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/. Embryo/, exp. Morph. Vol. 44, pp. 2J7-225, 1978
Printed in Great Britain © Company of Biologists Limited 1978
217
Scanning electron microscopy of the developing
chick anterior corneal epithelium
By PHILLIP R. WAGGONER 1
From the Department of Anatomy, Wayne State University
School of Medicine, Detroit
SUMMARY
The anterior corneal epithelium of the developing chick was observed with the scanning
electron microscope at various stages of development. In the earlier stages, up to about
15 days of incubation, the cells are characterized by regular polygonal outlines and a proliferation of microvilli on the surface. The microvilli then begin to coalesce and flatten so
that the surface is rather smooth by about 19 days of incubation. Just prior to hatching,
however, the cells begin to round up and once again become covered by microvilli. The cells
then lift off the surface and expose the underlying cells. After hatching, the surface cells lose
the synchrony of development that characterized the embryo and are found in various
stages of senescence. The cells eventually lose their regular polygonal outlines and the
corneal surface takes on a patchwork appearance.
INTRODUCTION
In relatively recent years many morphological studies of the developing
cornea have been carried out using the light microscope (Rones, 1932; Meyer
& O'Rahilly, 1959; Nuttall, 1976a, b), transmission electron microscope
(Miyashita, 1964; Brini, Porte & Stoeckel, 1966; Hay & Revel, 1969; Pei &
Rhodin, 1971) and the scanning electron microscope (Nelson & Revel, 1975;
Bard, Hay & Meller, 1975). From these studies and many other previous ones,
there has evolved a rather detailed description of the morphological events
encompassed by corneal development. Even so, there is a dearth of information
on the development of the surface layer of the anterior corneal epithelium the layer that is frequently referred to as periderm or epitrichium of the
embryonic ectoderm.
However, the adult anterior corneal epithelium as viewed with the scanning
electron microscope has been described by several authors (Blumcke &
Morgenroth, 1967; Hoffman, 1972; Pfister, 1973; Harding, Bagchi, Weinsieder
& Peters, 1974). These studies have dealt with adult mammalian and fish corneas
and in all cases the authors have described the corneal surface cells as exhibiting
1
Author's address: Department of Anatomy, Wayne State University School of Medicine,
Detroit, Michigan, U.S.A.
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P. R. WAGGONER
SEM of chick anterior corneal epithelium
219
a more or less regular pattern of microprojections either in the form of microvilli or microplicae. Other authors have dealt with the alterations of the surface
topography of the anterior corneal epithelium produced by alkali burns
(Henriquez, Pihlaja & Dohlman, 1976; Pfister & Burstein, 1976#) or ophthalmic
drugs (Mitsui, Takashima, Fujimoto & Kashiyama, 1976; Pfister & Burstein,
19766) but, to date, no one has used the scanning electron microscope to study
the development of the anterior corneal epithelium. The present investigation
describes the development of the chick anterior corneal epithelium as viewed
with the scanning electron microscope.
MATERIALS AND METHODS
Fertile chicken eggs were incubated at 38 °C and 85 % relative humidity in a
circulating air incubator. The eyes of chick embryos were excised at daily
intervals from 5 days of incubation through hatching. In addition, the eyes
were excised from chicks at 1, 5 and 10 days after hatching and from adult
chickens. The entire eye of the younger embryos was processed but only the
anterior segment of the eye of the older embryos and post-hatched chickens
was processed.
The tissue was fixed either in 2-5 % glutaraldehyde (in 0-1 M cacodylic buffer
or 0-1 M Sorensen's phosphate buffer, pH 7-2) for 2 h with postfixation in 1 %
osmium tetroxide for 1 h or in a mixture of 2-5% glutaraldehyde (0-1 M
cacodylic buffer, pH 7-2) and 1 % osmium tetroxide according to the method of
Hirsch & Fedorko (1968). After fixation, the tissues were washed in buffer and
dehydrated through a series of ethanols. Dehydrated tissues were critical point
dried using CO2 or Freon 13 (in which case it was first run through a series of
Freon 113) as the transitional fluid. The preferred procedure was fixation
according to Hirsch & Fedorko (1968) and critical point drying with CO2.
F I G U R E S 1-6
Fig. 1. The chick anterior corneal surface after 5 days of incubation. Note the regular
polygonal shape of the cells and the microvilli and marginal folds of plasma
membrane, x 3500.
Fig. 2. Anterior corneal surface of the chick after 5 days of incubation, x 10000.
Fig. 3. Anterior corneal surface of the chick after 9 days of incubation. Note that
there is an increase in the number of microvilli and a decrease in cell diameter
when compared to the situation after 5 days of incubation (Fig. 1). x 3500.
Fig. 4. Surface of chick anterior corneal epithelium after 9 days of incubation,
x 10000.
Fig. 5. Superficial layer of chick corneal epithelium after 15 days of incubation.
Note the greater number of microvilli than was observed at 9 days of incubation
(Fig. 3). x 3500.
Fig. 6. Cell surface from chick anterior corneal epithelium after 15 days of incubation. Notice that the microvilli appear to be interconnected, x 10000.
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P. R. WAGGONER
SEM of chick anterior corneal epithelium
221
The dried specimens were mounted on aluminium stubs, coated with gold in a
Hummer sputtering system and viewed either in a Philips 500 or an ETEC
autoscan scanning electron microscope.
RESULTS
The surface cells of the chick anterior corneal epithelium, after 5 days of
incubation, were not unlike the cells of the remainder of the embryonic ectoderm. They were polygonal in outline and exhibited scattered short microvilli
and cell boundaries demarcated by discontinuous folds of the plasma membrane (Figs. 1, 2). For the next 4 days of incubation there was no detectable
changes in the cell surfaces except for an increase in the number of microvilli
and a slight decrease in the cell diameter (Figs. 3, 4).
From 9 days of incubation to about 14 days of incubation there was an ever
increasing number of microvilli found on the surface cells of the chick anterior
corneal epithelium. After 15 days of incubation the microvilli had covered the
cell surfaces so that the cells had taken on a shaggy appearance (Fig. 5). With
closer examination it was found that the microvilli had begun to change. It
appeared as if interconnexions had formed between adjacent microvilli or else
the individual microvilli had simply become more tortuous (Fig. 6). The
number of microvilli and/or interconnexions or tortuosity continued to increase
for the next few days until the surface began to take on a sieve-like appearance
(Figs. 7, 8). It was not unusual during that time span (14-17 days of incubation)
to see cells in the final stages (telophase) of mitosis (Fig. 9). The cell surfaces
after 19 days of incubation became rather smooth with only occasional microvilli extending above the sieve-like surface (Fig. 10). Just prior to hatching (20 days of incubation) that monotonous landscape was broken by the
FIGURES
7-12
Fig. 7. Cell surface of chick anterior corneal epithelium after 16 days of incubation.
The number of microvilli has continued to increase and they continue to coalesce
with one another, x 10000.
Fig. 8. Anterior corneal cell surface after 17 days of incubation. There appears to
be a continued coalescence of microvilli. x 10000.
Fig. 9. A late telophase figure observed on the chick anterior corneal surface after
15 days of incubation, x 3500.
Fig. 10. The surface of the chick anterior corneal epithelium after 19 days of incubation. Notice the sieve-like appearance and that only occasional microvilli
extend above the surface, x 20000.
Fig. 11. The chick anterior corneal surface after 20 days of incubation. There are
cells with a variety of shapes and textures scattered over the surface, x 400.
Fig. 12. Higher magnification of part of the field seen in Fig. 11. Some of the cells
appear wrinkled (W) and are surrounded by 'stress furrows' while others are more
rounded (R) and are sitting in little depressions. The latter cells are covered with
microprojections. At other locations there are slight depressions (D) where second
layer cells can be seen, x 1000.
15
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44
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13
P. R. WAGGONER
14
SEM of chick anterior corneal epithelium
appearance of a plethora of polymorphic cells over the entire corneal surface
(Figs. 11, 12). Some of these cells were flattened and wrinkled while others were
rounded and covered with microvilli. In addition, depressions were observed
exposing second layer cells. The wrinkled cells were frequently surrounded by
radiating furrows and attachments to surrounding cells were observed (Figs.
13, 14). The rounded cells that were covered by microvilli were frequently seen
to be sitting in slight depressions (Figs. 13, 15 and 16). The second layer cells
found in depressions had surface morphologies similar to that observed at
17 days of incubation (Figs. 13, 14).
After hatching, the anterior epithelial surface took on a quilted appearance
with cell surfaces showing various textures and degrees of brightness. The cell
textures ranged from definite microvillous surfaces to rather smooth surfaces.
The cells with the surface projections appeared much lighter in the scanning
scope than the cells with the smooth surfaces (Fig. 17). This was in stark
contrast to the embryonic stages when all the surface cells had, in general, the
same appearance as if they were synchronized in their development. The adult
chicken corneal epithelial surface cells not only showed a lack of synchrony in
development but they no longer had regular polygonal outlines. The irregular
cell outlines and the short irregular microvilli on the lighter cells gave the adult
corneal surface a patchwork appearance (Fig. 18).
DISCUSSION
The significance of the increasing number of microvilli on the developing
chick cornea is not apparent, though other authors (Pedler, 1962; Ehlers, 1965;
Lemp, Holly, Iwata & Dohlman, 1970) have suggested that in the adult of other
species the microvilli may serve to anchor the tear film. This, most assuredly,
FIGURES
13-18
Fig. 13. Part of the field seen in the previous two figures. A wrinkled cell and three
round cells can be seen as well as two second layer cells, x 2000.
Fig. 14. The wrinkled cell seen in the three previous micrographs. Note the stress
furrows surrounding the cell and the points of attachment at the periphery of the
cell. x3500.
Fig. 15. Three round cells seen in Fig. 13. They are sitting in slight depressions
and on close observation points of attachment can be seen along the edge of the
center cell, x 3500.
Fig. 16. A round cell sitting on the surface of the chick anterior corneal epithelium
after 20 days of incubation, x 7000.
Fig. 17. Anterior corneal surface of the chicken 10 days after hatching. The cells
are still polygonal in shape with various surface textures and degrees of brightness
which gives the corneal surface a quilted appearance, x 1000.
Fig. 18. Adult chicken anterior corneal epithelium. The cells exhibit various surface
textures and degrees of brightness but have lost their regular polygonal shapes
which gives the surface a patchwork appearance, x 1000.
15-2
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P. R. WAGGONER
is not the case in the chick embryo. However, one must wonder if the microvilli
in the chick embryo are functioning in the dehydration and acquisition of
transparency of the chick cornea that has been found to occur in the chick
between incubation days 14 and 19 (Coulombre & Coulombre, 1958).
The telophase figures that were observed between days 14 and 17 probably
represented the terminal division for these peridermal cells since Nuttall
(1976#) has found that the synthetic index of these cells decline from day 14
after having reached a peak of about 9-5 %.
The reason for the microvilli being obscured as the chick nears hatching is
not entirely clear but other authors (Pfister, 1973; Harding et al. 1974) have
pointed out that some cells of the adult cornea in other species appear to be
covered by a coating material when observed with the scanning electron microscope. This has most often been ascribed to a mucus or mucous-like coating of
the cells. Pfister (1973) reported that acetylcysteine partially removed the
coating material from mammalian cornea but a pilot study by this author
found that acetylcysteine had no effect on the appearance of the chick embryo
cornea in the scanning electron microscope. Pei & Rhodin (1971) reported that
surface cells of the developing mouse cornea become smoother preparatory to
desquamation and it is very likely that a similar thing is occurring with the
chick cornea.
The appearance of the polymorphic cells on the anterior corneal epithelium
just prior to hatching has been interpreted as a sloughing or desquamating
process. The more flattened wrinkled cells with the stress lines around them
represent the first stages of rounding up and lifting off. As the cells round up
they simultaneously become covered with microvilli and are eventually lost
from the surface and thus expose the second layer cells found in the bottom of
the depressions. Whether this method of desquamation is unique to the embryo
or is also found in the adult remains to be determined. This method of desquamation is very different from that described by Pfister (1973) for the adult
mammalian cornea. He found that exfoliation was accomplished by the development of a full-thickness hole through the center of the cell followed by the
retraction of the edges of the hole toward the periphery of the cell.
The different degrees of brightness and the variability of the surface texture
that was observed after hatching has been reported for other adult species
(Hoffman, 1972; Pfister, 1973; Harding et al. 1974). This phenomenon has been
attributed to the number and/or length of microvilli and the presence or
absence of coating material. Harding et al. (1974) suggest that the variability
of surface cells in the fish corneal epithelium could be the result of various
states of differentiation or senescence and a variable affinity for a mucus
coating. The present data suggest that the different surface textures of chicken
anterior corneal epithelial cells is due to actual changes in the cell membrane
as a reflexion of the state of differentiation or senescence but it neither supports
nor refutes the idea of a differential adhesion of a mucous coating.
SEM of chick anterior corneal epithelium
225
The author wishes to thank Dr Clifford Harding for the use of facilities at the Kresge
Eye Institute and Dr Alfred Coulombre for helpful discussions during the study. The work
was supported by N.I.H. Grant number RR 05384.
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{Received 8 September 1977)