Development 99, 473-480 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
473
Rescue of developmental lens abnormalities in chimaeras of
noncataractous and congenital cataractous mice
A. L. MUGGLETON-HARRIS, K. HARDY and N. HIGBEE
MRC Experimental Embryology and Teratology Unit, Medical Research Council Laboratories, Woodmansteme Road, Canhalton, Surrey,
SM5 4EF, UK
Summary
In the study of the lens of a congenital cataractous
mouse mutant (CAT), it has been shown that a loss of
growth regulation at the cellular level causes gross
lens abnormalities. The phenotypic characteristics of
the cataractous mouse lens are similar to those seen in
human congenital cataract and thus serves as a model
system for medical research.
In this present investigation, we have demonstrated
that the abnormalities of the congenital cataractous
lens can be rescued by forming chimaeras between
DBA/2 (a noncataractous strain of mouse) and the
CAT mutant. This report describes the histological,
cellular and biochemical analysis of the resultant
chimaeric eyes, and discusses possible mechanisms by
which these results were achieved.
Introduction
doubling levels of the cataractous MLE is modified
(Lipman & Muggleton-Harris, 1982). These in vitro
studies suggested that a modification of the mitotic
activity of the Cafr lens epithelial cells and the
developmental defects in vivo might be possible.
Support for this concept is derived from the work
where a rescue of an abnormal developmental defect
in the retina (LaVail & Mullen, 1976) and the
formation of normal mosaics using (a) 'lethal' (t 12 /
t12) embryos, and (b) the X-linked lethal jimpy
(jpmsd) mutant, had been successfully achieved by
the aggregation of normal and mutant embryos to
form chimaeras (Mintz, 1964a; Eicher & Hoppe,
1973). A similar approach was used for trisomies 15,
16, 17 and 19, and the results demonstrated that the
trisomic cells could contribute to the normal tissues of
the developing chimaeric animals (Epstein, Smith,
Zamora, Sawicki, Magnuson & Cox, 1982; Cox,
Smith, Epstein & Epstein, 1984).
This paper describes how the dominant autosomally inherited congenital lens abnormalities of the
CAT mutant are obviated in the majority of live
young by forming chimaeric mice from the aggregation of early embryos of CAT and noncataractous
DBA/2 mice.
Hereditary cataracts are usually transmitted as an
autosomal dominant trait. Congenital cataracts are a
major cause of blindness in children, the prevalence
varying from 10-14% (Nelson, 1984). Several cataractous mouse mutants have been used to make
detailed studies at the genetic, cellular and molecular
levels. In a recent review the advantages of using
mouse mutants as model systems to study developing
cataracts in humans is discussed (Muggleton-Harris,
1986). One such mutant is the Cataract Fraser mouse
(Cfl^O; the inbred strain CAT is homozygous for the
Cafr gene causing congenital cataracts (MuggletonHarris, Festing & Hall, 1987). The nuclei of the deep
cortex fibres of the cataractous lens are pyknotic and
vacuolation and degeneration of the cortical fibres
occurs. The anterior epithelial lens cells show unusual
mitotic activity with the formation of multiple cell
layers that infiltrate into the fibres of the lens (Fraser
& Schabtech, 1962; Verrusio & Fraser, 1966; Zwaan
& Williams, 1968, 1969).
Somatic cell hybridization studies of cultured lens
epithelial cells from the cataractous mutant and
noncataractous mouse lens epithelial cells (MLE)
have shown that the phenotypic in vitro population
Key words: mouse, chimaeras, congenital cataractous
mice, lens defects.
474
A. L. Muggleton-Harris, K. Hardy and N. Higbee
Materials and methods
The CAT inbred strain of mouse is homozygous for the
Cafr gene causing congenital cataract. This strain had
been bred previously at WPI, Worcester, Mass., USA and
is now held at MRC Laboratories, Carshalton, Surrey, UK.
They are albino and have the strain-specific variant of the
glucose phosphate isomerase enzyme, GPI-1B, whereas the
DBA/2 noncataractous mouse is GPI-1A (Eppig, Kozack,
Eicher & Stevens, 1977). The lens abnormalities of the CAT
mutant begin to develop in utero at 15 days of gestation. The
inbred strain DBA/2 (noncataractous) mouse carries a
dilute brown coat colour, pigmented retina and has no
known lens anomalies (Altman & Katz, 1979).
(A) Recovery of embryos
Embryos were obtained from female CAT and DBA/2
mice after natural mating, or superovulation with intraperitoneal injections of 5i.u. pregnant mares' serum (PMS;
Intervet) followed 48 h later by 5i.u. of human chorionic
gonadotrophin (hCG; Intervet). The females were paired
overnight with CAT and DBA/2 males, respectively, and a
vaginal plug taken as an indication of successful mating on
the following day. 4- to 8-cell embryos were flushed from
the anterior portion of the uterine horns 48 h later with
prewarmed (37 °C) Medium 2, containing 4 mg ml" 1 bovine
serum albumin (M2 + BSA; Fulton & Whittingham, 1978).
The zona pellucida was removed by brief exposure of the
embryos to prewarmed acid Tyrode's solution (Nicholson,
Yanagimachi & Yanagimachi, 1975). The zona-free embryos were washed through three changes of fresh medium
before further manipulation.
(B) Aggregation of embryos
Techniques for producing chimaeras have been described
previously (Tarkowski, 1961; Mintz, 1962). In brief, two
embryos, one embryo of each genotype, were placed into a
small drop of 37°C Medium 16 + BSA (M16 + BSA, Whittingham, 1971) in a 60x15 mm plastic culture dish under
paraffin oil, previously equilibrated with medium. Contact
between the two embryos in each drop was accomplished
with the aid of a glass pipette. Embryos were examined
2—4h later to establish contiguity. In some instances phytohaemagglutinin (100 jig ml" 1 in M2, Type E, Sigma) was
used for 1-2 min, to assist aggregation of the embryos. The
following day the successfully aggregated morulae were
transferred into the uterus of pseudopregnant recipient Fj
(C57BL/6J female x CBA/Ca male) hybrid females. These
females were housed individually and allowed to deliver
their offspring.
(C) Examination of lenses in vivo
Hand-held slit lamp (Zeiss) observations were made on the
chimaeric CAT *-* DBA/2 offspring from 15 to 65 days
postnatally. Detection of vacuolation and minor disturbances of the lens fibres, plus abnormal proliferation of the
epithelial cells can be clearly detected. However, gross lens
abnormalities can be readily detected without the aid of a
slit lamp soon after the eyes are open, the lens is smaller
(shrivelled) and opaque in comparison with the DBA/2
mouse lens. Comparative observations with control CAT
and DBA/2 littermates, as well as CAT*-> CAT and
DBA/2 «-» DBA/2 offspring allowed an assessment of lens
abnormalities.
(D) Examination of the retinal pigmentation and lens
Following enucleation of the eye, the retina and lens of the
mice were examined by transmitted and reflected light on
the stage of a dissecting binocular microscope. A photographic record was kept of the distribution of pigment and
clarity of the lens. The lens could then be used for the
histological, cellular and biochemical analyses.
(E) Histological procedures
After cervical dislocation the eyes were enucleated from
control and experimental adult mice and fixed in Carnoy's
fluid (3:1 ethanol/acetic acid). The head region of embryos
at day 15 of gestation was similarly fixed. After dehydration
and paraffin embedding, serial sections (5 jjxn) of the tissue
were cut and stained with haematoxylin and eosin for
microscopical examination.
(F) Biochemical analysis of genotypes
The existence of chimaerism in tissues or cells was assessed
by analysis of strain-specific allelic variants of glucose
phosphate isomerase (GPI:EC5.3.1.9). These were analysed electrophoretically on small samples of blood, tissue
and cells of the donor, controls and experimental chimaeras. The isozymes were separated using a Titan HI
Zipzone cellulose acetate plate (Helena Laboratories,
Beaumont, Texas) with 0-025 M-Tris base, 0-192M-glycine at
pH8-5 for lh at 200 V. Following the application of 2 ml
1-5% agar overlay containing 2-0 ml of lM-Tris-HCl at
pH8-0, 1-0 ml of l m g m r 1 NADP, 0-1 ml of lOOmgrnP1
fructose-6-phosphate, 0-1 ml of 5-41 g 100 ml" 1 magnesium
acetate, 0-lml of lOmgrnl"1 dimethyl thiazolyl diphenytetrazolium bromide, 0-lml of 2-5mgmF 1 phenazine
methosulphate and 3 ji\ of glucose phosphate dehydrogenase, the GPI isozymes could be visualized.
(G) Photography
Photographs of the lens and retina pigmentation were taken
on the binocular microscope with Pan F film. Histological
specimens were photographed with Pan X with a Zeiss
photomicroscope.
Results
From a total of 334 aggregations formed between
cataract mutant and control embryos (CAT<->
DBA/2), 126 coat colour chimaeras were obtained
(Table 1). 71 of these had a coat colour ratio
distribution of each genotype of approximately 40:60,
50:50 or 60:40. The remainder of the chimaeras
obtained showed a wide range in the distribution of
each genotype which varied from 70:30 or 90:10 in
favour of one genotype or the other as shown in
Table 2. At least 61 % of the 71 overt chimaeras who
reached 60 days of age had two normal-sized clear
Rescue of lens defects in chimaeras
475
Table 1. Number of chimaeras obtained from aggretations of mutant and control embryos
No. of embryos
transferred
into recipients
334
20
20
83
67
No. of
live young
DBA/2 <->CAT
DBA/2 <-> DBA/2
CAT<-> CAT
CAT
DBA/2
No. of living young
showing coat colour
chimaerism
147 (44 %)
5 (25 %)
6 (30 %)
32 (39 %)
27 (40 %)
126(38%)
0
fl
0
0
Table 2. Approximate distribution of coat colour in
the 126 chimaeras
Nos
Ratio DBA/2 to CAT
9
24
26
28
17
4
IS
90:10
70:30
60:40
50:50
40:60
30:70
10:90
overt chimaeras
(71)
7%
3%
29%
B
Clear lenses in both eyes
1 clear lens, 1 lens developing
abnormalities after 60 days of age
1 clear lens, 1 congenital
cataractous lens
Congenital cataractous lenses
in both eyes
Fig. 1. Diagram showing the degree of rescue from the
congenital cataractous state in the adult (over 60 days of
age) CAT<-» DBA/2 chimaeras.
\
Fig. 2. (A) Three CAT**DBA/2 chimaeras selected to
show the range of coat colour distribution, with a
DBA/2«-» DBA/2 experimental control animal on the
left and a CAT littermate on the right. (B) A direct
comparison of the normal clear lenses of a
CAT <-» DBA/2 adult chimaera, which were rescued from
the congenital cataract, can be made with the small
'shrivelled' opaque lens of a CAT littermate.
noncataractous lenses, and 29 % had clear noncataractous lenses, with one, or sometimes both, lenses
developing abnormalities after 60 days of age. A
small percentage (3 %) of the mice had one clear
normal-sized noncataractous lens at 60 days of age,
and one small congenital cataractous lens. 7 % of the
chimaeric mice had congenital cataracts in both
lenses (Fig. 1). To control for the methods used for
the aggregation technique and embryo transfers, a
series of aggregates was made of DBA/2 «-* DBA/2
476
A. L. Muggleton-Harris, K. Hardy and N. Higbee
and CAT<-» CAT embryos. Control, nonaggregated
DBA/2 and CAT embryos were also transferred into
pseudopregnant recipients. The DBA/2 mice always
had clear normal lenses and the CAT mice developed
congenital cataracts (Table 2).
The range of coat colour distribution of the chimaeric mice can be appreciated when comparing them
with the control DBA/2 or CAT mice in Fig. 2A.
The eyes of a chimaera with clear normal-sized lenses
can also be compared with those of a congenital
cataractous littermate (Fig. 2B).
The histological studies showed a number of differences between the embryonic congenital cataractous
lens and the normal DBA/2 lens as can be seen in
Fig. 3A,B. The nuclei of the mitotic bow are disturbed. Early vacuolation and swelling of the fibres
can be clearly discerned at 15 days gestation. Adult
lenses are difficult to section without some tearing of
the tissue; however, these artifacts are easy to identify in comparison with the vacuolated pathology of
the cataractous lens. Fig. 3C shows the vacuolated
and swollen cells of the cortex fibres. Fig. 3D shows
the pyknotic nuclei and multilayering cells of the
anterior epithelial cells. The cortex fibres are nonaligned, the nuclei of the lens have dissolved and in
many places have been replaced by large aqueous
filled areas.
Fig. 4A shows the GPI isozyme distribution in a
representative sample of 39 clear lenses from adult
chimaeric mice that had been rescued from the
congenital cataractous state. The lenses were of
normal size and slit lamp observations did not detect
any defects prior to 60-70 days of age. The ratio of
GPI isozymes of both the CAT and DBA/2 genotypes is well represented in a number of the lenses. It
is interesting to note that those adult chimaeric lenses
Fig. 3. (A) Histological section of the eye region of a 15 days gestation DBA/2 embryo. The ectoderm (e), which will
form the anterior epithelium of the cornea in the adult mouse, overlies the anterior epithelium of the lens (ae). The lens
cavity (Ic) has developed and the retina has been formed from the outer layer of the optic cup (r). The elongated lens
fibres stretch from one lens surface to the other (/). The nuclei (n) of the fibres form a broad curved row across the
lens. Bar, 120fan. (B) Cross section of the eye region of a 15 days gestation CAT embryo. The abnormalities of the lens
structure are already evident, the fibres are irregular and there is evidence of swelling in the cortex region (sf). The
nuclear bow pattern is disorganized. Bar, 120/an. (C) Cross section of an eye of an adult (60 days of age) CAT mouse.
The nucleus of the lens has dissolved, degeneration of the fibres has occurred and vacuoles (v) are evident. The nuclei
of the central fibres are pyknotic, but in the periphery, at the germinative bow region (gvb), normal fibres can be seen.
The anterior lens epithelium (ae) cells are multilayering in the central region, however they still maintain a monolayer
closer to the germinative bow region. Bar, 600 pm. (D) An enlargement of the central anterior area of the lens shown in
C. The multilayering of the anterior epithelial cells (mle) and the dissolved cortex fibres (dc) are very distinct. Pyknotic
nuclei (pn) of the disorientated fibres are found in the cortex. Much of the lens lacks cell structure with large vacuoles
(v) throughout. There are some fibres (/) in the periphery of the lens. Bar, 120/im.
Rescue of lens defects in chimaeras
o
Z
n±ls.D.
I
Ml
B
1
a/GPI (DBA)
b/GPI (CAT)
Ratio of GPI/a to b
Fig. 4. (A) Shows the glucose phosphate isomerase
(GPI) distribution in a representative (72 %) clear lenses
from adult CAT*-*DBA/2 chimaeras. The ratio of
DBA/2 (GPI-1A) to CAT (GPI-1B) isozymes is well
represented in a number of lenses. (B) The ratio of
CAT: DBA/2 isozymes in those lenses from the
chimaeras that developed abnormalities after 60 days of
age was approximately evenly distributed in a sample
population of 16 lenses.
that developed lens abnormalities beyond 60 days of
age tended to cluster in one area of the GPI distribution pattern, where both CAT and DBA/2 GPI
isozymes are well represented (Fig. 4B). In those
chimaeras with congenital cataracts no GPI-1A
(DBA/2) was detected.
The degree of DBA/2 pigmentation of the retina in
the overt chimaeric mice with clear lenses was noted
when the eye was enucleated prior to GPI analysis or
histological procedures (Fig. 5). There appeared to
be an approximate correlation between the ratio of
GPI isozymes in the lens and the distribution of
pigmented and nonpigmented retina epithelial cells.
The degree of pigmentation of the iris and retina
could also be seen in the histological sections of the
chimaeric lenses. Those lenses that had remained free
of any congenital defects had a normal morphology
and patches of pigment can be seen in the iris
Fig. 5. The degree of DBA/2 pigmentation of the retina
in the overt CAT <H> DBA/2 chimaeras was noted prior to
analysing the lens for GPI isozyme distribution. The
anterior and posterior orientation of these sample lenses
shows the pigmentation patterns.
(Fig. 6A,B). Those chimaeras rescued from congenital cataracts but developing lens abnormalities in later
life (60-70 days of age) show a number of the typical
cataractous lens defects, e.g. disturbed lens fibres,
pyknotic nuclei, vacuoles and/or multilayering of
the lens epithelial cells (Fig. 6C). A section of the
CAT lens at 70 days of age allows a comparison
to be made of the abnormal congenital cataractous
cell morphology; the pigmentation and normal lens
morphology of the DBA/2 mouse can be seen in
Fig. 6D,E.
Discussion
We have produced phenotypically normal chimaeras
with significant proportions of the congenital cataractous genotype in a variety of tissues including the
lens, retina, also in blood and coat colour. Furthermore, the majority of CAT «-> DBA/2 chimaeras did
not develop lens anomalies that could be detected by
slit lamp observations or histological studies. Ultrastructural or biochemical anomalies, if any, did not
result in a loss of transparency, biological function or
cell regulation. The normal diffraction properties of
the lens indicate that the delicate osmotic balance and
crystallin synthesis is being maintained by the chimaeric lens cells. Although the lens differentiates from
the surface ectoderm by a series of inductive processes culminating in an association with the optic
vesicle, the developing lens grows through division of
478
A. L. Muggleton-Harris, K. Hardy and N. Higbee
epithelial cells initially throughout the epithelium.
Later it is only the germinative zone located in the
equatorial region of the epithelium which continues
to replicate and the initial cells of the developing lens
are retained within the inner cortex fibres of the adult
lens. We therefore conclude that the correction of the
developing lens congenital defects in the chimaeras
occurred during organogenesis. Cells of the lens
epithelia, fibres and retina of the embryo and adult
chimaera eye contain both CAT and DBA/2 genetic
cell markers. This is not surprising in that the melanocytes of the pigmented epithelium are formed from
the outer layer of the optic cup which is derived from
the optic vesicle.
Control of cell division in the lens remains an
important developmental problem and has been
Fig. 6. (A) Normal morphology of an adult lens from an overt 'rescued' chimaera can be seen in this section. The
mitotic bow nuclei (mbn) are well regulated and of normal size. The monolayered lens epithelial cells (me) are
undisturbed and the lens fibres (//) intact. The patches of DBA/2 pigment can be seen in the iris (p). Bar, 120fan. (B)
High-power photograph of another 'rescued' chimaeric lens shows the details of pigment (p) in the cells of the iris and
lens epithelial cells (me). Bar, 48 fan. (C) A 'rescued' chimaeric lens that developed abnormalities after 60 days of age.
The cortex fibres (cf) are disturbed, multilayering of the lens epithelial cells (mle) and pyknotic nuclei (pn) are typical
of cataractous lenses. Bar, 120 fan. (D) A section through a DBA/2 lens showing the normal morphology at 60 days of
age. The intense pigment (p), monolayer of lens epithelial cells (ec), and normal nuclei (n) and fibres (cf) can be seen.
Bar, 48fan. (E) A similar section through a CAT lens at the same age. Lack of pigment, abnormal lens fibres (cf),
vacuoles (v) and pyknotic nucleic (pn) are seen. Bar, 48 fan.
Rescue of lens defects in chimaeras
shown to be a significant factor in hereditary cataract.
The manner in which the regulation of cell replication
has been achieved in our experimental chimaeras has
not been determined. The data presented in this
paper suggest a positive regulating process. Possibilities that may be considered are (1) that there are
sufficient numbers of noncataractous cells and/or
components in the developing chimaeric lens which
induce the congenital cataractous cells to replicate in
the normal manner, or (2) that the CAT cells could
have been at a proliferative disadvantage and the
DBA/2 cells had colonized the tissue. However, our
results have shown that the CAT cells form a significant proportion of the adult chimaeric lens. The high
proportion of the GPI-1B (CAT) genotype in the
clear chimaeric epithelium suggests that the abnormal
phenotypic replication associated with the cataractous lens MLE has been modified.
As the noncataractous and CAT embryonic lens
cells do not hybridize in vivo, the exchange of cellular
components and information would have to be
achieved by intercellular communication. The receptors on the cells' surface or the extracellular matrix
may play a role in regulating the cells' behaviour. We
have recently shown that subpopulations of cultured
MLE cells can respond to the stimulus of a piece of
the collagen lens capsule and synthesize the collagen,
glycoproteins and proteoglycan components required
for further lens cell differentiation to take place in
vitro (Muggleton-Harris & Higbee, 1987).
It has been suggested that gap-junctional communication plays an important role in regulating and
coordinating cellular growth (Pitts & Finbow, 1977;
Lowenstein, 1979; Sheridan, 1977; Wolpert, 1978;
Spray, Harris & Bennett, 1982). Preferential coupling
may have taken place in the cells of the chimaeric lens
during embryogenesis, and play an important role in
regulating cell growth, lens morphogenesis and response to growth factors (Rothstein, Worgul & Weinsieder, 1982; Goodall, 1985). Further data suggesting
that a positive regulating process has contributed to
the rescue of the experimental lens from the congenital cataractous state are the results that show that the
lens epithelial cells of the adult chimaeric lens have a
large proportion of CAT cells present. These cells
would have proliferated during embryogenesis similarly to the cells of the lens in the dominant congenital
cataractous mouse, had their replication not been
regulated by the presence of the noncataractous cells
and/or their components.
With regard to those adult chimaeras whose lenses
developed abnormalities at various times after 60
days of age, the results have indicated that these
occur when approximately equal distributions of
CAT/DBA/2 components are present (Fig. 4B).
Epstein (1985) has recently speculated on a variety of
479
ways in which imbalance of a locus could occur; he
suggests for example that changes in the concentrations of gene products, such as rate-limiting
enzymes, could play a role. Regulatory molecules,
involved in surface recognition and adhesion
phenomena, and receptors concerned with intercellular communication may disturb the functions of
the locus. If such molecules could alter or be diluted
with age in the adult chimaeric lens, then lens
abnormalities could occur.
The results from these experiments have shown
that a rescue from the congenital cataractous state has
been achieved in the majority of chimaeras. This
rescue must have taken place at the embryonic
cellular level, because those cellular defects associated with the congenital cataractous lens are easily
detected at the slit lamp and histological level. The
unusual mitotic activity of the epithelial cells and
vacuolation of the fibre cells, associated with the
embryonic CAT lens epithelial cells, would be
detected very easily. Also, a high percentage of the
chimaeras have a substantial amount of the CAT GPI
genotype in their lens tissue, therefore CAT cells
form a significant part of the epithelial and fibre cells
and yet cannot be differentiated morphologically
from the DBA/2 noncataractous lens cells.' The
'rescued' chimaeric lens differentiates and develops
in a normal manner without the dominant congenital
cataractous defects occurring.
We wish to acknowledge the partial support for this work
by a grant made to A. L. Muggleton-Harris from the
National Eye Institute, NIH, USA. We also thank Andy
Brammall and Eleanor Rawlings for their technical help.
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