Download Experimental Embryology of the Vertebrate Eye

Experimental embryology of the vertebrate eye
Alfred J. Coulombre
The size, shape, position, and orientation of the tissues of the vertebrate eye relative to each
other fall within the narrow geometric tolerances compatible with the optical function of this
organ. During embryonic development the establishment and maintenance of appropriate
relationships among the several ocular tissues remit from an orderly complex of specific
interactions among the tissues. Each tissue may be studied as a source of influence on other
tissues or, alternatively, as a target of influences arising from other tissues. This paper focuses
attention on the lens. The lens may be considered as a target of influences which emanate
from the eyecup and neural retina and which are involved in lens induction, lens fiber differentiation, lens suture orientation, lens growth, and the orientation of the lens relative to the
optic axis. The lens, in its turn, is a source of influence in the induction of corneal anterior
epithelium from ectoderm and in the control of the accumulation of the vitreous substance.
The accumulation of vitreous substances importantly influences the size and shape of the
pigmented epithelium, choroid coat, and sclera. The analysis of each tissue as both a source
and a target of influences permits the construction of flow sheets of tissue interactions in the
developing eye. These floio sheets provide a rational basis for understanding the teratology of
this organ and represent causal chains which must connect at many points with events at
the molecular and chromosomal levels.
T
to study the developing lens include extirpation, transplantation, explantation, autoradiography, and the confrontation of lens
precursors with other tissues. Intervention
at specific stages during lens development
has revealed many of the successive steps
which initiate and channel the differentiation of the lens cells. At the morphogenetic
level such studies are beginning to identify
and to characterize the mechanisms which
continually regulate the position, size, orientation, and shape of the developing lens.
During embryogenesis the vertebrate
lens is both the target of influences which
arise from surrounding tissues and a source
of highly specific influences which are essential for the normal differentiation and
morphogenesis of some of the other ocular
tissues. Enough information has accumulated concerning the influences which operate upon the lens at each stage of development, so that it is possible to con-
.he lens of the vertebrate eye has a re±h
markably
small number of components.
Within the acellular fibrous capsule which
surrounds it there is a single type of cell
and possibly a modest amount of extracellular space. The lens cells are at several
stages of cytodifferentiation. In the anterior epithelium they are cuboidal and
synthesize proteins of the alpha and beta
crystallin groups; at the equator of the
lens they begin to elongate and to manufacture proteins of the gamma crystallin
group.1'2 In the lens cortex they complete
their maturation as lens fibers; then as they
become buried deeper in the lens fiber
mass they lose their cell nuclei. The experimental techniques which have been used
From the Laboratoiy Neuroanatomical Sciences,
National Institute of Neurological Diseases and
Blindness, National Institutes of Health,
Bethesda, Md.
411
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412 Coulombre
Investigative Ophthalmology
August 1965
Chorda
Mesoderm
PRESUMPTIVE
NEURAL ECTODERM
ECTODERM
Notochord•
Axial
Mesoderm—»-•
NEURAL PLATE
NEURAL TUBE
Mitosis
Ventricular
»-l
PressureW
OPTIC VESICLE
NON-NEURAL
" ECTODERM
^ BASEMENT
MEMBRANE
Archenteron Roof.
»W
Heart Mesoderm
*-f
PRESUMPTIVE
LENS ECTODERM
PRESUMPTIVE
"NEURAL RETINA"
LENS PLACODE
VITREOUS
COMPONENTS
LENS CUP
Cell Death
LENS VESICLE-
1 '
LENS FIBERS
LENS
EPITHELIUM
LENS CAPSULE
Fig. 1. This flow sheet represents the major steps in lens development (heavy arrows) and
indicates some of the influences (light arrows) which impinge on, or are exerted by, the
embryonic lens. Dotted lines indicate uncertain relationships.
struct a flow sheet (Fig. 1) which (1)
tentatively delineates the sequence of individual steps in the differentiation and
morphogenesis of this structure; (2) focuses attention upon specific gaps in our
knowledge of the developing lens system;
and (3) provides a rational basis for understanding defects of the lens and of the
eye which arise from interference with
specific steps in the chain.
The lens as a target
A. Induction. The lens is a target of outside influences even in its origin, which deserves brief recapitulation here. Competence to form lens, which is widespread
in the primitive embryonic ectoderm,3 becomes restricted to the head region under
the influence of the foregut roof and the
presumptive heart mesoderm.3'10 The pre-
sumptive neural retina at the tip of the
optic vesicle plays the definitive role in
the induction of lens from overlying ectoderm.11"17 This induction requires proximity but not contact of the optic vesicle and
the overlying ectoderm.is"22 Lens induction
continues for a relatively prolonged period
during development,23"25 and the ability of
the neural retina to support the regeneration and growth of the lens persists even
in the adults of some salamanders.20 During its earlier stages the neural retinal influence results in a palisading of the ectodermal cells to form a lens placode,17
which invaginates to form the lens cup.
Closure of the edge of the lens cup cuts
off the lens vesicle internally, re-establishes continuity in the overlying ectoderm
(presumptive corneal epithelium), and
completely envelops the lens vesicle in
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Experimental embryology of vertebrate eye 413
the basement membrane (primitive lens
capsule) which had underlain the lens
placode.27
B. Lens fiber differentiation. The cells
of the lens vesicle which lie toward the
optic cup elongate progressively to form
lens fibers which eventually touch the posterior surface of the lens epithelium and
obliterate the cavity of the lens vesicle.
This elongation to form lens fibers is an
uninterrupted continuation of the cell
elongation which began during palisading
in the placode. The cells of the lens vesicle which lie away from the optic cup do
not elongate but form a simple cuboidal
epithelium which covers the fiber mass anteriorly and is continuous with it at the
equator of the lens. From the outset mitotic activity ceases in the lens fibers and is
confined to the lens epithelium. The cells
near the equatorial edge of the lens epithelium develop larger nuclei, more prominent nucleoli, a higher concentration of
cytoplasmic RNA, more numerous mitochondria, and increased height. It is in the
equatorial region that epithelial cells are
progressively recruited into the lens fiber
mass. As the lens grows, the axes of the
cells of the equator rotate through 90 degrees and take their place at the periphery
of the contiguous fiber mass. At later stages
the lens fibers which are generated at the
equator grow around the previously deposited fiber mass until they meet anteriorly and posteriorly with other fibers in
planes of junction which are called sutures.28
C. hens sutures. The patterns of suture
planes vary from a single plane to many
radiating planes of conjuncture depending
on the species. The orientation of these
planes with respect to the eye are constant in a given species and become determined during a specific period in embryonic development.20 Prior to this time,
experimental rotation of the embryonic
lens about its axis results, during ensuing
development, in the appearance of suture
planes which are normally oriented relative to the eye. Experimental rotation of
the lens about its axis following this critical period in embryonic development results in the development of suture planes
which are inappropriately oriented with
respect to the eye because their orientation has become fixed with respect to the
lens.
D. Lens growth. At any stage in development the volume of the lens is given
by the relationship: V = N7+S+C, where
V is lens volume, N is the number of lens
cells, v is the mean volume of the lens
cell, S is the volume of the extracellular
space, and C is the volume of the lens
capsule. Both S and C make extremely
small contributions to the total lens volume at all stages during development and
will not be considered further in this treatment. There is no information currently
concerning the volume of individual cells
(v) as a function of their position in the
epithelium, the equatorial region, or at
various depths of the fiber mass, or as a
function of developmental time. We do
know, as mentioned, that the influence of
the neural retina is necessary for the increase in cell volume which occurs when
lens cells are transformed into lens fibers.
This transformation is of central importance in the growth of the lens, since the
lens fiber mass makes the largest single
contribution to the increase in lens volume.
When the neural retina is removed from
the eyes of four-day chick embryos, the
growth of the lens ceases in a short time.
When the same operation is performed,
but a small piece of neural retina is reintroduced into the eye cavity, subsequent
growth of the lens is intermediate between
that in eyes without neural retina and that
in untreated eyes.30 The growth of the
lens appears to be independent of the
growth of all other tissues of the eye with
the exception of the neural retina. By inserting drainage tubes through the eye
wall of the four-day chick embryo, a portal
of egress was provided for the vitreous
substance as it was produced. As a consequence of this operation the eye as a
whole grew more slowly than normally
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414 Coulombre
and became microphthalmic. The neural
retina continued to grow independently
and was thrown into increasingly more
complex folds. The outer coats of the eye
(cornea, sclera, choroid coat, and pigmented epithelium) grew at rates which
were markedly below normal. Despite the
subnormal growth of the superficial coats
of the eye, the lens grew at a normal rate.31
Not only can the growth rates of the lens
and the outer coats of the eye wall be uncoupled in this manner, but the presence
of the outer coats does not seem to be essential for normal growth of the lens provided that the neural retina is present in
sufficient quantity. For example, removal
of the cornea at five days of incubation in
the chick embryo has no detectable effect
on the growth, shape, or internal organization of the developing lens.
At any stage in development N is exhaustively described by the following function: N = n+(M+mr-mo-D)t, where n is
the initial number of cells in the lens primordium; M is the number of cells added
to the population per unit time as a result
of mitosis; m-, is the number of cells added
to the population per unit time as a result
of migration of cells into the lens population from other tissues; m0 is the number
of cells lost to the cell population per unit
time as a result of migration of cells out
of the lens population; D is the number of
cells lost to the cell population per unit
time as a result of cell death and dissolution; and t is time. In all but certain experimental situations the terms mx and m0
have no bearing, and can be eliminated
from the equation as long as the capsule
is intact and the lens remains a closed
system. Mitosis is confined to the cells of
the lens epithelium and does not occur,
at any stage in development, in cells which
have traversed the equatorial region to
become part of the lens fiber mass. The
mitotic activity must be so balanced with
respect to the other factors affecting cell
number and cell volume that cells are
generated at a rate just sufficient to account for the addition of new fibers on the
Investigative Ophthalmology
August 1965
one hand and the increase in the number
of epithelial cells on the other. It is well
worth emphasizing that throughout development the lens epithelium has an area
which is precisely appropriate to the size
of the underlying fiber mass. In the normally developing lens it neither buckles
nor becomes so stretched that gaps develop.
The contribution of D to the growth of
the lens deserves more attention than it
has received. It is known that in all but
the earlier stages of development the cells
at the center of the fiber mass (lens nucleus) lose their cell nuclei and, in this
sense, die. However, it remains to be established whether or not any of these cells
undergo dissolution followed by removal
of the breakdown products from the lens.
Despite the fact that mitosis continues in
the lens epithelium throughout life in most
species, and that fibers continue to be
added to the lens cortex at the equator,
the increase in the volume of the lens ultimately becomes negligible. This paradox
could be resolved by a compensatory loss
in cell volume in the mature fibers (the
widely held concept of central compression), a compensatory loss of cells, or both.
The value of n is a function of the size
of the cells in the presumptive lens ectoderm at the time of lens induction, and of
the area of contact between the tip of the
optic vesicle and the overlying ectoderm.17' 32'3'1 The number of cells in the
primitive lens population is not constant;
rather, this population is increasing from
the very outset. The size of the eye which
develops is directly correlated with the
area of contact between the vesicle and the
ectoderm, and therefore with the number
of cells in the lens placode.10'34 However,
since the area of the neural retina also depends upon the area of this contact,35' 3G
it is impossible to say whether the initial
size of the lens population has any importance independently of the role played by
the neural retina. During later development there is, in experimental situations,
a correlation between the size of the lens
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Experimental embryology of vertebrate eye 415
and the surface area of the neural retina.34
The simple algebraic formulations given
previously are accurate, but they can be
misleading unless it is realized that each
of the terms can, and probably does, vary
as a function of developmental time. These
relationships focus attention on the major
components which contribute to lens size,
and invite attention to the mechanisms
which control the rate at which each of
the components increases during embryonic development.
E. Orientation of the lens. Given the
optical function of the lens, and the stringent geometric limits set by the laws of
optics, this structure can function effectively only if it is appropriately oriented
with respect to the optic axis and suitably
aligned with the other dioptric media. It is
a matter of observation that the lens is
usually properly positioned and oriented
throughout its entire development. The
initial alignment of the lens with the eyecup is assured because its definitive induction is earned out in the zone of contact
between the presumptive lens ectoderm
and the tip of the optic vesicle. It has been
demonstrated in a number of ways that
this mechanism continues to operate and
continuously adjusts the orientation of the
lens as development proceeds. If, in certain salamanders, the iris with its subtended lens is surgically removed and reversed so that its anterior surface now
faces the vitreous body, it will heal in
place. Subsequent removal of the lens from
such animals is followed by the regeneration of a lens from the dorsal iris. The
regenerated lens is oriented appropriately
with respect to the eye and in reversed
orientation with respect to the iris.30 The
system which orients the lens during lens
regeneration is probably similar to that
which has been demonstrated during normal development in the chick embiyo. If
the lens of the five-day chick embryo is
removed from the eye, rotated 180 degrees,
and replaced in such a manner that the
lens epithelium now faces the vitreous
body rather than the cornea, there is a
Fig. 2. This is an axial section of an eleven-day
lens of a chick embryo which had been reversed
at five days of incubation. The polarity of the
lens has become reversed, a new epithelium is being reconstituted anteriorly, and a new fiber mass
is developing posteriorly from the original lens
epithelium. The lens is of a size, shape, position,
and orientation appropriate to its age.
complex reorganization of the lens cells.
The old lens fiber mass, which is now
situated anteriorly, stops growing. The lens
epithelium, which is now placed posteriorly, rapidly increases in height as all its
cells differentiate into lens fibers. The lens
epithelial cells close to the equator continue to divide and reconstruct a new lens
epithelium anteriorly at the same time that
they contribute fibers to the new fiber
mass which has developed from the origi-
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416 Coulombre
nal epithelium. As a consequence, the
polarity of the lens is reversed 180 degrees (Fig. 2). During this process the
lens continues a normal increase in volume, and, despite the complex changes
occurring internally, maintains an appropriate shape and orientation.37 If a fiveday lens is removed and replaced with
two five-day lenses, the ensuing changes
obey the following rules regardless of the
orientation in which the lenses are introduced: (1) all of the lens epithelial cells
which are on the vitreal side of a plane
passing through the ciliary processes elongate to form lens fibers; (2) the lens epithelial cells located on the corneal side of
this plane continue to divide, form lens
epithelium on the entire corneal surface
of the combined lens mass, and reconstitute new equatorial zones adjacent to the
ciliary processes, which contribute fibers
to the new fiber mass. In the process the
combined lens mass achieves an appropriate internal organization (epithelium toward the cornea and lens fiber mass toward the vitreous body), and gradually
becomes regulated in its combined shape,
orientation, and total volume until these
properties match those appropriate to a
single lens of the host age. Even if the
embryonic lens is replaced not with a
whole lens but with an isolated embryonic
lens epithelium attached to its adjacent
capsule, a reasonably well-shaped lens will
develop in which the epithelium invariably faces the cornea and in which the
lens fiber mass lies toward the vitreous
body. This occurs regardless of the initial
orientation of the implant. Even after a
six-day chick embryonic lens epithelium
has been maintained for five additional
days in culture, it will reform a reasonably
good and appropriately oriented lens if it
is returned to a lentectomized eye, but
will not do so if it is transplanted into the
coelom of a host embryo.3S This experiment provides a convincing demonstration of the specificity of the eye environment. It has recently been demonstrated
in the mouse that the retinal factor(s)
In vcstigative Ophthalmology
August 1965
which contribute to this mechanism can
act across a Millipore filter, and are, therefore, probably diffusible.39 This system appears to have a relatively low taxonomic
specificity. If the lens of the five-day
chick embryo is replaced with an isolated
lens epithelium and capsule from a mouse
embryo of 18 days' gestation, the host eye
supports the differentiation of the implant
into a well-formed lens that becomes appropriately oriented with respect to the
chick eye.
The lens as a source of influence
During an orderly morphogenesis the
tissues of the vertebrate eye develop sizes,
shapes, positions, orientations, and cellular
structures which, in the aggregate, admirably suit this organ for optical function.
It is becoming increasingly clear that this
highly coordinated development rests upon
a complex of prolonged interactions among
the tissues of the eye, which assures that
they will develop appropriate relationships. It has been fruitful to consider each
tissue of the eye not only as a target for
influence from surrounding tissues, but
also as a source of influences which help
to control the development of one or more
of the other ocular tissues in highly specific ways. Two influences which the lens
exerts deserve mention.
A. Induction of the anterior corneal epithelium. The ectoderm which remains on
the surface overlying the embryonic eye
following closure of the lens pore is destined to form the anterior epithelium of
the cornea. At first, competence to form
the cornea is widespread throughout the
embryonic ectoderm. The lens vesicle, and
later the lens, provides the principal inductive influence which assures that the
cornea will differentiate over the eye.40"49
The precise alignment of the cornea with
the lens is achieved and maintained because of the continuing inductive relationship between the lens and the comeal
anterior epithelium.11' "• 40>4S"50 The ability of the lens to induce cornea is gradually lost with age.515'1 Concomitantly the
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Experimental embryology of vertebrate eye 417
Volume 4
Number 4
anterior epithelium of the cornea becomes
increasingly independent of its inductor.2r>>
-10, ii, 4a, 4-1, T.), 54-5G j
n s o m
e forms,
however,
this dependence persists throughout life.
B. The control of the accumulation of
vitreous substance. Experiments in the
chick embryo suggest that the presence
of the lens is necessary for the normal
accumulation of the vitreous substance.
Removal of the chick embryonic lens at
five days of incubation terminates the
accumulation of vitreous substance. If the
lens is removed from the eye and immediately replaced, the accumulation of vitreous substance is normal. If the lens is
removed, boiled, and returned to the eye,
vitreous substance does not accumulate.
The gross increase in the size of the eye
during development is attributable in
large part to the accumulation of vitreous
substance within the eye.57> 5S For this reason the control of the growth of the vitreous body by the lens has important consequences in that this growth, in its turn,
is a necessary condition for the normal
growth of the cornea,59'60 the sclera,57 the
choroid coat, and the pigmented epithelium.G1 The mechanism by which the accumulation of vitreous substance is controlled by the lens remains to be explored.
Conclusion
The complex interactions of the ocular
tissues during development, in which the
neural retina plays a key role, assure an
orderly organogenesis. We know an increasing amount concerning which tissues
exert influences, during what period they
influence, which tissues are competent to
respond, and in what ways they respond.
Despite this increasing body of information, we do not know the physical basis
of any of these numerous influences. The
most economical hypothesis is that diffusible substances are released from specific
tissues of origin according to a precisely
programmed sequence, and that these substances alter the number, size, or state of
differentiation of cells in target tissues
which are competent to respond. Yet the
matter is not so simple. We know, for
example, that mechanical tension generated by one tissue can alter the development of a neighboring tissue. We have
seen something of the role that the lens
plays in this complex of interactions both
in the influence which it exerts on surrounding tissues, and in the responses
which it gives to specific stimuli from its
neighbors. Each tissue of the eye, in its
turn, may be analyzed with this same approach. The flow sheet of interactions
which emerges from such analyses provides not only a rational basis for understanding the teratology of this organ, but
also represents a causal chain which must
connect at many points with the events
taking place at the chromosomal and molecular levels.
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Volume 4
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