment of a prominent necrotic
focus. It has also been pointed out that two distinct components, proximal and
distal (marginal) can be distinguished within the chick INZ (Pautou, 1975). Our
observations show that the marginal part of the INZ appears simultaneously with
AER degeneration, suggesting a causal relationship between the two processes.
The extracellular matrix of the mesenchymal tissue of the IM also shows
dramatic changes during the degeneration process. Until day 8 of incubation the
morphological characteristics and the pattern of ruthenium red-staining of the
extracellular matrix are similar to those reported in other stages of limb development (Kelley, 1975; Sawyer, 1982). At day 8-5, however, an enormous accumulation of collagen fibrils is observed. A similar process has been reported in the
Fig. 16. Large mesenchymal macrophage showing a segment located between the
basal ectodermal cells (e). Arrows show the limits of the ectodermal cleft. Note the
abundance of collagenous material in the intercellular space. 8-5-day IM. Scale
Fig. 17. Electron micrograph of the ectodermal layer of a 9-day IM showing a large
macrophage in course of being incorporated into the epithelium. Arrow shows the
presence of a ectodermal cell process contacting the subepithelial segment of the
macrophages. Scale bar = 2 /xm.
Fig. 18. Semithin section of a 10-5-day IM. Note the prominent peridermal marginal
ridge and the abundance of extracellular matrix component in the subepithelial zone.
Arrows show a large macrophage located within the epithelial tissue. Scale bar =
Fig. 19. Semithin section of the marginal zone of a 11-day IM showing the presence
of prominent peridermal cell cords protruding towards the amniotic sac. Scale
206
J. M. HURLE AND M. A. FERNANDEZ-TERAN
regressing IM of human embryos (Kelley, 1973). Both the origin and the significance of these collagen deposits are intriguing questions unsolved in our
study. Neither the structure of the mesenchymal cells which are poor in rough
endoplasmic reticulum, nor that of the ectodermal cells, support the possibility
of an intense secretion process accounting for such a quick appearance of large
clumps of collagen. In consequence, the only possibility which could be suggested is that the origin of collagen may be due to the polymerization of procollagen
molecules.
The possible involvement of the ectodermal tissue in the regression of the IM
has received little attention (Kelley, 1973; Hurle & Corvee, 1982). The first
morphological change in the ectodermal tissue observed in this study was the
degeneration of the AER. The basal ectodermal cells of the ridge degenerate and
gap junctions which are a distinctive feature of the AER (Fallon & Kelley, 1977)
disappear. By day 8-5 the basal cells of the marginal epithelium flatten and the
peridermal cells of the IM become rounded. At the same time a prominent ridge
consisting mainly of peridermal cell cords appears at the marginal zone of the
regressing membrane. The change of shape of the peridermal cells and the
abundance of oriented microfilament bundles within these cells have been interpreted as suggestive of an active invagination of the ectodermal epithelium
during the morphogenesis of the digits (Kelley, 1973; Hurle & Colvee, 1982).
Our observations do not lead us to discard such a possibility, however alternative
explanations can be proposed. It has been observed that the growth patterns of
the epithelial and mesenchymal components of the developing limb bud differ
significantly. While the mesenchymal tissue grows by proliferation or directed
outwards migration of the marginal tissue due to the inductive effect of the AER
(Reiter & Solursch, 1982), the epithelial tissue, especially the peridermal component, undergoes a proximodistal sliding process (Roncali, 1973; Milaire,
1973). Based on these facts it can be suggested that in the regressing IM the
disappearance of the mesenchymal tissue is followed by a process of sloughing
off of the ectodermal cover at the margin of the membrane. In this respect it
could be suggested that the AER, in addition to the inductive effect on the
underlying mesenchymal tissue, might also play a stabilizing role, synchronizing
the proximodistal sliding process of the limb ectoderm with the distal outgrowth
of the mesenchyme.
There are several reports suggesting that the cessation of limb-bud outgrowth
involves changes in the epithelial-mesenchymal interface (Jurand, 1965;
Kelley, 1973, 1977). Alterations in the epitheliomesenchymal interspace were
also reported in chick mutants with defective limb-bud development (Sawyer,
1982). In our study we have observed that the epithelial and mesenchymal
modifications present in the regressing IM are correlated with conspicuous
changes in the epitheliomesenchymal interface. As reported in the IM of the
human embryo (Kelley, 1973, 1977), we found that the amount of collagen
increases under the marginal ectoderm; but in contrast with the human embryo,
Structure of the regressing interdigital membranes
207
this was accompanied by prominent interruptions of the basal lamina. It is difficult from our study to ascertain the cause of the disruption of the basal lamina.
However, the presence of matrix vesicles in zones of basal lamina interruption
is a significant feature. During the development of the tooth organ, the basal
lamina of the inner enamel epithelium appears to be degraded by the liberation
of proteolytic enzymes from morphologically similar matrix vesicles formed by
budding from the plasma membrane of the preodontoblast cell processes (see
Slavkin, Trump, Brownell & Sorgente, 1977). If we compare our observations
with those obtained during tooth organ development, it can be suggested that the
disruption of the basal lamina of the IM could be related to the presence of the
matrix vesicles. These vesicles presumably originate from the disintegration of
the dying mesenchymal cells.
An interesting question in the present study is the significance of the ectodermal cell processes which pass across basal lamina discontinuities to establish
contacts with the underlying mesenchymal components. Several cases of
epithelial-mesenchymal interaction involve epitheliomesenchymal cell contact,
which is assumed to play a role in inductive process (see Saxen, Ekblom &
Thesleff, 1980). However, since the IM is in fact a regressing structure, the
changes at the epitheliomesenchymal interface may well be a consequence of the
controlled regression rather than reflecting active tissue inductive interaction.
This consideration does not discount the possibility that such modifications might
result in a change in the shape of the IM. The rupture of the basal lamina plus
possible compositional changes in the subepithelial matrix might well explain the
presence of ectodermal cell processes projecting into the mesenchymal space
(see Hay, 1982). On the other hand, it appears that most embryonic cells in the
presence of necrotic cells develop a phagocytic activity, surrounding such
degenerating cells by means of cytoplasmic processes and eventually internalysing them (see Hurle, Lafarga & Ojeda, 1978). The presence of contacts
between ectodermal cell processes and underlying cell fragments might well
indicate a phagocytic stimulus. Epithelial cell processes passing through disruptions of the basal lamina and participating in the phagocytosis of extracellular
fragments have been reported during the formation of the chick lens primordium
(Garcia-Porrero, Collado & Ojeda, 1979). However, we found no evidence that
the contacts between the epithelial cell processes and the dead cells result in the
engulfment of these cells.
The association of large macrophages with the ectodermal tissue is a striking
feature which merits detailed discussion. Macrophages were observed in the
following positions: i) under the ectodermal layer establishing close contacts with
the ectodermal cell processes; ii) located partly between the basal ectodermal
cells and partly in the mesenchymal space; iii) between the basal ectodermal
cells; iv) between the basal ectodermal cells and the peridermal cells; v) within
the peridermal cell cords of the marginal epithelium of the IM. In many cases
evidences of a process of detachment of these cords into the amniotic sac were
208
J. M. HURLE AND M. A. FERNANDEZ-TERAN
observed. Cell junctions between the epithelial cells and the macrophages were
never observed, nor was there any evidence of an intense phagocytic activity of
the epithelial cells supporting the possibility of a transformation of the epithelial
cells into macrophages. All these facts suggest the existence of a migration
process of the mesenchymal macrophages through the epithelial tissue being
finally detached into the amniotic sac along with the epithelial cells.
In conclusion, the present observations show that the disappearance of the chick
IM involves major alteration of the ectodermal-basal lamina-mesenchyme complex. Due to the descriptive nature of our observations, the possible cause-effect
relationships between the changes observed in different components of the IM
cannot be established, nor can their precise role in the elimination of the IM be
determined. However, it seems clear from the present study that cell death is
only one of the factors involved in the detachment of the chick digits.
Our thanks are due to Mrs Pilar Elena-Sinobas for her excellent assistance in making the
electron microscopic sections. One of us (M. A.F-T.) wishes to acknowledge receipt of a grant
'Para la formaci6n de personal investigador' of the Spanish Ministry of Education and Science.
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JURAND,
{Accepted 18 July 1983)