Download Neuroectoderm of the early embryonic rat eye. Scanning

Neuroectoderm of the Early Embryonic Rot Eye
Scanning Electron Microscopy
Dennis E. Morse and Patricia S. McCann
A more accurate description of the changes that occur in the neuroectodermal portion of the developing
eye is possible if the surface ectoderm and its underlying mesectoderm are dissected away prior to
scanning electron microscopic analysis. A clean preparation of the basal surface of the neuroectoderm
with its basal lamina can be prepared by this method. The primitive eyes form during day 11 as lateral
diverticula from the forebrain in the rat embryo. These optic vesicles initially have a broad attachment
to the diencephalon. By day 12, a true optic stalk connects the optic vesicle to the brain. As the vesicle
approaches the surface ectoderm, it involutes to form the optic cup. During day 13, the cup deepens
and creates a prominent rim on all but its ventral side. This cleft in the ventral portion of the optic
cup is known as the optic fissure. Three portions of the neuroectodermal eye are apparent at this stage:
the optic cup, optic stalk, and a short narrow region that joins these two—the collum. The optic fissure
extends into the collum but ends abruptly at the junction of the collum with the stalk. The fissure
closes on day 14. Its only remnants at this time are a shallow groove in the optic cup and a small
patent portion in the collum that permits passage of the intraocular vessels. Invest Ophthalmol Vis
Sci 25:899-907, 1984
The developing vertebrate eye serves as the model
system for studies of such diverse morphogenetic
problems as induction,1 neuron development,2 cell
death, 34 melanogenesis,5 and extracellular matrix production by epithelia.6'7 Formation of both the optic
cup and fissure involves all of these processes. Of the
two, the optic cup has received more of the attention
in published works.
The optic fissure is a prominent, albeit transitory,
groove on the ventral aspect of the optic vesicle in all
but the most primitive vertebrates.8 In mammals, this
groove typically is described to extend the entire length
of the optic stalk, which connects the optic vesicle to
the forebrain.
The purpose of the fissure is twofold. First, it creates
a channel whereby the intraocular vessels can enter
the optic cup. Second, while the retinal neurons do
not actually enter the fissure, it is established that these
neurons do not successfully extend their processes to
the diencephalon in the absence of the optic fissure.29"12
In each case, the formation of the fissure provides
direct access between the cup and stalk. 213 Within a
short period of time after its formation, the optic fissure
is obliterated by the fusion of its two lips. Fusion begins
near the midpoint of the fissure and proceeds in either
direction. 814
Electron microscopic study of the fissure is limited
to that of Geeraets.15 Her observations are based on
thin sections from the golden hamster eye. Presently,
there is no published report that uses scanning electron
microscopy to study the formation of the optic fissure.
By microdissection, we are able to remove the surface
ectoderm and subjacent mesectoderm to expose the
neuroectoderm, which gives rise to the optic vesicle,
stalk, cup and fissure. This method avoids the treatment
with enzymes and provides the entire structure for
study rather than relying on random fractures or razor
cuts through the head region to expose the deeperlying neuroectodermal portion of the embryonic eye.
This paper describes the scanning electron microscopy
of normal rat optic fissure formation.
Materials and Methods
Female Sprague-Dawley rats (approximately 200 g)
were caged overnight with a male rat of the same strain.
The presence of sperm in a vaginal smear performed
the following morning was used as an indication of
pregnancy. The day of the positive smear was termed
day 0. A pregnant female rat was placed in a separate
cage and maintained until the desired stage of gestation.
Ages used in this study were 11-, 12-, 12.5-, 13-,
13.5-, and 14-days gestation. All animals used in this
study were handled in accordance with the ARVO
Resolution on the Use of Animals in Research.
From the Department of Anatomy, Medical College of Ohio,
Toledo, Ohio.
Submitted for publication: November 9, 1983.
Reprint requests: Dennis E. Morse, PhD, Department of Anatomy,
Medical College of Ohio, C.S. 10008, Toledo, OH 43699.
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Fig. 1. The neuroectoderm gives rise to two lateral diverticula on day 11. These early optic vesicles (OV) have a broad attachment to the
forebrain at this stage. The midline neural groove is seen in its final stages of closure in this anterior view of the head region. The surface
ectoderm and mesectoderm have been removed. A few surface ectodermal cells are adherent in the neural groove region (arrows). Bar = 100 j*m.
The rats were injected intraperitoneally (IP) with
sodium pentobarbitol just prior to surgery. After the
rat was fully under the effect of the anesthesia, a laparotomy was performed. The uterine horns were exposed, excised, and placed immediately in cold (4°C)
2.5% glutaraldehyde-2% paraformaldehyde buffered
with 0.2 N cacodylate. The final solution pH was 7.4.
Embryos were dissected free and then placed in fixative
at least overnight. Following initial fixation, the embryos were rinsed in 0.2 N cacodylate buffer, postfixed
in 2% osmium tetroxide in 0.144 N cacodylate buffer
and then rinsed with 0.144 N cacodylate buffer. Specimens were dehydrated in an ascending series of ethanols and critical point dried with liquid carbon dioxide
as the transitional fluid. Tissue was mounted on aluminum stubs using silver conductive paste and sputtercoated with gold-palladium.
Exposure of the study area was accomplished by
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partial dissection of the specimen while in the initial
fixative. In most cases, the surface ectoderm was
stripped off and various amounts of the mesectoderm
were teased away at this stage of preparation. At the
11-12.5 days gestation stages, the lens placode was
still a part of the surface ectoderm and, thus, was removed with that layer. The lens separated from the
surface ectoderm at later stages. In some of these specimens, the lens and other contents of the optic cup
and fissure were dissected out. After the specimens
were dried and mounted, additional mesectoderm was
either picked, brushed or blown away to adequately
expose the neuroectoderm.
Results
The neural portion of the developing visual system
is evident morphologically on day 11 of gestation in
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NEUROECTODERM OF THE EMBRYONIC EYE / Morse and McCann
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Fig. 2. A posterior view of the left portion of the specimen in Figure 1 shows a well-delineated optic vesicle (OV) and the early indication
of the narrower optic stalk (OS). It is common for mesectodermal elements to be more adherent to the optic vesicles than to other portions
of the neuroectoderm at this stage. Bar = 100 nm.
the rat embryo. Mechanical removal of surface ectoderm and its underlying mesectoderm at this stage
reveals that a pair of lateral diverticula are budding
from the primitive forebrain. These optic vesicles initially have a broad attachment to the brain, but very
soon acquire a narrowed connection termed the optic
stalk (Figs. 1, 2). Cells of the mesectoderm over the
optic vesicles and neuropore adhere to the neuroectoderm.
On day 12, two important processes occur. The optic
stalk lengthens considerably so that the optic vesicle
comes to lie farther from the brain and closer to the
surface ectoderm. Synchronously, the optic vesicle begins its characteristic involution to create the optic cup
(Figs. 3, 4). A shallow furrow forms on the ventral
surface of the optic cup. The furrow, the presumptive
optic fissure, is bounded on either side by folds of
neuroectoderm and filled by mesectodermal tissue.
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In the living state, the optic cup is occupied by the
forming lens and mesectoderm. The lens vesicle develops from the surface ectoderm at this time. It is
still connected to the surface ectoderm by the lens stalk
and thus is most often removed with that layer. The
smooth texture of the neural portion of the eye at these
early stages is most likely caused by the basal lamina
which covers the exposed (basal) aspect of cells of the
neuroectoderm.
Several changes are noted by the end of day 13. The
optic stalk lengthens farther. A slightly narrower neck
region connecting the stalk to the optic cup is evident
(Fig. 5). We refer to this region as the collum. The
lens vesicle separates from the surface ectoderm and
typically remains in the optic cup when the surface
ectoderm is removed. Mesectoderm occupies the remainder of the cup and also fills and camouflages the
optic fissure (Figs. 5, 6). Removal of the lens and much
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1984
Fig. 3. The surface ectoderm and mesectoderm are removed from
the optic cup (OC) and ventral aspect of the stalk (OS) in this specimen
from a 12-day embryo. The optic fissure is present on the ventral
portion of the cup. The fissure is wide and shallow with the lips of
the cup which form its border (asterisks) far apart. Bar = 100 Atm.
of the mesectoderm reveals the fissure as a deep slit
in the area of the optic cup (Figs. 7-9). As it extends
to the collum from the optic cup, the fissure becomes
more shallow and ends abruptly at the junction of the
collum with the optic stalk (Figs. 7, 8). The opposing
lips of the fissure establish contact during day 13
(Fig. 9).
On day 14, the optic fissure closes completely. All
that remains of the fissure at this time is a shallow
furrow on the ventral aspect of the eyeball (Fig. 10).
Intraocular vessels enter the optic cup through a small
persistent portion of the optic fissure in the collum
(Figs. 10, II).
Discussion
Analysis of sectioned material for descriptive purposes always introduces the difficulty of relating the
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Fig. 4. A lateral view of the optic cup (OC) in a 12-day embryo
demonstrates the early optic fissure (OF) in the ventral wall of the
cup. The lens stalk is still attached to the surface ectoderm at this
stage and is removed with that layer. Mesectodermal tissues also
have been removed except at the outermost rim of the cup (arrows).
The basal lamina of the neuroectodermal cells of the cup give the
surface a homogeneous appearance. Bar = 100 /im.
information found in a given section to the entire organ
or tissue. Factors contributing to interpretive problems
in sectioned material include specimen orientation relative to the cutting edge, section thickness, and staining
characteristics. In many instances, correlative scanning
electron microscopy helps minimize some of these difficulties by presenting the three-dimensional morphology.
This paper utilizes scanning electron microscopy to
study the optic fissure of the rat embryo. Two contributions to the knowledge of visual system development are presented. One is technical in nature and
the other clarifies a major discrepancy in the literature
regarding the extent of the optic fissure.
Previously published studies of the neuroectodermal
portion of the developing eye, particularly at the electron microscopic level, concentrate predominantly on
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NEUROECTODERM OF THE EMDRYONIC EYE / Morse and McConn
Fig. 5. During day 13, the optic stalk (OS) continues to elongate.
The optic fissure is obscured by mesectodermal tissues (M) in this
preparation. The fissure is more of a slit now as the opposing lips,
which form its margins (asterisks), come closer together. The lens
vesicle (L) has separated from the surface ectoderm and now typically
remains in the optic cup when surface ectoderm is removed. Bar
= 100 fim.
the optic cup and its derivatives. The optic fissure is
mentioned in passing or not at all. The dense collection
of mesectodermal tissue surrounding the optic cup and
stalk has forced the scanning electron microscopist to
resort to random razor cuts or fractures through this
organ16 or to "view" the structures based on features
reflected by the surface ectoderm.17 The present paper
describes a technique to remove tissues that cover the
neuroectodermal portion of the forming eye. Microdissection permits a clean preparation ofjust the intact
optic cup and stalk and obviously allows a much more
accurate description of these structures as they change
with development. This technique has been applied
to descriptive studies of other developing systems with
equal success.18 It may provide a mechanism for the
analysis of the eye using specific labels and markers
for cellular and matrical components such as those
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Fig. 6. Mesectodermal cells (M) remain in the optic fissure (OF)
during the time of closure on day 13. Bar = 5 fim.
recently used by Hilfer and Yang7 and Yang and
Hilfer.16
Most modern as well as classic textbooks of embryology and ophthalmology describe the optic fissure
to extend not only through the optic cup but along
the ventral surface of the optic stalk. The degree to
which the fissure is affiliated with the stalk varies as
much among authors as species studied. On the one
hand, the opticfissureis said to extend the entire length
of the stalk to reach or nearly reach the lateral wall of
the forebrain in man,81319"22 chick,23 pig,24 or all vertebrates.25 Others report that the fissure extends only
onto the distal portion of the stalk in man,14-26 chick,27
and rat and mouse.2-4 A substantial number of publications are nonspecific in their descriptions of the
optic fissure relative to the optic stalk. Most simply
state that the fissure extends along the ventral surface
of the stalk.1015-28'32
Care must be taken in defining the parts of the neuroectodermal portion of the eye. It is the ventral portion
of the optic cup that is retarded in its "growth," which
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Fig. 7. Three neuroectodermal regions of the eye can be recognized at 13 days gestation: optic cup (OC); collum (C); optic stalk (OS). Some
of the mesectoderm has not been removed from the proximal portion of the optic fissure (OF). The fissure extends through the cup and
collum portions only. No evidence of the fissure is seen on the optic stalk. The lens has been removed from the optic cup. Bar = 100 nm.
Fig. 8. The eye from a 13-day embryo with surface ectoderm, lens, and mesectoderm removed demonstrates that the optic fissure (OF) is
restricted to the optic cup (OC) and collum (C). No evidence of the fissure is found on the optic stalk (OS). Bar = 100 jjm.
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NEUROECTODERM OF THE EMBRYONIC EYE / Morse and McConn
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Fig. 9. The surface ectoderm, mesectoderm, and lens have been removed from this 13-day specimen, which is viewed from the lateral
aspect. The optic cup (OC) is deep and lips of the optic fissure (OF) appear to be in contact at some points on the ventral portion of the cup.
Bar = 100 pm.
leads to the formation of the optic fissure. If one observes only the ventral aspect at these stages, the initial
tendency is to classify the region that contains the
proximal portion of thefissureas optic stalk. However,
careful analysis of the entire optic cup/optic stalk transition region reveals that the great majority of the fissure
is associated with the cup.
The, narrow segment, which connects the optic cup
and stalk, has been recognized only recently. Its formation is quite likely due to a focus of cell death since
this region corresponds to the constricted and necrotic
zone (necrotic area 6) in the C57BL-6J mouse embryo.9
We have termed this region the collum for two reasons.
First, this portion of the neuroectodermal outgrowth
from the diencephalon possesses the characteristics
implied by this term, ie, a constricted portion of any
organ or structure that connects two parts. Second,
this is the only portion of the neuroectoderm outside
of the optic cup that contains optic fissure. This distinguishes it from the optic stalk.
With this classification of the parts of the early neuroectodermal eye, some of the apparent disparity in
the literature regarding the optic fissure is explained.
This is especially true for those authors who describe
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a fissure only on the distal stalk.2<4>14-26-27 it is likely
these authors were referring to the collum.
The possibility of variation in the optic fissure location due to species differences must be considered.
This potential is less credible, however, when it is realized that major discrepancies exist for descriptions
of the same species by different laboratories. Planes of
section may account for some of the disagreement.
The direction of the fissure in the collum is very
oblique. Thus, the fissure is a shallow groove in the
proximal neck and a deep cleft in the distal portion.
This produces a furrow through which vasoformative
tissues that give rise to the intraocular vessels enter
the developing eyeball without crossing the rim of the
optic cup and without piercing epithelial layers. As
indicated by Mann13 and Silver and Robb,2 the same
effect is created for the axons of retinal neurons that
leave the optic cup to form the optic nerve and establish
connections in the brain.
The mechanism by which the central canal of the
optic stalk is occluded is unknown. It is implied in
most works that the infolding of the stalk to create the
opticfissurecontributes to the narrowing and eventual
loss of this communication between the cavity of the
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1984
Fig. 10. The optic fissure is closed by day 14. The remnant of the
fissure is a shallow groove (arrowheads) on the ventral aspect of the
optic cup. The optic stalk (OS) is fractured. An intraocular vessel
(arrow) enters the optic cup through a persistent portion of the optic
fissure in the collum. This area is illustrated in Figure 11. Bar = 100 jim.
Fig. 11. Higher magnification of the area indicated by the arrow
in Figure 10 demonstrates a cross-sectional fracture of an intraocular
vessel as it traverses a small patent portion of the optic fissure to
enter the optic cup. This patency is in the collum. Bar = 10 ^m.
optic vesicle and primitive ventricular system of the
brain. Evidence from the rat embryo in this paper
shows that is not the case. A reevaluation of the components of the optic stalk and their fate and contribution to eye morphogenesis is needed.
6.
Key words: optic cup, optic fissure, scanning electron microscopy, rat embryo, neuroectoderm
8.
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