Download Abnormal eye development associated with Cat4a, a

Abnormal Eye Development Associated with Cat4a, a
Dominant Mouse Cataract Mutation o n Chromosome 8
Patricia A. Grimes,1 Brigitte Koeberlein,1 Jack Favor,2 Angelika Neuhduser-Klaus,2
Dwight Stambolian1
and
PURPOSE. Cat4a, one of four mutant alleles at the mouse Cat4 locus, causes central corneal opacity
and anterior polar cataract in heterozygotes and microphthalmia in homozygotes. The Cat4 locus
has been mapped to chromosome 8, 31 cM from the centromere. In this study ocular development
of Cat4a mutant mice was investigated to characterize the defects in eye morphogenesis.
METHODS. Serial sections from eyes of wild-type, heterozygous, and homozygous littermates were
examined by means of light microscopy at selected intervals from embryonic day 11 to postnatal
day 1. Eyes of adult heterozygous and homozygous mice also were evaluated histologically.
RESULTS. Failure of separation of the lens vesicle from the surface ectoderm was the earliest
structural defect observed. In heterozygous embryos, the abnormality was limited to persistent
connection of the anterior pole of the lens to the cornea. Adult heterozygotes had defects in the
central corneal stroma and endothelium and anterior polar cataracts with or without keratolenticular adhesion. In homozygous embryos, the persistent connection of lens to surface ectoderm
was associated with aborted lens development, failure of closure of the optic fissure, and impairment of growth of the eyecup. Microphthalmic eyes of adult homozygous mice had a poorly
developed cornea, and the anterior chamber and vitreous compartment were absent. An extensively folded retina and remnants of a degenerated lens filled the interior of the globe.
CONCLUSIONS. A developmental defect inhibits separation of the lens vesicle from surface ectoderm
in mice heterozygous or homozygous for the Cat4a mutation. In homozygotes subsequent lens and
eye morphogenesis are also severely affected. Cat4a shows phenotypical similarity to several other
independent mouse mutations including Small eye, a mutation of the Pax6 gene. Cat4 may be one
of several genes involved in a common developmental path and may be part of the fVo;6-regulated
gene cascade governing eye morphogenesis. (Invest Ophthalmol Vis Set 1998;39:1863-1869)
M
ouse mutants with congenital ocular abnormalities
are an important resource for identification and investigation of genes involved in normal eye development. Characterization of such genes in lower animals can aid
in isolation of their human homologues and increase understanding of congenital ocular defects in humans.
The mouse Cat4 locus includes four independent mutant
alleles, all of which are expressed in heterozygous carriers as
anterior polar cataract and central corneal opacity.1 Three of
these mutations (Cat4b, Cat4c, and Cat4d) are homozygous
lethal, and heterozygotes display moderate microphthalmia in
addition to cornea and lens abnormalities. Carriers of the re-
maining allele (Cat4a) have eyes of normal size whereas homozygotes, which are viable and fertile, show severe microphthalmia with closed eyelids. Although in an earlier study we
suggested linkage of Cat4 to chromosome 2, 2 more extensive
mapping has localized Cat4 to the central region of chromosome 8 at position cM31 3 No other known mutations resulting
in cataract or microphthalmia map to chromosome 8, and Cat4
mutants have no visible abnormalities other than eye defects.
In this histologic study, we describe the abnormalities of ocular
development in heterozygous and homozygous mice expressing the Cat4a mutant allele.
METHODS
From the department of Ophthalmology and Scheie Eye Institute,
University of Pennsylvania School of Medicine, Philadelphia; and the
2
GSF-Institut fiir Saugetiergenetik, Neuherberg, Germany.
Supported by Research Grant EY10321 from the National Institutes of Health, Bethesda, Maryland; the Nina and Paul Mackall Trust,
and an unrestricted grant from Research to Prevent Blindness, New
York, New York.
Presented in part at the annual meeting of the Association for
Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May
1995.
Submitted for publication November 26, 1997; revised April 13,
1998; accepted May 6, 1998.
Proprietary interest category: N.
Reprint requests: Patricia A. Grimes, D-603 Richards Building,
University of Pennsylvania, Philadelphia, PA 19104-6075.
Investigative Ophthalmology & Visual Science, September 1998, Vol. 39, No. 10
Copyright © Association for Research in Vision and Ophthalmology
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The animals used in this study were descendants of congenic
C5H-Cat4a/+ mice maintained in Neuherberg. Progeny of mutant animals were examined at intervals between embryonic
day (E)ll and birth. The day of vaginal plug detection was
designated E0. The heterozygous phenotype was characterized
at E l l , E13, E15, and E17 in litters of obligate heterozygotes
derived from mating homozygous to wild-type mice. The homozygous phenotype was identified by comparison with the
established heterozygous phenotype at the same intervals in
litters resulting from homozygote X heterozygote or heterozygote X heterozygote mating. Pregnant females were killed with
an overdose of carbon dioxide. Embryos were fixed in Carnoy's
1863
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Grimes et al.
IOVS, September 1998, Vol. 39, No. 10
solution (3 parts 95% ethanohl part glacial acetic acid) for 24
hours and then transferred to 70% ethanol. The heads were
embedded in glycol methacrylate (Historesin; Leica Instruments, Heidelberg, Germany) and sectioned coronally at 3 5
jLtm to 6 jam. Serial sections through the eyes were collected
and stained with toluidine blue. The eyes of six heterozygous
and four homozygous adult mice (4-6 months old) were also
examined, fixed in 4% paraformaldehyde for 24 hours, and
similarly processed. All experimental procedures in this study
conformed to the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
RESULTS
External E x a m i n a t i o n o f E m b r y o s
On gross examination, heterozygous embryos did not differ
from normal littermates during gestation, but homozygous embryos could be unambiguously identified from El3 onward by
inspecting the eyes (Fig. 1). In homozygous mutants at El3 the
pigmented anterior margin of the optic cup was abnormally
heart shaped with a narrow ventral gap at the site of the optic
fissure (Fig. IC). At later stages of development, extension of
the dorsal and lateral margins of the optic cup became more
prominent, until by El7 the pupillary area was reduced to a
narrow Y-shaped opening (Fig. ID). The diameter of the optic
cup was consistently smaller than normal in the homozygous
embryos.
Early D e v e l o p m e n t : E l l t o E13
At El 1, the earliest stage examined, the lens vesicle was normally separated from surface ectoderm (Fig. 2A). In all heterozygous and homozygous mutant embryos, however, a cellular strand connected the surface ectoderm and the anterior
wall of the lens vesicle (Fig. 2B, 2C). In homozygous embryos,
the lens vesicle and the optic cup were also notably smaller
than normal, and the ventral wall of the optic cup was poorly
developed.
At El3, the optic cup and lens of normal (Fig. 3A) and
heterozygous embryos (Fig. 3B) were similar in size and differentiation despite a persistent connection between corneal and
lens epithelium in the heterozygotes. More severe abnormalities were evident in homozygous embryos (Fig. 3C, 3D). A
small mass of primary lens fibers formed from the posterior
wall of the lens vesicle, but neither an organized anterior lens
epithelium nor an equatorial bow region was present. The
small optic cup had defects consistent with those observed in
intact embryos (Fig. IC). An abnormally extended and folded
region of the dorsal margin of the optic cup was bordered
laterally by shortened and blunted regions (Fig. 3C, 3D), and
the optic fissure, closed at this stage in normal and heterozygous embryos, persisted as a narrow cleft in the ventral optic
cup (Fig. 3C).
Late Development a n d Adult Phenotype of
Heterozygous Mutants
Abnormalities of heterozygous eyes in subsequent development were limited to the site of contact between lens and
cornea. At El5 and El7, a full-thickness defect extended
through the otherwise normally formed corneal stroma and
endothelium. In some eyes the defect was filled with cells
connected to and resembling the corneal epithelium with only
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FIGURE 1. External phenotype of fixed heterozygous (+/—)
and homozygous (—/—) embryos at embryonic day (E)13 (A,
C) and E17 (B, D). Eyelids were removed from El7 specimens
before photography; all lenses are opaque because of fixation.
Heterozygous embryos (A, B) show no visible defects. The
eyes were indistinguishable from those of normal littermates.
In homozygous embryos (C, D), abnormal eye development is
clearly evident. At El3 (C), the anterior margin of the eyecup
shows a distinctive heart shape. At El 7 (D), extension of the
eyecup margin almost completely occludes the lens, leaving
only a narrow Y-shaped opening. Scale bar, 1 mm.
a small protrusion of the anterior lens pole contacting the
internal corneal surface (Fig. 4A). More commonly, the stromal
defect was filled with a large conical protrusion of the lens pole
(Fig. 4B), and sometimes (El7 only) lens fiber material extruded through the cornea into the conjunctival sac (Fig. 4C).
These patterns show that the strand of cells connecting the
lens to the surface ectoderm at earlier developmental stages
may differentiate into corneal epithelium or lens epithelium.
Lenses with a prominent extension into the cornea, in particular those that lost lens fiber material into the conjunctival sac,
were smaller at El7 than those attached only to the inner
corneal layers. The defect in the surface epithelium and loss of
lens mass associated with extrusion of fiber material through
the cornea seemed to be repaired before birth, because in all
newborn and adult heterozygotes examined, corneal epithelium always covered the central stromal defect, and the lenses
were of approximately normal size.
IOVS, September 1998, Vol. 39, No. 10
Eye Development in a Mouse Cataract Mutation
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FIGURE 2. Eye structure of normal ( + / + ) , heterozygous (+/—), and homozygous (—/—) embryos at embryonic day 11. The lens
vesicle, completely separated from surface ectoderm in normal embryos (A), remains joined to the surface by a strand of cells
(between arrows) in heterozygous (B) and homozygous (C) embryos. In homozygous embryos, the lens vesicle is small, poorly
developed, and lies deep within the eyecup. The eyecup also is small, and differentiation of its ventral portion is retarded. Toluidine
blue; scale bar, 100 jixm.
The patterns of lens-corneal attachment evident in El7
heterozygotes presaged the abnormalities observed in mature
eyes. The lens remained attached to the cornea in 75% of the
adult eyes examined, and in most instances of persistent attachment a small projection of lens epithelium and capsule was
entrapped in the central corneal stromal defect (Fig. 4D). Lens
tissue incorporated in the cornea was continuous with a multilayered plaque of epithelial cells and capsulelike material
overlying an area of anterior cortical fiber disintegration. The
structure of the remaining lens was usually normal, but in a few
eyes, swelling and disorganization of posterior cortical fibers
were evident. Sometimes the lens was only superficially attached to the cornea at the level of the gap in Descemet's
membrane and corneal endothelium. In these cases, the defect
in the corneal stroma was filled with a downward growth of
corneal epithelium or fibrous scar tissue. The lens was attached
by reflection of Descemet's membrane and corneal endothelium from the lateral margin of the corneal defect onto the
intact lens capsule, by fine fibrous connections between the
corneal stromal scar and the lens capsule, or by both. In eyes
in which the lens was separated from the cornea, the structure
of the central corneal lesion showed that the original attachment of the lens and cornea had been of the more superficial
type. The stromal defects, which showed no entrapped lens
tissue, were filled with a plug of corneal epithelium or fibrous
scar tissue (Fig. 4E), and the lens capsule was intact over the
region of the anterior polar cataract (Fig. 4F).
Late Development and Adult Phenotype of
Homozygous Mutants
The lens and eyecup of homozygotes were markedly abnormal
at El5 and El7. Although both structures enlarged with increased gestational age, they remained smaller than those of
age-matched heterozygous or normal embryos. A thick column
of closely packed cells originating from the corneal epithelium
joined the lens as a disorganized cluster of abnormal lens
epithelium. By El7 the lens epithelium surrounded the lens
fiber mass and extended from it in multilayered cords or tubes
(Fig. 5A, 5B). In sections stained with the periodic acid-Schiff
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method, a thin lens capsule surrounded the ectopic cords of
epithelial cells (not shown). No organized region of lens fiber
formation from epithelial cells was present. Where the epithelial covering of the lens was incomplete, lens fibers bulged
from the surface or erupted into the vitreous cavity (Fig. 5B).
The eyecup extended anteriorly to occlude the pupillary area
and enclose the lens. A layer of condensed mesenchyme extending from the developing choroid fused with the overlying
corneal stroma, and no anterior chamber formed. The optic
fissure remained partially open (Fig. 5A), but differentiation of
the neural retina and pigment epithelium otherwise appeared
normal.
Adult homozygous eyes consistently were very small and
located deep in the orbit behind permanently closed lids. The
average diameter of the fixed globes was 1 mm, approximately
one third the diameter of normal adult eyes and approximately
the same size as the eyes of homozygous embryos at El7. The
microphthalmic eyes were characterized by a poorly developed cornea overlying an almost completely closed eyecup
(Fig. 5C). Neither corneal endothelium nor anterior chamber
was present. In all eyes, a strand of corneal epithelial tissue
penetrated the disorganized corneal stroma and subadjacent
densely pigmented uveal tissue to fuse with remnants of the
lens lying within the eyecup. Lens elements in the adult eye
consisted only of isolated or clustered bladder cells sometimes
surrounded by lens capsule. The ectopic strands of lens epithelial cells prominent in developing eyes at El7 were not seen
in the adult eyes. The remainder of the eye was filled with an
extensively folded but well-differentiated retina. Retinal pigmented epithelium and choroid were normal in appearance
except for colobomata near the optic nerve.
DISCUSSION
The Cat4a mutation caused a developmental failure of lens
vesicle separation from surface ectoderm. In heterozygous
embryos, subsequent growth and differentiation of the lens
and other ocular structures was not inhibited despite persis-
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Grimes et al.
IOVS, September 1998, Vol. 39, No. 10
FIGURE 3. Eye structure of normal ( + / + ) , heterozygous (+/—), and homozygous (—/—) embryos at embryonic day 13. The lens
and optic cup of normal (A) and heterozygous (B) embryos are similar in size and organization except for the connection of corneal
and lens epithelium (arrows) in the heterozygous mutants. In homozygous embryos (C), the lens consists of a small ball of primary
lens fibers capped anteriorly by a long cellular stalk (arrows) continuous with the surface ectoderm. There is no organized anterior
lens epithelium or zone of differentiating fibers. The dorsal margin of the optic cup is shortened and blunted in this plane of
section, and the open optic fissure is visible as a narrow cleft (arrowheads) in the ventral wall of the optic cup. In a deeper plane
of section through the same homozygous eye (D), the optic cup extends anteriorly to encompass the lens; a prominent fold
(arrowhead) in the dorsal arm of the optic cup indicates that the eye is not enlarging normally. Toluidine blue; scale bar, 100 /xm.
tent ectodermal connection. The resultant abnormalities of the
mature eye, including defects in the central corneal stroma and
endothelium and anterior polar cataracts with or without keratolenticular adhesion, arose directly from the preexisting epithelial connection between the lens and surface ectoderm. In
homozygous embryos, defective lens vesicle separation led to
abortive lens development, failure of closure of the optic fissure, and inhibited growth of the optic cup that resulted in
microphthalmia.
Lens vesicle-ectoderm separation in mouse eye morphogenesis involves localized cell death in the epithelial layer,4 but
the mechanism triggering this event is unknown. Expression of
the Cat4a mutation in the ectodermal cells or adjacent mesenchyme could inhibit necessary interactions between the ectoderm, mesenchymal cells, or extracellular matrix required for
separation. In homozygous embryos, the lens vesicle not only
remains attached to the surface ectoderm by a long stalk but is
positioned deep in the optic cup and does not form an orga-
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nized anterior epithelium. This defect and the ensuing abnormalities of lens development may arise from a failure of the
inductive interactions between the lens and the mesenchymal
and neural tissue at the margin of the eyecup that control size,
shape, and orientation of the developing lens. 5 6 The abnormal
expansion of the lens epithelial cell population noted in El7
homozygous embryos suggests a transient stimulation of cell
proliferation, but the increased epithelial cell population at this
stage could also result from a decreased rate of epithelial cell
differentiation into lens fibers. The small size of the lens in
homozygous embryos during development may account for
the associated microphthalmia; normal lens growth is required
for appropriate enlargement of other eye structures with the
notable exception of the retina. 7 9 A direct effect of the homozygous mutation on development of the optic cup cannot
be excluded.
At least four other independent mutations result in persistent lens-ectoderm connection and microphthalmia in the
IOVS, September 1998, Vol. 39, No. 10
Eye Development in a Mouse Cataract Mutation
1867
FIGURE 4. Embryonic day (E)17 and adult heterozygotes. At El7 (A, B, C), attachment between cornea and lens is always present
but varies in extent as indicated in sections from three representative embryos. (A) The intact anterior pole of the lens touches
the cornea at the site of a defect in the corneal endothelium. An overlying defect in the corneal stroma is filled by corneal epithelial
cells (arrows). (B) A conical protrusion of the anterior lens bulges through the corneal stroma. The lens epithelium is interrupted
at the tip of the protrusion, but the overlying corneal epithelium is intact. (C) Lens fibers rupture into the conjunctival sac
(asterisks) through a defect in the lens epithelium and all corneal layers. In a representative adult heterozygous mouse, the lens
is connected to the cornea in one eye (D) but is separated in the other eye (E, F). (D) At the cornea-lens attachment, lens cells
and capsule embedded in the stromal defect are continuous with the anterior lens pole where a multilayered plaque of epithelial
cells overlies an area of cortical disintegration. Corneal endothelium and Descemets membrane terminate on either side of the
fusion point (arrows). (E) In the cornea of the other eye, fibrous scar tissue fills the stromal defect and extends irregularly through
the gap in the corneal endothelium and Descemets membrane (arrows). (F) The separate and normally positioned lens has an
anterior polar cataract similar to that seen in the attached lens. Toluidine blue; scale bar, 100 jixm.
mouse. These include the dominant mutations Small eye
(Pax6)H)J l and Coloboma,12,1* both located on chromosome
2, and the recessive mutations, dysgenetic lens1•'1'15 and aphakia. 16 The phenotypical similarities suggest that the affected
genes function in the same essential steps of eye morphogenesis. The affected gene responsible for the Small eye phenotype in mice has been identified as Pax6, and mutations of the
human PAX6 gene have been identified as a cause of congenital aniridia. 1720 Pax6 encodes a highly conserved and developmentally expressed transcription factor that has been proposed to regulate the cascade of genes that participate in eye
morphogenesis. 21 In mouse development Cat4, Coloboma,
dysgenetic lens, and aphakia may all be part of the Pax6
regulatory cascade.
The phenotype of Cat4ci/+ mice resembles Peters' anomaly, a human congenital condition characterized by defects in
the central corneal stroma, Descemet's membrane, and corneal
endothelium sometimes associated with keratolenticular adhesion and cataract. 22 Identification of PAX6 mutations in two
cases of Peters' anomaly11 is consistent with the presence of a
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comparable phenotype in Small eye mice; PAX6 mutations,
however, may only be a rare cause of Peters' anomaly in
humans. 23 Peters' anomaly occurs with and has been grouped
with Rieger's anomaly, Axenfeld's anomaly, iridogoniodysgenesis, sclerocornea, and congenital endothelial dystrophy, all
developmental malformations of the anterior chamber attributed to mesenchymal dysgenesis of the iris and cornea. 22 24
These disorders show phenotypic overlap, and affected members of the same family may display one or the other malformation 24 suggesting variable expression of the same gene defect. Conversely, genetic heterogeneity is indicated in some
phenotypically similar conditions, such as Rieger's syndrome
with linkage to 4q25 25 and 13ql4. 26 The gene involved in
4q25-linked Rieger's syndrome (RIEG/PITX2)27 and the mouse
homologue (Rieg/Pitx2)z ,2H have recently been cloned and
characterized as a transcription factor belonging to the class of
bicoid-vel'Ated homeo proteins. A mutation of RIEG/PITX2 has
also been identified in 4q25-linked autosomal dominant iris
hypoplasia, a condition that does not have several of the
features characteristic of the Rieger's syndrome pheno-
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IOVS, September 1998, Vol. 39, No. 10
G r i m e s et al.
FIGURE 5. Embryonic day (E)17 and adult homozygotes. At E l 7 (A, B) n o anterior c h a m b e r is present, and the optic c u p encloses
the abnormal lens. (A) In this peripheral section, a column of cells (arrows) projects from the anterior lens epithelium into the
corneal stroma through a n a r r o w opening in the optic c u p . The cell column fuses with t h e corneal epithelium in adjacent sections.
The lens epithelium forms a multilayered covering around t h e lens fiber mass and extends as cords of cells into the vitreous cavity.
The optic fissure (arrowheads)
persists as a n a r r o w cleft in the ventral optic cup. (B) A central section through the lens of the same
eye shows the absence of any region of lens fiber differentiation and the disorganization of existing fibers. At gaps in the
multilayered epithelial covering (arrowheads),
lens fiber material erupts into the vitreous cavity. In an adult homozygous mouse
(C), the cornea of the microphthalmic globe is pierced centrally by a strand of corneal epithelial cells (arrows) that connects in
adjacent sections with lens remnants c o m p o s e d of bladder cells (le), scattered epithelial cells, and lens capsule. The anterior
c h a m b e r is absent. Densely pigmented tissue continuous with the choroidal layer extends forward and is fused with the irregularly
organized corneal stroma. The vitreous cavity is also absent, and the globe is filled with folded retina (re) that is well differentiated
in some areas. Toluidine blue; scale bar, (A, B) 100 jam; (C) 200 jum.
RIEG together with PAX6 and other as yet unidentype.'
tified genes at loci associated with anterior c h a m b e r dysgenesis
are likely to be part of a c o m m o n developmental pathway.
The gene affected at the Cat4 locus seems to have an early
function in eye morphogenesis, and its isolation and characterization should provide information about the genetic regulation of ocular development in the mouse and possibly in
humans. No other mutations resulting in cataract or microphthalmia have b e e n m a p p e d to m o u s e c h r o m o s o m e 8, and
n o likely candidate genes are evident in the region near the
Cat4 locus. 3 K n o w n homology correlations b e t w e e n the
m o u s e and h u m a n g e n o m e indicate that the proximal and
central portions of m o u s e c h r o m o s o m e 8 are syntenic with
portions of h u m a n c h r o m o s o m e s 8 ( 8 p 2 1 - p 2 3 ) , 19 ( 1 9 p l 3 1 13.2; 1 9 . 2 c e n - q l 3 , 4 ( 4 q 2 8 ~ q 3 1 ) , and 16 ( I 6 q l 3 - q 2 2 ) . 3 1 The
only h u m a n cataract or eye defect m a p p e d to these regions is
anterior segment ocular dysgenesis at 4 q 2 8 - q 3 1 3 2 Carriers of
this autosomal dominant disorder express corneal opacities,
iris adhesions, and cataract. 3 3 The region of the Rieger's synd r o m e locus at 4q25 is homologous to a region of mouse
c h r o m o s o m e 3 3 4 and is unrelated to Cat4\ Rieg/Pitx2
has
b e e n m a p p e d to this portion of m o u s e c h r o m o s o m e 3 2 8 At
present, based on similarities in p h e n o t y p e , m o d e of inheritance, and chromosomal position, mouse Cat4 may be homologous with h u m a n anterior segment ocular dysgenesis. Additional studies of Cat4 are required to establish its role in eye
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development and its association with congenital ocular abnormalities in humans.
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