Vol. 88, Issue 5, 1614-1622, May 2000
Embryonic quail eye development in microgravity
Joyce E.
Barrett*,
Diane C.
Wells*,
Avelina Q.
Paulsen, and
Gary W.
Conrad
Division of Biology, Ackert Hall, Kansas State University,
Manhattan, Kansas 66506-4901
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ABSTRACT |
The US-Russian joint quail embryo project
was designed to study the effects of microgravity on development of
Japanese quail embryos incubated aboard Mir. For this part of the
project, eyes from embryonic days 14 and 16 (E14 and
E16) flight embryos were compared with eyes from several groups of
ground-based control embryos. Measurements were recorded for eye
weights; eye, corneal, and scleral ring diameters; and numbers of bones
in scleral ossicle rings. Transparency of E16 corneas was documented,
and immunohistochemical staining was performed to observe corneal
innervation. In addition, corneal ultrastructure was observed at the
electron microscopic level. Except for corneal diameter of E16 flight
embryos, compared with that of one of the sets of controls, results
reported here indicate that eye development occurred normally in
microgravity. Fixation by cracking the shell and placing the egg in
paraformaldehyde solution did not adequately preserve corneal nerves or
cellular ultrastructure.
cornea; immunohistochemistry; neurofilament; spaceflight; ultrastructure
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INTRODUCTION |
ONE OF THE OBJECTIVES of the current
international space program is to observe possible effects of
microgravity on animal and plant development. The joint US-Russian
quail embryo development project was designed to study Japanese quail
(Coturnix coturnix japonica) embryos after they had been
incubated in space. On the basis of several criteria, quail embryos
provide an effective model for animal developmental experiments in the
space program. Fertilized eggs require only 17 days at 37°C for
development. In addition, quail eggs are small and more easily
transported and incubated than are present alternative vertebrate
models, and they do not require care of a pregnant adult animal to
support embryonic development. In addition, quail meat and eggs have
been considered as a potential high-protein food source for use in long-term spaceflight. It is critical to understand what effects microgravity has on physiological processes, including embryonic development, growth, and reproduction (15, 19, 22).
Some abnormalities have been observed in eyes of animals flown on
previous spaceflights, but it is unclear whether they arose as a result
of microgravity. Guryeva et al. (15) reported a higher frequency of
abnormally developed eyes in quail embryos flown aboard Mir, but no
data were provided for this flight group or for the synchronous or lab
control groups. Philpott et al. (29) observed retinal cell changes in
rats flown in space, including the presence of unusual nuclei, spaces,
or channels and the presence of macrophages in the retinal layers.
In chicks and, by extrapolation, also quail, the cornea develops as the
primitive ectoderm overlying the optic cup and lens vesicle synthesizes
a primary stroma of orthogonally ordered plies of collagen fibrils that
then serves as a substratum for the migration of the first wave of
neural crest cells, thereby forming a monolayer on its proximal
surface, the corneal endothelium (20, 32). Shortly thereafter, a second
wave of neural crest cells migrates between the plies of the primary
stroma and differentiates as corneal stroma fibroblasts, the
keratocytes. The keratocytes synthesize the bulk of the subsequent
corneal stroma, whose orderly polymerization uses the primary stroma as
a template and scaffold (4, 14, 17). Last to enter the developing
cornea are the axons of the sensory nerves, whose cell bodies reside
mostly in the trigeminal ganglia. In chicks, the growth cones of those
nerves encircle the cornea until a complete ring is formed and then
enter the corneal stroma along all radii (3). Transparency of the chick cornea appears rapidly, approximately two-thirds of the way through embryogenesis, in a thyroxine-dependent process in which dehydration of
the stroma, caused by pumping of the corneal endothelial monolayer, is
associated with a thinning of the cornea and the appearance of
transparency (2, 9, 25, 26). During the period when neural crest cells
are entering the corneal stroma, a set of ~14 focal thickenings
appears in the conjunctival epithelium surrounding the cornea. These
thickenings induce some of the underlying neural crest mesenchyme cells
to form the membrane bones of the scleral ossicle ring, with each
epithelial papilla inducing formation of one bony plate (10, 11, 16,
28). No cartilage model precedes the formation of the scleral ossicles.
As participants in the quail project, we were interested in effects of
microgravity on eye development and on corneal innervation in
particular. Because corneal innervation is dense and easily visualized
in whole-mount preparations (21), it can provide an excellent system
for studying effects of microgravity on nerve formation and on corneal ultrastructure.
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MATERIALS AND METHODS |
Launching, egg incubation, and embryo dissection.
All materials and procedures for egg handling and fixation were
selected and conducted in accordance with protocols previously established by the Russian space agency. Forty-eight freshly laid, fertilized Japanese quail eggs were obtained from the Avian Physiology Laboratory, University of Wisconsin-Madison, and shipped to the Kennedy
Space Center. They were launched on the US STS-76 at 14°C in a
commercial refrigerator/incubator module and then transferred to Mir
after docking (time between laying, shipping, launching, and beginning
of incubation on Mir 6-9 days, temperature 14-20°C). The
eggs then were transferred immediately into a Slovakian-made incubator
(type 1M-02) for incubation; eggs were not turned but were
free-floating in microgravity (recorded temperature was
39-40°C) (Table 1). On specified
days ranging from embryonic days 0-16 (E0-E16), eggs
were removed from the incubator, squeezed gently to introduce cracks in
the shell, and placed into triple-layered zipper-type plastic bags (one
egg per bag) containing 75 ml fixative solution (4% paraformaldehyde
in 0.2 M sodium-potassium-phosphate buffer) [National Aeronautics
and Space Administration (NASA) protocol: SLM quail experiment
procedures established for use in the Mir 21/NASA 2 Avian Developmental
Biology Flight Experiments]. The fixed embryos, still inside
their shells, remained in the fixative solution without agitation at
ambient temperature (Table 1). Embryonic tissues, therefore, underwent
fixation as paraformaldehyde diffused through the cracked shell and
into the egg interior. These eggs, designated as Flight eggs, were
returned to Earth aboard STS-79 and were shipped immediately to the
NASA-Ames Research Center, Moffett Field, CA, where they were stored
until dissection.
Control eggs were obtained from the same flock as the Flight eggs. Two
groups of 48 fertile quail eggs were shipped to the Ames Research
Center 12 days after the Flight eggs were shipped to the Kennedy Space
Center, stored at 14°C, and incubated in a commercial Lyon RX-2
incubator (Lyon Electric, Chula Vista, CA). A 7-day incubation and
fixation time delay were introduced to allow for downloading of flight
data from Mir. The first group of synchronous control eggs (called
Synch-1) was incubated at similar temperatures to those of the Flight
eggs (39-40°C), and the first group of laboratory control eggs
(Lab-1) was incubated at the standard egg incubation temperature of
37.5 ± 0.5°C (Table 1). Before placement of eggs into the
fixative solution after incubation, shells of the control eggs were
crushed in a manner similar to that used with the Flight eggs. In
addition, in the control eggs a small hole was cut in the shell above
the air cell to allow them to sink into the fixative solution. Such a
hole was not made in the shells of Flight eggs because, in
microgravity, eggs are totally immersed in the solution. Both sets of
controls were put into fixative bags containing fresh 4%
paraformaldehyde and remained at room temperature until dissection.
The first two groups of control eggs (Synch-1 and Lab-1) did not
undergo simulated launch stress factors before incubation (Table 1).
Therefore, five additional control groups of quail eggs were subjected
to launch simulation conditions of acoustics, hypergravity, and
vibration [Synch-2, Lab-2, Synch-3, Lab-3, and Lab-3HI (HI
temperatures were a higher range to mimic those of flight); Table 1].
They then were incubated in conditions simulating the Flight and
original two control groups.
All embryos were dissected by Russian and US researchers at NASA-Ames.
Tissues were examined grossly and microscopically to determine quality
of fixation, sex, and stage of embryonic development. For our part of
the team investigations, we removed the fixed eyes from their orbits
after the embryos had been transferred into saline. Eyes were put
immediately into individual vials of fresh, room temperature 4%
paraformaldehyde. Eyes and all other dissected tissues then were
shipped in the vials of fixative to the various individual
investigators for further analyses.
Additional experiments on E16 quail eyes were conducted in our
laboratory at Kansas State University to judge the stability of eyes
that had been removed from the embryos immediately after death, fixed
directly in paraformaldehyde, and stored for several months in the
fixative solution (Table 1; indicated as KSU group). Animal rearing and
fixation procedures were conducted according to the National Research
Council's animal use guide (27). Fertilized Japanese quail eggs,
obtained from Oak Ridge Game Farm (Maysville, AR), were stored at
17°C for an average of 7 days and then incubated for 16 days at 37 ± 1.0°C. Embryos were removed from their shells, and the spinal
cords were severed at the base of the skull. Nonpunctured eyes were
removed from the orbit, freed of eyelid tissue, and immediately placed
into paraformaldehyde solution (Sigma Chemical, St. Louis, MO)
[4% (wt/vol) in Na-K-PO4 buffer (0.062 M
KH2PO4 + 0.109 M
Na2HPO4), pH 7.2]. They were stored at
room temperature (20-22°C) in the same solution for 2-6 mo,
with occasional swirling for agitation to simulate storage conditions
on Mir.
Physical measurements and observations.
Eyes were handled with forceps to grasp the optic nerve, and care was
used to avoid touching the corneas. Eyes were trimmed free of eyelid
and membranous noneyelid tissue in saline G (30), quickly blotted to
remove excess moisture, weighed, and then returned to saline G. Eye and
corneal diameters were measured along the dorsal-ventral and
nasal-temporal axes with the use of a drafting compass with fine points
and a ruler. Eye fronts were removed and placed into saline G, and the
remaining posterior eye region was returned to 4% paraformaldehyde.
Lens and iris tissue were removed from eye fronts to expose the corneas
and surrounding scleral rings.
Corneal transparency, electron microscopy, and
immunohistochemistry.
Corneal transparency was documented by photographing a fine wire mesh
viewed through the corneas immersed in saline G, with their concave
sides oriented upward. The clarity and uniformity of the grid image
were judged visually and independently by two individuals. Intact
corneas were removed while in saline G, and a wedge-shaped section that
included the corneal center as well as the limbus was excised from
each. The wedges were stored at room temperature in individual vials of
2.5% glutaraldehyde in 0.1 M Sorensen's sodium-potassium-phosphate
buffer, pH 7.2 (Electron Microscopy Sciences, Fort Washington, PA;
catalog no. 15980), for 3 days. They were then postfixed in 2.0%
osmium tetroxide and processed for routine electron microscopy.
Remaining corneal tissue was stained immunohistochemically with
antibodies against neurofilament protein to reveal corneal nerve
patterns by using a whole-mount procedure for highly fixed tissue
recently devised in our lab (1). Corneas were given three sequential
pretreatments designed to achieve antigenic unmasking (microwave
incubation, incubation in SDS, and then digestion with testicular
hyaluronidase) and then given three 20-min rinses of 0.3%
H2O2 to inactivate endogenous peroxidases (21).
The tissues were then incubated in the primary antibody
[antineurofilament 200-kDa IgG, developed in rabbits (Sigma
N-4142), diluted 1:1,500 in blocking solution: saline G + 0.2% Triton
X-100, 5% (wt/vol) powdered milk (Carnation natural nonfat dry milk,
Nestle Food, Glendale, CA), 1% (wt/vol) BSA (Sigma), and 1% (vol/vol)
normal filtered goat serum (Hazelton Research Products, Denver,
PA)] at 0-4°C overnight with constant movement and then
rinsed 4 × 15 min in blocking solution. Corneas were then
incubated in the secondary antibody [goat anti-rabbit IgG
peroxidase conjugate (Sigma A-0545), diluted 1:200 in the blocking
solution] for 3 h at room temperature with constant movement.
They were rinsed for 15 min in blocking solution and then rinsed in
saline G plus 0.2% Triton X-100 (4 × 15 min). To reveal the
nerves, they were incubated in 3,3'diaminobenzidine
tetrahydrochloride (DAB) (Sigma D-5905, 0.238 mg/ml saline G + 0.2%
Triton X-100) for 30 min in the dark, with constant movement, followed
by incubation in metal-enhanced DAB (Pierce 34065, prepared according
to package instructions) for 5-15 min with movement. Corneas were
rinsed (2 × 15 min) in saline G + 0.2% Triton X-100 and then
stored at room temperature in 100% glycerol. After a minimum of 24 h
storage, corneas were photographed with a Leica MPS 52 camera mounted
on a Wild M5 stereo dissecting microscope (Leica, Heerbrugg, Switzerland).
Scleral ossicle arrangement.
Corneas of birds are surrounded by a ring of overlapping plates of
membrane-type bone (10, 11). After removal of the cornea, remaining
scleral tissues were stained to reveal the number and orientation of
individual bones in each scleral ossicle ring surrounding the cornea.
The scleras were given two 15-min rinses in saline G to remove the
fixative, dehydrated in acetone for 15 min, and then stained for 2 h at
room temperature in 0.02% (wt/vol) Alizarin red S (Sigma A-3757) in
1% (wt/vol) KOH with continuous movement. After staining, the tissues
were rinsed repeatedly in glass-distilled water for at least 2 min
until no stain remained in the rinse solution. They were cleared in a
solution of benzyl alcohol, 95% ethanol, and glycerol (1:2:2
vol/vol/vol) overnight with agitation and then stored in glycerol at
room temperature. Ossicle numbers were counted, and inner and outer
diameters of ossicle rings were measured along the dorsal-ventral and
nasal-temporal axes. The rings were photographed with the same camera
and dissection microscope as described under Corneal transparency,
electron microscopy, and immunohistochemistry.
Examination of E14 embryo eyes.
In addition to the observations made on E16 eyes, measurements were
also recorded for eyes from E14 quail embryos. Eye weights, eye
diameters, and corneal diameters were recorded for eyes from the Flight
and the seven groups of E14 control embryos.
Statistical analysis.
A completely randomized experimental design was used. For each of the
two embryonic ages analyzed (E14 and E16), data were analyzed
separately with a one-way ANOVA by use of the GB-STAT statistical
program by Dr. Philip Friedman, Howard University (version 5.4.1 for
Macintosh by Richard Taylor, 1995; Dynamic Microsystems, Silver Spring,
MD). Additional comparison to determine significance of treatments was
performed as needed by using Dunnett's procedure for one-way analysis.
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RESULTS AND DISCUSSION |
Incubation parameters for the Flight and each of the control groups of
E16 embryos are given in Table 1. Only one additional Slovakian-made
incubator (type 1M-02), as used for in-flight incubations on Mir, could
be obtained for use in ground control incubations. Therefore, Lyon RX-2
incubators were used for the additional ground controls.
It has been shown that incubated eggs should be turned frequently
throughout development, particularly during the first week of
incubation (12, 13, 18, 33). A hen on the nest rotates the eggs with
her beak and also moves them with her other normal body movements.
These changes in orientation aid in gas exchange between the embryo and
the outside air and allow for adequate nutrient distribution to the
embryo. The yolk-filled cell and associated multicellular blastoderm
(both surrounded by the vitelline membrane) float above the more dense
albumen, allowing the blastoderm to remain near the shell membrane and,
later in development, the chorioallantoic membrane, where efficient gas
exchange can occur (31). The Slovakian-built incubator had no mechanism
to rotate the eggs automatically, but eggs in the Lyon incubator were
rotated at preset time intervals. The Synch-3 eggs incubated in the
Slovakian incubator were manually turned three times per day (Table 1).
Physical measurements of E16 eyes.
Tables 2 and 3
present the results of quantitative measurements of physical attributes
of E16 Flight embryo eyes compared with those of each control group.
There were no significant differences in eye weights or eye diameters
(Table 2) between the Flight group and any of the control groups.
However, there were statistically significant differences between
corneal diameters (both dorsal-ventral and nasal-temporal) in the
Flight embryos compared with those of the Synch-3 control embryos
(Table 2). The Synch-3 eggs underwent acoustic and hypergravity launch
simulations. They were incubated at 37-38°C in the same type
of incubator as were the Flight embryos and were manually turned three
times per day. No other group of control tissues showed a statistically
significant difference when corneal diameters were compared with those
of Flight corneas.
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Table 3.
Number of bones per ossicle ring and inner and outer diameters of
ossicle rings in eyes from E16 embryos
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Corneal transparency, ultrastructure, and nerve
immunohistochemistry.
Qualitative observations made for corneal clarity indicated no
consistent differences between transparency of Flight embryo corneas
compared with that of the controls. Within each of the groups, some
corneas had a slightly cloudy, nontransparent appearance, whereas
others appeared clear and fairly transparent (data not shown).
Electron microscopic examination of corneas from the Flight, Synch-1,
and Lab-1 groups showed evidence of poor fixation in the epithelium and
the stromal and endothelial layers (Figs.
1, 2, and
3). Specifically, the component cells of
those tissues were irregular in size and contained numerous large
vesicles. Moreover, both the epithelial (Fig. 1, a-c) and
the endothelial (Fig. 3, a-c) layers were often detached
from the stromal layer (Fig. 3, a and c). In
comparison, corneas from eyes fixed directly in
paraformaldehyde solution showed epithelial and stromal cells of normal
ultrastructure, with the epithelial and endothelial layers remaining
attached to the stroma (Figs. 1d, 2d, and
3d). The paraformaldehyde-fixed corneas from the Flight,
Synch-1, and Lab-1 groups did not attain the normal dark color of
osmicated tissues when subsequently postfixed with glutaraldehyde and
osmium tetroxide in a standard protocol for preparation of
paraformaldehyde-fixed tissue for electron microscopy. In contrast,
corneal ultrastructure in corneas from eyes fixed by direct immersion
in 4% paraformaldehyde (KSU lab) (Figs. 1d, 2d, and
3d) showed better fixation than did the Flight, synchronous
control, and laboratory control groups. These results suggest that
detailed comparisons of corneal ultrastructure in Flight and control
embryos require a fixation technique that introduces the
paraformaldehyde fixative to the region around the eye more quickly
than was possible in the protocol used here. The demonstration of good
ultrastructural preservation in eyes subjected to direct immersion in
4% paraformaldehyde indicates that such solutions are capable of
fixing tissue well if access is provided.
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Fig. 1.
Corneal epithelial tissue from embryonic day 16 (E16) quail
eyes. Evidence of poor fixation in embryos from eggs flown aboard Mir
(Flight; a) and from synchronous (Synch-1; b) and
laboratory (Lab-1; c) control eggs includes detachment of
epithelium from stroma and presence of many large, empty vesicles in
cytoplasm. In contrast, in corneas fixed by direct immersion in
paraformaldehyde fixative solution from embryos studied at Kansas State
University lab (KSU; d), epithelial layer remained attached
to stroma and displayed normal ultrastructure. See Table 1 for
incubation conditions. Bar = 10 µm.
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Fig. 2.
Corneal stroma from E16 quail eyes. Evidence of poor fixation in
Flight, Synch-1, and Lab-1 groups (a-c, respectively)
includes extensive vesiculation of virtually all stromal cytoplasm and
nuclei. In contrast, KSU corneas fixed by direct immersion in
paraformaldehyde fixative solution (d) display normal
ultrastructure for nuclei and cytoplasm of stromal keratocytes,
including abundant rough endoplasmic reticulum and a flat cellular
shape sandwiched between the collagenous plies. Bar = 4 µm.
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Fig. 3.
Corneal endothelial tissue and neighboring Descemet's membrane and
stroma from E16 quail eyes. Evidence of poor fixation in Flight,
Synch-1, and Lab-1 groups (a-c, respectively) includes
detachment of the endothelium from Descemet's membrane in a
and c and presence of extensively vesiculated cytoplasm in
a-c. In contrast, KSU corneas fixed by direct immersion in
the paraformaldehyde fixative solution (d) remain attached to
Descemet's membrane and display normal ultrastructure. Bar = 10 µm.
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Corneas from the Flight, Synch-1, and Lab-1 groups generally showed
poor nerve staining compared with that of corneas fixed by direct
immersion in our lab (Fig. 4). There was a
small amount of nerve staining in Flight corneas (Fig. 4a) and
only slightly improved staining in the synchronous and laboratory
controls (Fig. 4, b and c). In contrast, corneas fixed
in our lab by direct immersion in 4% paraformaldehyde solution for at
least 2 mo showed normal staining of well-branched nerves (Fig.
4d). The poor fixation of the Flight corneas and two sets of
controls is likely to have arisen from the slow diffusion of the
paraformaldehyde through the shell and chorioallantoic membrane and
finally into the embryo. Although the proportion of gas exchange
performed by the chorioallantoic membrane begins to decline, in
comparison with that performed by the lungs, after piping into the air
cell has occurred during the day before hatching, the chorioallantoic
membrane still totally envelops the embryo and presents a formidable
barrier to a diffusion front of fixative entering from outside the
shell and reacting with all tissue it encounters as it enters. Thus the
embryo per se, in the egg interior, can be predicted to be among the
last tissues to encounter fixative, by which time tissue autolysis has
begun. Such autolysis would have contributed to poor preservation of
corneal cell ultrastructure and of corneal neural antigens, even though
the overall shape and morphology of the eye, scleral ossicles, and
cornea were grossly normal.
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Fig. 4.
Whole-mount corneal nerve staining from E16 quail eyes. Evidence of
poor fixation in Flight, Synch-1, and Lab-1 groups includes detection
of very few nerves in Flight corneas (a) and the presence of
only major nerve trunks in Synch-1 (b) and Lab-1 (c)
corneas. In contrast, KSU corneas fixed by direct immersion in the
paraformaldehyde fixative solution (d) display very strong
staining of nerve trunks, as well as visualization of fine branches.
Bar = 300 µm.
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Ossicle arrangement.
Normal ossicle appearance after staining with Alizarin red S stain is
dark and shows overlapping ossicles (usually 14), which form a
complete, closed circle surrounding the corneal margin (10, 11). In the
Flight eyes, as well as in the eyes from the control groups, the
pattern of overlapping ossicles appeared normal and the bones were
darkly stained (Fig. 5). Bone numbers in
the ossicle rings varied slightly within each group, although there
were no consistent differences between the Flight eye ossicle rings
compared with those of the controls (Table 3). Table 3 also shows the
ossicle ring diameters along the dorsal-ventral and nasal-temporal
axes. No significant differences in ring diameters were observed
between Flight and any of the control groups.
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Fig. 5.
Ossicle ring in the scleral tissue of E16 quail eyes, showing 14 overlapping bones. a: Flight; b, Synch-1; c:
Lab-1. Alizarin red staining. Cornea has been removed. Bar = 750 µm.
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Prolonged exposure to microgravity results in significant changes in
skeletal long bones. However, scleral ossicles normally do not appear
to bear comparable types of stresses. Thus lack of effect on scleral
ossicle development was hypothesized.
E14 eye and corneal measurements.
We observed no statistically significant differences between eye
weights or eye and corneal diameters of the Flight eyes compared with
eyes from either of the two groups of E14 control embryos with which
they were most closely matched (Synch-1 and Lab-1) (Table
4). Additional control groups incubated at
later times displayed some quantitative differences from the original
Flight group, mostly in terms of corneal diameters (Table 4). Because these differences were not seen in the original sets of control embryos
with which the Flight embryos were most closely matched, it seems
possible that the differences may have arisen because of slight
differences in the later batches of eggs used. However, it should be
noted in comparing Table 2 with Table 4 that whatever differences may
have existed between the E14 embryos of groups-2 and -3 (i.e., Synch-2,
Lab-2, Synch-3, Lab-3, and Lab-3HI) vs. Flight apparently disappeared
during the subsequent 2 days of development to E16 (Table 2). A
technical detail may provide a more likely explanation, for we noted
that, at the time of dissection, the fixed E14 eyes were somewhat
misshapen compared with the normal smoothly spherical fixed E16 eyes.
This misshapenness made measurements of E14 eye and corneal diameters
more difficult to obtain accurately. We, therefore, suggest that
differences between the E14 Flight and group-2 and -3 control eyes may
have arisen as a measurement artifact rather than from a real
difference among the eyes of these three groups (E14 eyes were used by
other investigators and therefore were only available to us during team
dissections and only for quickly making measurements of eye weights and
diameters and corneal diameters).
Eye development is known to require intraocular pressure (5-8).
Even brief exposure to microgravity causes changes in intraocular pressure (23, 24); thus it was reasonable to propose that subjecting
developing embryos to persistent microgravity might cause changes in
intraocular pressure in the developing eye and, thereby, cause changes
in eye development. The fact that no changes in eye development were
observed in this study suggests that normal intraocular pressure may
have reappeared during the period of microgravity incubation. It will
therefore be of interest to determine the short-term and long-term
changes in intraocular pressure in humans and animals as a result of
persistent microgravity. It remains unclear why Guryeva et al. (15)
reported a higher frequency of abnormal eye development in Japanese
quail embryos incubated on Mir. However, no quantitative data on eye
development were provided in that study for this flight group or for
the synchronous or laboratory controls. The observation of normal eye
development in the present study suggests that some factor other than
microgravity deleteriously affected eye development during the study by
Guryeva et al. (15).
There are two substantive conclusions from this study. First, the
overall, macroscopic structural dimensions and physical properties
(transparency) of E16 Flight eyes were not significantly different from
those of the various control eyes [with only the exception of
corneal diameters of one set of controls (Table 2)]. The
demonstration of essentially normal eye development under conditions of
microgravity therefore represents a strongly positive experimental
result. It is unlikely that the normal development seen here in Flight
embryos arose as an artifact. The growth of the embryonic eye appears
to require a steady increase in intraocular pressure (5-8).
Moreover, it is known that exposure of adult humans to brief episodes
of microgravity causes sharp increases in intraocular pressure (23,
24). Thus it was reasonable to hypothesize in designing the present
experiment that prolonged exposure of embryos to microgravity might
have a very significant effect on the growth of several tissues in the
eye as well as the overall size and shape of the eye. The data obtained
in the present study, however, strongly suggest that embryonic eye
development occurs normally during prolonged periods of microgravity.
Second, our observations of cellular ultrastructure and corneal
innervation were limited by the poor quality of tissue fixation. Therefore, this study demonstrated the need for development of other
methods for fixing tissues in future microgravity research experiments.
Data presented herein suggest that better fixation would occur if
embryos could be quickly and directly exposed to the fixative solution,
rather than requiring fixative to permeate slowly through the
shell and extraembryonic membranes (principally the chorioallantoic
membrane) before finally reaching the embryonic tissues.
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ACKNOWLEDGEMENTS |
This research was supported by National Aeronautics and Space
Administration (NASA) Grants NAG 2-1005 (to G. W. Conrad) and NAGW-2328 (to B. Spooner). We acknowledge the Kansas State University Biology Research Microscope and Image Processing Facility, supported in
part by Kansas National Science Foundation Experimental Program to
Stimulate Competitive Research (EPSCOR) Grant EPS9550487,
Kansas NASA EPSCOR Grant NCC5-168, and Kansas Agricultural
Experiment Station.
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FOOTNOTES |
*
The first two authors contributed equally to this work.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. W. Conrad,
Div. of Biology, Ackert Hall, Kansas State Univ., Manhattan, KS
66506-4901 (E-mail: gwconrad{at}ksu.edu).
Received 3 June 1999; accepted in final form 12 December 1999.
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