/ . Embryol. exp. Morph. Vol. 69, pp. 251-263,1982
Printed in Great Britain © Company of Biologists Limited 1982
251
Surface changes in the embryonic
interdigital epithelium during the formation of
the free digits: a comparative study in the
chick and duck foot
By J. M. HURLE 1 AND E. COLVEE
From the Departmento de Anatomia, Facultad de Medicina,
Universidad de Santander
SUMMARY
The formation of the free digits of the chick is accompanied by conspicuous surface
changes of the interdigital ectoderm. These changes were much less pronounced or absent
in the duck.
As early as the interdigital grooves were detected in the chick, the morphological features
of the ectodermal cells changed from a polygonal shape and flattened appearance to a
rounded shape and bulging appearance. These changes were not present in the webbed foot
of the duck. On the other hand the development of the interdigital commissures was accompanied by the formation of ectodermal ridges consisting of an accumulation of rounded
cells which were in some cases in course of detachment to the amniotic cavity. These ridges
were very prominent in all the interdigital commissures of the chick. In the duck they were
less pronounced and were only present in the first and third commissure. From these results
it is suggested that in addition to the well-known interdigital mesenchymal necrotic process
(INZ) the ectodermal tissue of the interdigits might also be actively involved in the formation
of free digits.
INTRODUCTION
The development of the free digits of most vertebrates, takes place by their
detachment from an initial hand or foot plate. A great number of papers have
shown that the process of digit detachment in reptiles, birds and mamnjals
involves the participation of conspicuous interdigital mesenchymal necrotic
zones (INZ) (Saunders, Gasseling & Saunders, 1962; Menkes, Delenu & lilies,
1965; Saunders & Fallon, 1967; Ballard & Holt, 1968; Pautou, 1974, 1975;
Fallon & Cameron, 1977; Hinchliffe, 1974). Inhibition of these interdigital
necrotic areas leads to soft-tissue syndactyly (Deleanu, 1965; Hinchliffe &
Thorogood, 1974; Pautou, 1976). However, there are several aspects of digit
morphogenesis which cannot be explained by such a simplistic interpretation
of that morphogenetic event: (i) the interdigital tissue to be eliminated
1
Author's address: Departmento de Anatomia, Facultad de Medicina (Poligotio de
Cazona), Santander, Spain.
252
J. M. HURLE AND E. COLVEE
Fig. 1-6. Vitally stained chick (Fig. 1-3) and duck (Figs. 4-6) feet, showing the
pattern of interdigital cell death. Note the relative high extension of the necrotic
zones in the interdigital spaces I-II and III-IV of the duck and the distal arrangement of INZ of the chick from day 8-5 (Fig. 3).
Figs. 1-3. Chick foot at days 7, 8 and 8-5 of development. Magnifications x25,
x 25 and x 22 respectively.
Figs. 4-6. Duck foot at days 9, 9-5 and 10 of development. Magnifications x28,
x 22 and x 22 respectively.
Interdigital surface changes during digit morphogenesis
253
Fig. 7. Panoramic SEM view of the chick leg bud at day 5. Note the presence of
the apical ectodermal ridge (arrows), x 60.
Fig. 8. High magnification SEM view of the dorsal surface of the chick leg bud at
day 5. Note the homogeneous polygonal shape of the epithelial cells, x 900.
Fig. 9. Chick embryo foot at day 7 of development. Note the presence of prominent
interdigital grooves (arrows) between the digital elevations, x 60.
Fig. 10. Epithelial surface of the second interdigital groove at day 7. Note that
the epithelial cells show a rather rounded appearance displaying abundant mierovilli and some blebs (arrows). Compare with Fig. 11.x 800.
Fig. 11. Epithelial surface of a digital zone of the chick foot at day 7. Note that
the epithelial cell surface at this zone -shows a morphology similar to that of the
preceding stages, x 800.
9
EMB 69
254
J. M. HURLE AND E. COLVEE
consists of a core of mesenchymal cells covered by a two-layered epithelium.
The INZ affects mainly the mesenchyme, so the fate of the epithelium must
be explained; (ii) in the duck and Herring gull a considerable amount of the
interdigital mesenchyme is removed by cell death, but the digits remain
joined by interdigital webs (Hinchliffe, 1974; Pautou, 1974; Saunders &
Fallon, 1967); (iii) in amphibians there are not necrotic processes associated with the development of free digits (Cameron & Fallon, 1977).
From these observations it can be deduced that in addition to the mesenchymal necrosis, the interdigital epithelial tissue might also play a role in the
digit detachment process.
To confirm that hypothesis we have undertaken a comparative analysis of
the development of the foot in the chick and in the duck. The evolution of the
necrotic zones was surveyed using neutral red vital staining and the possible
modifications of the epithelial layer were studied by scanning electron microscopy. Our results show that in addition to the differences in the intensity of
INZ, the epithelial layer of the interdigital spaces of the chick undergoes
significant changes which are not present in the duck, thus supporting the
idea of an epithelial factor in the detachment process of the digits.
MATERIALS AND METHODS
The foot of White Leghorn chick embryos ranging from day 5 to 10 of
development (stages 27-36 of Hamburger & Hamilton, 1951) and Royal Pekin
duck embryos ranging from days 6 to 12 of development were studied by the
following techniques:
Fig. 12. Panoramic SEM view of an interdigital space of a day 8 chick foot. Note
the rounded appearance of the epithelial cells at the proximal zone of the interdigital groove (arrows) and compare with the flattened appearance of the cells
of the digital zones (D). x 160.
Fig. 13. Panoramic SEM view of an interdigital space of a chick foot at day 9 of
development. All the epithelial cells of the interdigit have now achieved a rounded
shape. Note the more prominent rounded appearance of the cells of the interdigital commissure (arrow), x 140.
Fig. 14. Detailed SEM view of the epithelial cells of an interdigital groove at day
9 to show the rounded and fusiform shape of the cells at this stage, x 1000.
Fig. 15. Detailed SEM view of the epithelial cells of a digital zone of the chick foot
at day 9 of development to show the polygonal and flattened appearance of the
cells. xlOOO.
Fig. 16. Detailed SEM view of the first interdigital commissure of a chick foot at
day 9 showing the rounded and prominent appearance of the epithelial cells at
this zone. xl800.
Interdigital surface changes during digit morphogenesis
255
$-2
256
J. M. HURLE AND E. COLVEE
Interdigital surface changes during digit morphogenesis
257
(a) Vital staining
The interdigital necrotic areas were mapped in ovo by vital staining with
neutral red following the method of Hinchliffe & Ede (1973).
(b) Scanning electron microscopy (SEM)
The feet were fixed in 3 % glutaraldehyde buffered in 0-1 M sodium cacodylate at at pH 7-3 for 3-4 h, washed in buffer alone, dehydrated in a series
of acetones and dried by the critical-point method. The specimens were then
gold-sputtering coated and viewed in a Philips SEM 501 electron microscope.
RESULTS
General evolution of the INZ in the chick and duck
The evolution of the INZ of the chick and duck has been previously studied
by several authors (Pautou, 1975; Saunders & Fallon, 1967) and it is only
surveyed here with the purpose of emphasizing some aspects of their arrangement which suggest the existence of other mechanisms in the formation of the
free digits.
Figures 1-3 and 4-6 summarize the evolution of the INZ in the chick and
duck respectively. In the chick the interdigital necrosis commences at day 7
(stage 31) (Fig. 1) and progresses until day 8 (stage 34) (Fig. 2) in which the
necrosis reaches its climax and then undergoes progressive reduction (Fig. 3)
until day 9-5 (stage 36). In the duck the necrosis can be detected from day 9 to
day 11. As can be seen in Figs. 4-6 the first interdigital space shows the biggest
necrotic area, while in the second and third interdigital spaces the necrotic
foci are located only in the most distal zone.
An important feature to be noticed in the chick is that by day 9 the necrotic
Fig. 17. Panoramic SEM view of the third interdigital commissure of a chick foot
at day 10. The epithelial cells form now a prominent ridge of rounded cells
(arrows). x200.
Fig. 18. Detailed SEM view of the epithelial ridge showed in fig. 17. Note the'
irregular size and rounded appearance of the cells. Some cells make a great
prominence towards the amniotic cavity (arrow), x 1000.
Fig. 19. Panoramic SEM view of the duck embryo leg at day 7-5. x 30.
Fig. 20. Detailed view of the epithelial cells of the duck leg bud at day 7. Microvilli are marginally located and a central cilium is often observed (arrows), x 2400.
Fig. 21. Panoramic SEM view of the duck foot at day 9. Note the presence of
prominent interdigital grooves (arrows) similar to those of the chick foot at day 7
(Fig. 7). x30.
Fig. 22. Panoramic view of an interdigital groove of a duck foot at day 9. Note;
the homogeneous polygonal and flattened shape of the cells both in the groove
(G) and digital (D) zone, x 500.
258
J. M. HURLE AND E. COLVEE
" • • •Y^SBbM^EutB' * ^
"•
\
** J> * %
Interdigital surface changes during digit morphogenesis
259
zones are located at the most distal part of the interdigital spaces but in the
proximal part of the second and third spaces there is still a prominent interdigital membrane with very little degeneration. This fact suggests that other
complimentary mechanisms might be involved in the elimination of the interdigital membrane. On the other hand, it can be noted that the third interdigital
space of the duck displays an important necrotic zone in spite of the fact that
digit III and IV will be joined by a large interdigital web.
Scanning electron microscopy
The SEM observations allowed us to follow important surface modifications
in the chick foot which were much less prominent in the duck. The changes
were mainly detectable in the dorsal face of the foot, so the following results
only refer to the dorsal face of the feet.
Chick embryo foot
At day 5-5-5 the chick foot showed a rounded shape contoured by the
apical ectodermal ridge (Fig. 7). The epithelial layer appeared to be formed by
an uniform sheath of polygonal cells with distinct marginal folds and abundant
microvilli (Fig. 8).
At day 7, the interdigital spaces appeared as prominent grooves located
between the proximal zones of the digital elevations (Figs. 9, 10). At this stag;e
the cells of the dorsal surface of the digits were similar to those of day 5. They
Fig. 23. Duck foot at day 10 of development. Note that in addition to the interdigital grooves an initial formation of the interdigital commissures can be detected.
xl3-5.
Fig. 24. Detailed SEM view of the first interdigital commissure of a duck foot at
day 9-5, showing the presence of a small ridge of rounded ectodermal cells (arrow).
x320.
Fig. 25. Duck foot at day 10-5 of development. Note that digit I is mostly free
from digit II and that the third interdigital commissure is now very pronounced
(arrow), x 15.
Fig. 26. SEM micrograph of the third interdigital commissure of a duck foot at
day 11 showing the presence of a small interdigital epithelial ridge (arrows), x 200.
Fig. 27. Detailed SEM view of the epithelial cells of an interdigital commissural
ridge of a duck foot at day 10-5. Note that the cells are rounded and irregular both
in size and in the density of microvilli. x 1800.
Fig. 28. Duck foot at day 13-5 of development. Note that the foot has now
achieved its definitive appearance, x 13-5.
Fig. 29. SEM micrograph of the epithelial surface of the digital zones of the
duck foot at day 12 showing the polygonal and flattened appearance of the
epithelial cells. xl800.
Fig. 30. SEM micrograph of the epithelial surface of an interdigital space of the
duck foot at day 12. Note that the morphology of the cells is similar to that of
the digital zones (Fig. 29). x 1800.
260
J. M. HURLE AND E. COLVEE
displayed a polygonal shape and flattened surface, showing prominent marginal
folds and abundant microvilli (Fig. 11). In the interdigital grooves the cells
showed a rather fusiform appearance with a rounded central nuclear bulge.
In addition to microvilli these cells showed abundant surface blebs (Fig. 10).
At day 8, the bulging shape of the cells of the interdigital spaces was more
prominent, contrasting sharply with the smooth surface of the digital zones
(Fig. 12). These differences were mainly present in the proximal 2/3 segment
of the interdigital spaces. At higher magnifications the cells of the interdigital
spaces appeared fusiform with the long axis oriented parallel to the interdigital
groove. Microvilli and blebs were abundant as in the preceding stage. The cells
of the digital zones remained flattened and polygonal.
At day 9 (Fig. 13) the presence of rounded cells extended throughout the
entire interdigital space (Fig. 13,14) while in the digital zones the cells remained
polygonal (Figs. 13, 15). As can be seen in Fig. 13, the rounded appearance of
the interdigital cells was particularly prominent at the interdigital notch, forming
in most cases a conspicuous commissural ridge. The morphology of the cells
of these ridges appeared variable. At the first and second interdigital commissures, the cells appeared rounded and smooth (Fig. 16) giving in some
instances the impression of being in the process of detachment. At the third
interdigital commissure the cells showed an exaggerated degree of the rounded
appearance of the cells of the interdigital grooves, with extensive microvilli.
At day 10 the digits appeared extensively free except at their most proximal
zones in which a small triangular interdigital membrane was present (Fig. 17).
The epithelial cells appeared flattened in both the digits and webs but at the
third interdigital commissure there was still a prominent epithelial ridge
formed by rounded cells very rich in microvilli which appeared in some
instances to be in the process of detachment (Fig. 18).
Duck embryo foot
The interdigital spaces of the duck foot can first be detected by day 7-7-5
of development (Fig. 19). At this stage the epithelial cells showed an uniform
morphology throughout the foot. They were polygonal in shape. Microvilli
were abundant and tended to appear marginally located (Fig. 20). A central
cilium was often observed.
At days 8-9 the duck foot was similar to that of the chick at day 7 (Fig. 21).
As can be seen in Fig. 22 no remarkable differences were observed between
the epithelial surface of the digital and interdigital zones. The cells were
polygonal as in the preceding stages. Microvilli were abundant but their density
was irregular among the different cells, although it was always higher at the
cell margins. In some instances intercellular clefts were observed, giving a
stellate appearance to the cells. A central cilium was also a frequent feature.
At days 9-5-10 the digits remained joined by the interdigital membranes
but at the tip of the digits small interdigital notches were observed (Fig. 23).
Interdigital surface changes during digit morphogenesis
261
The morphology of the cells remained similar to that of the previous stages
but at the first interdigital commissure, which was the most prominent, an
epithelial ridge was present. As can be seen in Fig. 24 the cells of the ridge
were rounded and prominent as observed in the chick embryo.
At days 10-5-11 digit I was mostly free from digit II, and the interdigital
commissure of the third interdigital space was very accentuated (Fig. 25). The
morphological features of the dorsum of the digits and that of the interdigital
spaces was similar to that of the preceding stages. In the third interdigital
commissure a prominent ectodermal ridge was present (Fig. 26). The cells of
the ridge were rounded, irregular in size and very rich in microvilli (Fig. 27).
By day 12 the duck foot had achieved the definitive appearance (Fig. 28).
The cells both in the digital (Fig. 29) and interdigital zones (Fig. 30) were
polygonal with abundant microvilli and marginal folds, as observed in the
preceding stages.
DISCUSSION
As has been pointed out by Fallon & Cameron (1977), in spite of the fact
that massive interdigital cell death accompanies the formation of the free
digits of birds, reptiles and mammals, it is important to note that it may not
be the only mechanism accounting for the formation of the free digits. Kelley
(1970) has suggested that the mesenchymal cells may migrate from the intefdigital spaces to the digits. On the other hand, Cameron and Fallon (1977) in
amphibians, and Pautou (1975) in birds, have proposed the existence of an
important mechanism of differential growth involved in the formation of free
digits. The results of the present study support the previous study by Kelley
(1973) suggesting that the epithelium of the interdigital space might also play
an active role in the formation of the digits.
From the SEM observations the formation of the digits can be described
in two main steps: First, the formation of interdigital grooves can be detected.
This is followed by the development of interdigital commissures. We observed
morphological changes in the epithelium associated with each process. The
grooving process is much more prominent in the chick and it is concomitant
with a conspicuous change in the surface morphology of the interdigital epithelium of the dorsal face of the foot. On the other hand the development of
the interdigital commissures is correlated with the formation of a prominent
epithelial ridge which might serve a detaching function of the interdigital
epithelial cells.
The changes in the surface morphology of the interdigital spaces were
observed only in the chick foot and consisted of the transformation of the
apical surface of the epithelial cells. As soon as the interdigital grooves were
detected in the chick, the epithelial cells changed from a polygonal shape and
flattened appearance to a fusiform shape and rounded appearance with the
long axis oriented longitudinally to the interdigital space. These changes could
262
J. M. HURLE AND E. COLVEE
be due to a passive deformation of the cells produced by the growth of the
skeleton of the digits; but since they were not observed in the duck foot, we
suggest that they are active modifications of the ectodermal cells. A great
number of papers have revealed the active involvement of the epithelial tissues
in morphogenetic invagination processes (see Burgess & Schroeder, 1979 for a
review). The epithelial interdigital tissue consists of a two-layered tissue, and
the modifications observed here could be due either to the active change of
the outer epithelial cells (periderm) or their adaptation to modifications in the
inner epithelial cells. Our unpublished ultrastructural studies show that the
shape modifications are also present in the inner epithelial cells.
The interdigital ridges are epithelial thickenings of rounded cells, which are
heterogeneous in size, located at the interdigital commissures. In some instances
some cells appeared to be in the process of detachment towards the amniotic
cavity. Furthermore, it is possible that some cells are actually degenerating
cells since Pautou (1975) reported the existence of a focal epithelial regression
zone in the third interdigital space of the chick. The ridges appeared during
the development of the interdigital commissures. They were present in all the
interdigital spaces of the chick and only in the first and third spaces of the
duck which are precisely the zones in which the digits of the duck are most
detached from each other. These facts suggest that the ridges might play the
role of eliminating the superfluous epithelial cells once the mesenchyme has
been eliminated. There are some examples of elimination of embryonic tissue
by means of detachment of epithelial cells, such as the formation of the
foramina secunda in the chick embryo heart (Hendrix & Morse, 1977), the
fusion of the endocardial tubes (Ojeda & Hurle, 1981) or the elimination of
the oral membrane in the chick (Waterman & Schoenwolf, 1980). In all
these cases the epithelial cells progressively take on a more rounded appearance,
as observed here, and the processes are accompanied by a discrete amount
of cell degeneration.
From our results we concluded that the detachment of the digits from the
foot plate takes place not only by mesenchymal cell death and selective growth
of the digits as suggested by Pautou (1975) but also that there is an active
contraction and elimination of the epithelium of the interdigital spaces. With
respect to this it should be noted that the administration of Janus green to
chick embryos produces soft-tissue syndactyly. This syndactyly has been interpreted as an inhibition of the phagocytosis in the INZ (Pautou, 1976), but
Janus green also produces a marked alteration of the ectodermal tissue (Pautou
& Kieny, 1971) which could also be a factor in the genesis of that syndactyly.
REFERENCES
BALLARD, K. J. & HOLT, S. J. (1968). Cytological and cytochemical studies on cell death
and digestion in the foetal rat foot: the role of macrophages and hydrolytic enzymes.
J.CellSci. 3,245-262.
Interdigital surface changes during digit morphogenesis
263
D. R. & SCHROEDER, T. E. (1979). The cytoskeleton and cytomusculature in
embryogenesis. An overview. In Methods and Achievements in Experimental Pathology^
vol. 8 (ed. G. Jasmin and M. Cantini), pp. 171-189. Basel: Karger.
CAMERON, J. A. & FALLON, J. F. (1977). The absence of cell death during development of
free digits in amphibians. Devi Biol. 55, 331-338.
DELEANU, M. (1965). Toxic action upon physiological necrosis and macrophages reaction
in the chick embryo leg. Rev. Roum. Embryol. Cytol. Ser. embryol. 2, 45-56.
FALLON, J. F. & CAMERON, J. (1977). Interdigital cell death during limb development of the
turtle and lizard with an interpretation of evolutionary significance. /. Embryol. exp.
Morph. 40, 485-489.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in development of
the chick embryo. J. Morph. 88, 49-92.
HENDRIX, M. J. C. & MORSE, D. E. (1977). Atrial septation. I. Scanning electron microscopy
in the chick. Devi Biol. 57, 345-363.
HINCHLIFFE, J. R. (1974). The patterns of cell death in chick limb morphogenesis. Lib.
J. Sci. 4 A, 23-32.
HINCHLIFFE, J. R. & THOROGOOD, P. V. (1974). Genetic inhibition of mesenchymal cell
death and the development of form and skeletal pattern in the limbs of Talpid8 (Ta*)
mutant chick embryos. / . Embryol. exp. Morph. 31, 747-760.
HINCHLIFFE, J. R. & EDE, D. A. (1973). Cell death and the development of limb form and
skeletal pattern in normal and wingless (ws) chick embryos. /. Embryol. exp. Morph. 30,
753-772.
KELLEY, R. O. (1970). An electron microscopic study of mesenchyme during development
of interdigital spaces in man. Anat Rec. 168, 43-74.
KELLEY, R. O. (1973). Fine structure of the apical rim-mesenchyme complex during lirrtb
morphogenesis in man. /. Embryol. exp. Morph. 29, 117-131.
MENKES, B., DELEANU, M. & IILIES, A. (1965). Comparative study of some areas of physiological necrosis at the embryo of man, laboratory mammalians and fowl. Rev. Roum.
Embryol. Cytol. Ser. embryol. 2, 161-172.
OJEDA, J. L. & HURLE, J. M. (1981). Establishment of the tubular heart. Role of cell death.
In Perspectives in Cardiovascular Research, vol. 5 (ed. T. Pexieder), pp. 101-113. N^w
York: Raven Press.
PAUTOU, M. B. (1974). Evolution comparee de la necrose morphogene interdigitale dans le
pied de l'embryon de poulet et de canard. C r. hebd. Seanc. Acad. Sci. Paris D 278,
2209-2212.
PAUTOU, M. P. (1975). Mbrphogenese de l'autopode chez l'embryon de poulet. /. Embryol.
exp. Morph. 34, 511-529.
PAUTOU, M. P. (1976). La morphogenese du pied de l'embryon de poulet etudiee a l'ajde
de malformations provoquees par le vert Janus. /. Embryol. exp. Morph. 35, 649-665.
PAUTOU, M. & KIENY, M. (1971). Effect du vert Janus sur la necrose morphogene interdigitale chez le poulet: analyse histologique et cytologique. C. r. hebd. Seanc. Acad. Spi.,
Paris D 272, 2378-2381.
SAUNDERS, J. W. JR. & FALLON, J. F. (1967). Cell death in morphogenesis. In Major Problems
in Developmental Biology (ed. M. Locke), pp. 289-314. New York: Academic Press.
SAUNDERS, J. W. JR., GASSELING, M. T. & SAUNDERS, L. C. (1962). Cellular death in morphogenesis of the avian wing. Devi Biol. 5, 147-178.
WATERMAN, R. E. & SCHOENWOLF, G. C. (1980). The ultrastructure of oral (bucopharyngeal)
membrane formation and rupture in the chick embryo. Anat. Rec. 197, 441-470.
BURGESS,
(Received 17 November 1981, revised 23 December 1981)