Surface changes in the embryonic interdigital epithelium during the formation of the free digits: a comparative study in the chick and duck foot

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 mammals 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 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.

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 :

The interdigital necrotic areas were mapped in ovo by vital staining with neutral red following the method of Hinchliffe & Ede (1973).

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.

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 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.

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.

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 stage the cells of the dorsal surface of the digits were similar to those of day 5. They 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).

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).

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.

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 intetdigital 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 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.