Chapter-I

Review of literature and genesis of the Thesis

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1. Cataract and its Etiology

Cataract can be simply defined as loss of transparency of the crystallin lens, in

which the refractive index of the lens differ significantly over distances approximating

the wavelength of the transmitted light. Variation in the refractive index over these

distances can result from changes in lens cell structure, changes in lens protein

constituents, or both. Cataracts are generally associated with alteration of the lens

micro-architecture. When mutations in crystallin proteins are enough to cause

aggregation they usually lead to congenital cataract, however if they merely make

them susceptible to environmental insults such as light, hyperglycemic or oxidative

damage they might attribute to age-related cataract (1). The ongoing epidemiological

studies have figured out few risk factors such as UV-B exposure, low antioxidant

intake, certain medications, cigarette smoking, diabetes, gout as well as family history

in the development of cataracts (2). All these mechanisms result in the structural

changes in the α-crystallins. From αA-crystallins, Cys 131 and Cys 142 are present in

transparent human lenses as a mixture of cysteine sulfhydryl and half-cysteine

disulfide groups, whereas cataractous lenses were lacking cysteine sulfahydryl group

(3).

In contrast to age related forms of cataracts, early childhood cataracts occur in

developed countries with a frequency of 30 cases in 100, 000 births; with further 10

cases being detected by the age of 15 years (mainly as dominant forms). In developing

countries these rates are likely to be higher because of rubella infections and

consanguinity (for the recessive forms; 4)

2. Classification of Cataracts

Cataracts are broadly divisible into two major groups based on etiological factors.

1. Developmental or congenital cataract in which the normal development of the lens

is affected by genetic, nutritional or inflammatory changes.

2. Degenerative cataract includes senile cataract and are associated with radiation or

systemic diseases.

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3. Subtypes of Cataracts

Cataracts are also classified according to the location of opacity (Fig.1) in the affected

lens and to the specific factors involved in their formation. The cataracts are further

subdivided into seven subtypes.

Fig: 1.1. Schematic illustration of mouse eye lens showing locations of the lens opacity at different parts of the lens. The cataracts are named as per their location in different parts of the lens. Anterior cataract (AC), Posterior cataract (PC), Nuclear cataract (N), and Equatorial cataract (EC).

1. The opacities are located at the anterior or posterior pole of the lens in anterior-

polar and posterior-polar cataract respectively.

2. In Zonular nuclear cataract, opaque cells are deposited in the lens surrounded by

clear zones. This type of cataract may lead to slight or significant impairment of the

vision depending on the density of the opacity.

3. Nuclear or central cataract is a rare type in which the opacity affects the embryonic

nucleus of the lens and cause little damage to the vision.

4. Small round opacities scattered irregularly throughout the nucleus and cortex in the

punctuate type of cataract. Occasionally these opacities appear light blue in color and

consequently, this type is sometimes called caerulean cataract.

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5. The galactosemic cataract is associated with a hereditary anomaly of lactose

metabolism. In this type a central portion of the lens is visible as a transparent portion

within a largely opacified lens.

6. The development of cataract is associated with a mutation of the L-ferritin gene

which codes for an iron storage protein in hereditary hyperferrintinaemia cataract

syndrome (HHCS).

7. Senile cataract is the commonest type to develop after the age of 50. This type of

cataract is usually bilateral and begins either in the superficial cortex or close to the

nucleus of the lens.

4. Therapeutic Measures of Cataracts

Medical remedies have been inaccurately advertised as effective for the treatment of

cataracts. In fact there is no proven medical treatment available to reverse or slow the

progression of, or prevent the formation of a cataract. Surgical removal of the opaque

lens is the only possible remedy both in animals and humans, and often provides a

return of functional vision. Various surgical procedures available require meticulous

and precise microsurgical techniques. Surgery is performed using an operating

microscope and sophisticated microsurgical instruments. Two techniques are currently

used to remove cataract from the eye. One method involves making a large incision

into the eye and removing the affected lens. A newer advance method i.e.

phacoemulsification, allow for small incision surgery, and may prove a better result.

This technique involves placing a small ultrasonic instrument into the eye. This

instrument generates about 40,000 tiny vibrations per second to fragment the cataract.

The lens material is then aspirated out of the eye, much like a small intraocular

vacuum (5). This sophisticated technique has been associated with a higher success

rate for many cataract surgery patients.

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5. Mutant Mice Models in Cataract study

Significant progress in understanding the genetics of human congenital cataracts could

be possible due to the intense cellular and molecular analysis of the animal models

with congenital eye defects. Murine cataract mutants are excellent models for detailed

studies on the process of lens opacification. These mouse models allow one to study

all stages of the embryonic development. Hereditary mouse cataract models have great

relevance to humans because it is estimated that congenital cataracts comprise

approximately 10% of visual loss in humans.

For decades the target of the developmental biologists has been to focus the

development of ocular structures. With the advent of modern genetics, now it becomes

possible to characterize mutation that causes human disorder and their comparison

with mutations in animal models. The outcome of this comparative study revealed that

there is common genetic network that underlies eye development in flies, mice and

human.

Over past decades few spontaneous mutant animal models with congenital eye

diseases have been identified and studied for their phenotypic and genotypic details.

With the availability of recombinant DNA technology several targeted mutations were

created in mice to study the function of genes in the mammalian eye development.

Extensive characterization of these models has contributed significantly towards the

understanding of vertebrate lens development, lens physiology and mechanism that

underlie the developments of cataracts in humans and animals.

A variety of mouse mutagenesis technologies, both gene- and phenotype-driven, are

being used to explore systematic and comprehensive approaches to mammalian gene

function studies (6). One of the major groups at the GSF - National Research Center

for Environment and Health, Institute of Developmental Genetics, Germany, has been

actively involved in mutagenesis program for past several years and used

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ethylnotrosourea (ENU) as a mutagenic agent to generate mutation in mice (7). They

initiated a systematic approach to identify murine cataract mutations (8) and collected

about 150 lines of independent origin and distinct phenotypes (9, 10). Some of them

have been characterized for the molecular alterations. The Cat2 mutant family

represents the largest group among Neuherberg cataract collection center.

Table-1.1. Mutant alleles coding for α-Crystallin proteins involved in congenital cataracts. Name Allele

Symbol Phenotype Molecular

Changes Protein Functions Ref.

Cryaa CryaaL Recessive progressive

opacity

Targeted deletion Loss of function 11

Lop18 Recessive nuclear & cortical

cataract

R54H n.d. 12

Aey7 Nuclear cataract V124E n.d. 13

CRYAA Recessive cataract W9X Loss of function 14

CRYAA Dominant nuclear cataract R49C Abnormal nuclear cataract 15

CRYAA Dominant nuclear cataract R116C Increased membrane

binding capacity

16

Cryab CryaaL No ocular phenotype Targeted deletion Loss of function 17

CRYAB Myopathy with cataract R120G Irregular structure 18,19

Posterior polar cataract 450delA Aberrant protein 20

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Table-1.2. Mutant alleles coding for β-Crystallin proteins involved in congenital cataracts. Name Allele

Symbol Phenotype Molecular

Changes Protein Functions Ref.

Cryba1 Po Progressive cataract Splicing intron6

W168R;∆W168

4TH Greek key motif 21

CRYBA1 Zonular cataract Splicing intron 3 n.d. 22

CRYBA1 Pulverulent cataract Splicing intron 3 n.d. 23

CRYBA1 Lamellar cataract G91del Reduced solubility 24,25

CRYBA1 Cataract Splicing intrin n.d. 26

CRYBB1 Pulverulent cataract G220X Reduced solubility 27

Crybb2 Philly Progressive cataract 12-bp deletion in

exon 6

4TH Greek key motif 28

Aey2 Progressive cataract V187E 4TH Greek key motif 29

CRYBB2 Cerulean cataract Q155X n.d. 30

CRYBB2 Coppock-like cataract Q155X n.d. 31

CRYBB2 Suture cataract & Cerulean

opacity

Q155X n.d. 32

CRYBB2 Central nuclear cataract W151C Loss of solubility? 33

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Table-1.3. Mutant alleles coding for γ-Crystallin proteins involved in congenital cataracts. Name Allele

Symbol Phenotype Molecular

Changes Protein Functions Ref.

Cryga-f Several

alleles in

mice

Various types of cataract Missense,nonsense

,deletion/insertion

Mainly Greek key motif

affected

34

CRYGC Coppock-like cataract T5P Folding Impairment 35

CRYGC Zonular-Pulverulent cataract Insertion, 52 new

amino acids

Hybrid protein 36

CRYGC Lamellar cataract R168W 4TH Greek key motif 37

CRYGD Punctate Progressive cataract R14C Alter surface protein 38

CRYGD Lamellar cataract P23T n.d. 37

CRYGD Cerulean cataract P23T 1st Greek Key motif, altered

folding & solubility

39

CRYGD Cataract P23T n.d. 26

CRYGD Coral-shape cataract P23T Less soluble 40

CRYGD Fasciculiform cataract P23T n.d. 41

CRYGD Prismatic crystals R36C crystallization 42

CRYGD Aculeliform cataract R58H Folding Impairment 35

CRYGD Central-nuclear W158X 4TH Greek key motif 37

n.d., not detected. (Courtesy, Graw, J. 2004. Int. J. Dev. Biol. 48: 1031-1044).

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6. The Eye Development in Vertebrate Animals

The eye is a very complex structure that originates from primordial tissues derived

from a number of sources, including the wall of diencephalon, the overlying surface

ectoderm and immigrating neural crest cells. Normal eye development begins with an

evagination of neural ectoderm to form the early optic vesicle. The optic vesicle

approaches the surface ectoderm leading to the neural tissue and head ectoderm

appears to be in close contact. Lens development starts by the invagination of the lens

pit at the places of the lens placode at both sides of the prospective forebrain. This

initial process takes place in the mouse embryo at day 9.5 of embryonic development.

The lens placode begins to form the lens pit and subsequently the lens vesicle (LV) at

E11.5. From the posterior wall of the LV primary lens fibers (PLF) cells grow into

lumen and fill over the lens cavity by the end of 13th day. From that time on, a life

long process of formation of secondary fiber cells is initiated. The equatorial zone has

been established and secondary lens fibers (SLF) are being laid down. As long as the

lens grows, new SLF move in from the equator onto the outer cortex of the lens. The

lens continues to develop throughout life. During the process of terminal

differentiation of the lens fiber cells, all cell organelles are degraded, resulted finally

to cells without nuclei and mitochondria in the center of the lens. The development

process is completed for the mouse lens two weeks after birth, when eye lids are

separated (43).

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A B

Fig. 1.2: Histological view of vertebrate eye lens and lens fiber cells. The lens nucleus is located at center place, surrounded by lens cortex. The single layer of epithelial cells is present at the anterior site of the lens which is mitotically active (A). The primary lens fibers are arranged in the center of the lens and surrounded by secondary lens fiber cells (B).

7. Molecular Biology of Eye and Lens Development in Mice

Lens-cell differentiation occurs at a fairly early stage of embryogenesis and results in

very simple tissue architecture. These features allow the embryonic lens to provide a

paradigm of tissue development starting from tissue induction to tissue maturation

(44). Over the past decade, numbers of genes and transcription factors participating

during the various stages of lens development, along with their actual functions have

identified by using modern genetic and tissue manipulating tools.

7. 1. Transcription Factors in Eye Development and their Mutations

The eye develops through a temporally and spatially regulated pattern of

differentiation, coordinated by several growth and transcription factors. Several

transcription factors are implicated in the regulation of these processes. Some are

discovered as regulators of crystallin expression whereas others are found in different

context.

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PAX6

PAX6 is a transcriptional protein, crucial for early eye determination (45), the

specification of ocular tissue and normal eye development in vertebrates (46). Pax6,

mutation affects various alleles of Small eye (Sey) in mouse and rat as well as in the

human inherited diseases known as Aniridia (45, 47, 48) and Peter’s anomaly (49).

Walter Gehring’s group in 1995, showed ectopic expression of the mouse Pax6 that

induces formation of functional ommatidal eye in Drosophila antennae or legs (50).

This suggests that the homologous genes, i.e. eyeless from Drosophila and Pax6 from

the mouse share the same functions. Pax6 function seems fairly well conserved

through the animal kingdom as Pax6 can rescue genetic defect of eyeless, a

Drosophila homologue of small eye (pax6), required for development of the

compound eyes which are analogous to retinas of vertebrates (45, 50). Pax6 is

expressed both in the optic vesicle/retina and in the lateral head ectoderm to receive

inductive signals from the optic vesicle. Expression of Pax6 in the latter is

independent of the former, initiating far earlier than the optic vesicle apposition, and is

not affected by the removal of the optic vesicle (51). Pax6 continue to be expressed in

the entire lens vesicle and when the lens mature, its expression is largely confined to

the lens epithelium (51, 52), arguing that the pax6 function in the lens is related

primarily to the nature of the epithelial cells.

Pitx3

The second important transcription factor involved in early eye development is Pitx3.

In the mouse mutant aphakia (53), the promoter of the Pitx3 is affected by two

deletions (54, 55). The phenotype is characterized by a small lens vesicle with a stable

contact to the cornea, progressed with degradation of lens vesicle leading to eye

without lens. In contrast to the mouse condition, mutation of pitx3 gene caused

congenital cataract in the human counterpart (56). Pitx3 is strongly expressed in the

developing lens vesicle at day 11 of embryonic mouse eye development, and later in

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the anterior epithelium and equator region (57). There are recent reports that Pitx3 is

also expressed in the domaminergic neurons of the substatia nigra in the brain (58).

Maf, Sox, Fox and Eya

Some other genes coding for transcription factors involved in the eye and lens

development are Maf, Sox1, Sox2, FoxC1 and FoxE3. Particularly, Maf and Sox 1 act

as transcription factors on the promoters of the γ-crystallin encoding genes. It was

demonstrated by several authors that L-Maf is involved in the regulation of crystallin

expression in chicken and Xenopus. L-Maf is first expressed at the lens placode and is

maintained in specifically in lens cells (59). The targeted deletion of c-Maf in the

mouse, the third mammalian member of the Maf family, stops primary lens fiber

elongation at the lens vesicle stage (60).

Sox

The Sox family of transcription factors has a high mobility group (HMG) domain in

common. The Sox1, Sox2 and Sox3 genes belong to subgroup B of Sry gene (61). Sox2

is expressed during early eye development in the lens placode; a portion of ectoderm

invaginates to form the lens vesicle. This invagination coincides with the onset of

Sox1 expression in the mouse lens placode. At latter stages, Sox2 is regulated and Sox1

come into action (62). A targeted mutation of Sox1 caused microphthalmia and

cataract in mice. Mutant phenotype is characterized by the failure of fiber cells

elongation associated with complete absence of Cryg transcripts (63).

Eya

Another mammalian family of genes has close relationship to a Drosophila gene: eyes

absent. In mouse and man four members belong to this family (Eya1-4). Only one

mutation reported in human Eya gene leads to formation of cataract together with

Peter’s anomaly and nystagmus (64).

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Six

This factor is encoded by the vertebrate homologue of Drosophila’s Sine oculis which

is expressed from the lens placode stage till the development of lens and it’s

expression decreases as lens develops (65,66).

In addition, other several genes, e.g. Shh, rx/eyeless, Lhx, Bmp4 or Bmp7, though to be

involved in the early stages of eye development, however, the phenotype produced in

targeted deletion are showing anophthalmia or microphthalmia but no cataracts.

Fibroblast growth factors

The presence of transcripts of three subtypes of fibroblast growth factors (FGF1,2,3)

in the developing optic cup and vesicle is critical for the lens fiber differentiation and

their subsequent survival (67).

7. 2. Crystallin Genes in Eye Development and their Mutations

The vertebrate eye lens is a unique, highly transparent and flexible organ, derived

from only one type of cell. The eye lens contains large amount of proteins (~30-35%),

reducing the water content to ~60-70% which is usually approximately 95% in the

cells. Lens cells synthesize abundant amount of crystallin proteins. In the adult lens,

crystallins are accumulated at high concentration in soluble form which keeps the

transparency and high reflectivity of the lens. The crystallins are classified into major

sub classes, i.e. α, β, γ and δ-crystallins, according to their decreasing molecular

weight and increasing isoelectric point of the native proteins. Delta crystallin is

present in the lens of avian and reptilian animals replacing γ-crystallins found in other

vertebrate species. These crystallins are very tissue specific and expression of most of

the crystallins is limited to lens and few other tissues and expression of the genes

coding for crystallins is good indication of lens differentiation (68). The crystallins

have been characterized with respect to their genetic organization, the regulation of

their expression pattern and their participation in several diseases. Evolutionary

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analysis has demonstrated the relationship of crystallins to proteins, implicated in the

protection against stress.

Expression of α--crystallin genes begins at the lens vesicle stage, whereas β and γ-

crystallin usually expressed with the lens fiber differentiation. Expression of Delta

crystallin is initiated at the placode stage and, hence, serves as a marker for

transcriptional regulations occurring early in the lens cell differentiation in response to

the induction by the optic vesicle. The expression of β, γ and δ–crystallin genes is

presumably augmented through the same mechanism in the lens fiber cells.

Table-1.4. Major Crystallin genes in the vertebrate eye lens. Gene Mouse

Chr.

Proteins Mol.

weight

Expression in

Tissue

Functions

Cryaa 17 αA-Crystallin

αAins-Cryst.

20 kDa

25 kDa

lens, spleen Structural proteins,

Chaperone, Autokinase &

gene activator

Cryab 9 αB-Crystallin 22 kDa lens, heart, brain,

muscle & kidney

Heat shock proteins

Cryba1 11 (46) βA1/A3-crystallin 23/25

kDa

Lens Structural proteins

Cryba2 1 (41) βA2-crystallin 22 kDa Lens Structural proteins

Cryba4 5 (59) βA4-crystallin 22kDa Lens Structural proteins

Crybb1 5 (59) βB1-crystallin 28kDa Lens Structural proteins

Crybb2 5 (60) βB2-crystallin 23 kea Lens Structural proteins

Crybb3 5 (60) βB3-crystallin 24 kDa Lens Structural proteins

Cry b2 Pseudogene Lens Structural proteins

Crygs ? γS-crystallin 20 kDa Lens Structural proteins

Cryga-

Crygf

1 (32) γA-γF-crystallin 20 kDa Lens Structural proteins

*cM position according to the Mouse Genome Database (1997) in brackets.

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α-Crystallins

The α-Crystallin represent the major class of water soluble proteins in the lens

(~30%), which is composed of two related proteins, αA and αB crystallins and are

encoded by two genes, Cryaa and Cryab. The molecular mass ranges between

approximately 800-1000 kDa; the isolated subunits have molecular masses of 20 and

22 kDa (αA and αB crystallin respectively) and isoelectric points of the native

proteins were known ranging from 4.5 to 5.0 (69,70). The α-Crystallins are widely

accepted as the structural proteins and the object of a variety of post-translational

modifications, e.g. truncation, glycosylation, glycation, carbamylation and acetylation

(71).

Sequence comparison revealed homology between the small heat shock proteins from

Drosophila and the α-Crystallin proteins (72). The most exciting finding of (73)

concerns the function of α-crystallin as a molecular chaperone and demonstrated that

α-crystallin prevents thermal aggregation of several enzymes and β/γ-crystallins. It

binds denatured protein and keeps it in solution. The chaperone activity is necessary

for the lens because degradation and extrusion of defective proteins is not possible as

it is in other tissue. Moreover the lens is exposed to variety of hazardous agents, in

particular light of various wavelengths, which leads to perioxidative effects of quite a

number of lens proteins.

Variations in the response of Cryab expression were observed against heat-shock and

oxidative stress, in ocular trabecular meshwork. A transient change in mRNA

mobility was observed only after heat-shock (74). The other important aspect of α-

crystallin concerns its participation in the intracellular architecture via cellular

filaments. The lens fiber cells exhibit a unique filamentous polymer, called beaded

filaments, when analyzed by electron microscopy. It is formed by a lens specific

cytoskeletal protein, referred to as CP49, and α-crystallin (75). αA-crystallin also

interacts with tubulin (76) and actin (77). They encoded by two genes Cryaa and

Cryab, which are located on different chromosomes. Cryaa and Cryab are expressed

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in a variety of species, e.g. mouse, man, hamster, rat, chicken and rabbit (Table 1).

Both genes contain three exons of similar size.

It is now widely accepted that several diseases are accompanied by alterations in α-

crystallin. Brady et. al. 1997 (78) reported an opacifiction of the lens in mouse

knocked out for α- A-crystallin gene. The targeted disruption of α-A gene produced

lens opacities, resulting from the inclusion bodies containing α-B crystallin. This

finding was supported by reporting new lens opacity gene (lop18). In the homozygous

mutant animals, tiny vacuoles were visible in the lens cortex at E14; these vacuoles

become more prominent at E16, and continued the cataract development even after

birth. There was prominent degeneration of the cortex, migration of the lens epithelial

nuclei and formation of abnormal lens fiber at posterior pole of the lens at the final

stage 4 months after the birth (79). Alterations in α- B crystallin, which was also

expressed outside the lens are known to be associated with a broad variety of

degenerative neurological diseases. Duguid, et. al. (80) reported accumulation of α-B

crystallin in scrapie-infected hamster brain cells and latter in human brain cells having

Creutzfed-Jacob disease (CJD).

β-Crystallin

The β-crystallin polypeptide is a member of β-crystallin superfamily. According to

original finding of three main members of the lens crystallin proteins, the β-crystallin

were characterized as oligomers (the molecular mass of the monomers is between 22

and 28 kDa) with native molecular masses ranging up to 200 kDa for octameric forms

and general isoelectric point ranging between 5.7 and 7.0. Biochemically, β-crystallin

are also characterized by blocked N-termini. The β-crystallin is composed of two

domains built up by two Greek key motifs each and individual Greek key motifs are

encoded by separate exons (Fig.1.3). The β-crystallin can be divided into two

subgroup such as βA and βB-crystallin. Each subgroup is encoded by three genes

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Cryba1, -2, -4; Crybb1, -2, -3. Cryba1 encodes two proteins such βA1- and βA3-

crystallin.

The expression of the β-crystallin starts from the early eye development and continue

to increase after birth to accumulate at high concentration in the lens cortex. However,

the pattern of expression among the individual β-crystallin varies. Northern blot

analysis showed that all Cryb genes in rats were expressed from E13 onward; the

Crybb3 reaches it’s highest level around birth and drop down to background level

after 6 months; the Cryba1 transcript are maximally present two months after birth

reach back ground level at the age of 8 months (81). Recently βB2-crystallin was

detected outside of the lens in mouse and cat neural and pigmented retinas as well as

in cat iris (82).

Philly mouse is one of the well studied inherited cataract mutant mice which lack a

functional mRNA responsible for the Crybb2 (83). The corresponding cDNA from

Philly lenses contains an in-frame deletion of 12 bp resulting in a loss of 4 amino

acids. The deleted region is very close to carboxy-terminus and is responsible for the

formation of the tertiary structure of the βB2- crystallin (84) suggesting that the

morphological alterations produced in the Philly mouse are the consequences of

mutation in Crybb2 gene.

In human two cataract mutations are currently thought to be associated to the β-

crystallin encoding genes. A linkage of gene causing autosomal dominant zonular

cataracts and Y sutural opacities to human chromosome 17q11-12 is reported in three

generation family (85). The CRYBA1 gene is localized in this region and therefore

serve good candidate gene for this diseases. The second one, a cerulean blue cataract

is described in a large family as a dominant inherited disorder. This disease was

closely linked to the region of human chromosome 22, where two gene encoding β-

crystallin CRYBB 2 & 3, and a pseudo gene CRYBB2 I (86). In addition, βB2 –

crystallin is also known to be involved in age-dependent cataract formation. The

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formation of disulfide bonds between Cys37 and Cys66, only in human senile nuclear

cataract (Gade IV), but not in normal lenses have been demonstrated (87).

γ -Crystallins

The γ-crystallin polypeptide is a member of γ-crystallin superfamily. The native γ-

crystallins protein are characterized as monomers with a molecular weights of 20 kDa

and a free N-terminus. They are the most basic crystallins in mammals, with

isoelectric points ranging from 7.1 to 8.6 (88). A γ-crystallin protein is composed of

two domains built up by two Greek key motifs each, encoding two motifs by one exon

only (Fig. 1.3). The γ-crystallin encoding genes are (Cryg) are localized as a cluster of

6 genes (γA-γF-crystallin: Cryga-Crygf). This gene cluster is found on mouse

chromosome 1 (89) and on human chromosome 2 (90). The six genes are very similar

and the protein sequences, which are deduced from the mouse Cryge and Crygf genes,

are even identical (91). A Cryg gene is composed of three exons; the first exon

contains only 9 bp, followed by a short intron of about 100 bp. The second exon (243

bp) and the third exon (273-276 bp) are separated by a large intron of about 1-2 kb

(Fig.1.4). In mouse lens Cryg genes are expressed from E 13.5 onwards in primary

lens fibers and latter on in the secondary lens fiber cells (92, 93). At the first weeks

after birth, the expression of Cryg genes reaches to maximum level (94). The decrease

of Cryg genes expression could be related with an increase in methylation of the 5’

regions of various rat Cryg genes (95). In human, the expression of Cryg genes

observed during the prenatal development, because different time period of mouse and

human intra-uterine life.

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Domain I Domain II

MIotif I Motif II MIotif I Motif II

Fig.1.3. Greek Key motifs of β/γ-Crystallins. The general structure of the 4 Greek Key motifs in the β/γ-Crystallin superfamily is schematically indicated. The motifs are built up by symmetrically, twisted antiparallel β-sheets. The N- and C-terminal extension varies among the subfamilies. In the β-crystallin, each motif is encoded by one exon, whereas in the γ-crystallin each exon encodes one domain consisting of two motifs (Courtesy, Graw, J. 1997. The Crystallins: Genes, Proteins and Diseases. Biol. Chem.378:1331-1348).

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Exon 1 Intron Exon 2 Intron Exon 3

9 bp 100 bp 243 bp 1.2 Kb 275 bp

Fig.1.4. Structure of mouse Cryg gene. A typical Cryg is composed of three exons; the first exon contains only 9 bp and is followed by a short intron of 100bp. The second exon (243 bp) and the third exon (275 bp) are separated by a large intron of about 1.2 kb.

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Various mutations in the Cryg genes have been reported by mammalian lens research.

Currently, in mice eight independent mutations were known to be linked with the Cryg

gene cluster (Table 1). The Eye lens obsolescence (Elo) mutant represent with a single

nucleotide deletion in the Cryge gene. The reading frame of the gene and fourth Greek

key motif of the protein is destroyed by the mutation (96). Seven independently

observed and phenotypically distinct mutant lines under Neuheberg Cat2 mutant series

were demonstrated to be closely associated with the Cryg gene cluster (97, 98, 99).

The molecular analysis of Cat2nop have demonstrated a combined deletion and

insertion of 11 and 4 bp, respectively in the Crygb gene and Cat2ns as a deletion of the

entire 3’ end of Cryge gene. The maturation of primary lens fibers and differentiation

of secondary lens fiber cell are affected in the Elo and Cat2 mutants. The elongation

of the secondary fiber cells is not mature and they do not reach the poles of the lens.

In the case of the Cat2ns mutants the phenotype is appeared as a suture cataract in the

heterozygotes (100) and in the case of the Cat2nop mutants as a nuclear cataract. The

Elo mutant was first manifested with change as impaired elongation of the central

fibers at the basal cytoplasm on E12.5 day. Necrotic cells were found among the

central lens fibers, which never attained full maturation length and progressively

degenerated thereafter (101). This phenotype seems to be more severe than the Cat2nop

phenotype. In this mutant expression of Cryge genes decreased from E12, however,

morphological alterations can be observed by E15 (93). In humans, a mutation re-

activating the human Cryge pseudo gene was identified leading to the so-called

Coppock-like cataract. The Coppock-like cataract is nonprogressive and affects only

embryonic nucleus of the lens resulting in a very mild phenotype. A cluster of

sequence changes was found within and around the TATA box leading to its

reactivation (102).

Other than initiation of the crystallin expression, it is known that invagination and

separation of the lens vesicle from ectodermal layer is associated with expression of

the cell adhesion molecule N- cadherin, which eventually supplants E- cadherin

expressed in the ectoderm (103). Cell elongation in the lens fiber compartment is

associated with expression of intermediate filament proteins filensin CP49 (104).

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Entry into the post-mitotic phase of the fiber cells is dependent on the expression of

Cdk inhibitors p27 and p57, and mutant mice minus p27 and /or p57 affects the lens

fiber maturation (105,106).

7. 3. Lens Membrane Genes and their Mutations

Membrane Intrinsic Protein (MIP)

MIP form specialized junctions between the fiber cells and is expressed first in the

primary fiber cells of the early lens vesicle. In situ hybridization demonstrated highest

MIP expression in the elongating fiber cells in the bow region. MIP antiserum

specifically decorates fiber cell membranes (107,108). In Cataract Fraser (CatF r)

mutant, the cell nuclei in the deep cortext become abnormally pycnotic, degeneration

of cytoplasm and destruction of the lenticular nucleus follow (109).

Lim:

Mice heterozygous or homozygous for the Total opacity (To3) is mutation that display

a total opacity of the lens with a dense cataract. Additionally homozygotes display

microphthalmia and abnormally small eyes. Histological examination indicated

vacuolization of the lens and gross disorganization of the lens with posterior lens

rupture only in homozygotes. The molecular analysis of To3 revealed a single G-T

transversion of the Lim 2 gene coding for a lens specific integral membrane protein,

MP 19 (110). In human, a missense mutation in the LIM2 gene resulted into autosomal

recessive presenile cataract (111).

Connexins:

Connexins (Cx) are gap junction proteins that permit intercellular exchange of both

ionic and biochemical molecules. The connexin gene family encodes at least 20

different proteins, of which three are expressed in the lens (112, 113) that facilitate the

22

gap junctional coupling necessary for homeostasis and growth. The lens epithelium

predominantly expresses Cx43 (114, 115), however its expression is down regulated

and replaced by Cx46 and Cx50 during epithelium-to-fiber cell differentiation (116,

117, 118, 119).

The deletion of these connexin proteins demonstrated their diverse role in the lens

homeostasis. Cx43 knockout phenotype exhibits cardiac malformation and neonatal

death (120). Targeted mutation of Cx50 in mice resulted to milder nuclear cataracts

and significant reduction in the lens growth (121, 122), in contrast, deletion of Cx46

produced severe cataracts without altering ocular growth (123).

The other group at The Jackson Laboratory (TJL), USA, is involved in the

identification of new ocular phenotypes in mutagenesis programs at the Neuroscience

Mutagenesis Facility. Eye investigators at TJL are particularly interested in glaucoma,

ocular development, retinal degeneration, corneal disease, age-related macular

degeneration, and ocular neovascularization. Dr. Smith works with other staff

scientists at TJL to define ocular clinical and morphologic phenotypes in mice and to

compare them to similar human diseases. Here we attempted to establish and

characterize spontaneous congenital cataract mutant mice of the kind we have

observed. We therefore believe that our study will provide novel information

regarding genes implicated in eye development.

To our knowledge this is the first attempt to establish and characterize a mutant mouse

model for the congenital cataract and microphthalmia in this country. From the

morphological observations of dcm phenotypes, our studies have indicated that there

are four developmental anomalies such as cataract, microphthalmia, microphakia and

aniridia, in dcm mice. Histological examination of the mutant mice eyes during the

fetal life have shown normal formation of eyes in early part of the embryonic eye

development, however the major structural changes noticed in lens fiber cells (LFC)

were degeneration, loss of elongation and destruction of microarchitecture that

subsequently affect the survivability of the LFC. In high resolution 2 D separation of

23

the lens proteins, we have noticed few missed and over expressed proteins in cataract

mutant lenses when compared with that of wild type counterparts. The attempt

towards the identification of gene (s) responsible for these abnormalities in dcm

phenotype is conceptually logical that the identified gene (s) may probably have a

crucial role in the process of mammalian eye development.

The preliminary findings from the morphological, histological and breeding studies

revealed that appearance of dcm phenotype in mutant mice may be produced due to

genetic disorder and further explorations of the relationship between dcm phenotype

and its genetic origin could be highly informative in unraveling the mechanism

implicated in the development of mutant phenotype.

The eye is a very complex structure that originates from primordial tissues derived

from a number of sources and is formed via a number of developmental stages. Lens

formation is the result of a series of inductive processes. One of the most important

events in the eye development is communication between the lens placode and the

overlying surface ectoderm. Alterations in these processes lead to phenotypes that

primarily affect vision, and therefore provide an excellent model system in

developmental biology research for extrapolating the eye defects in humans.

In dcm mutation, it may be possible that some point mutation or truncation in a

crystallin may affect its solubility and therefore cause the cataract, or it may also be

able to arise due to a defective transcription factor that controls the expression of

many genes important for lens development. Hence to arrive at this, an extensive

characterization of this mutant mouse model at cellular and molecular level is

essential. The information generated from the molecular analysis would probably

explain the mechanism that underlies the formation of lens opacity in dcm phenotypes.

Upon characterization, the present mutant mouse model with congenital eye

abnormalities could be an excellent animal model in developmental biology research

for investigating the eye defects in humans and would enhance our understanding of

the pathophysiology of congenital cataractogenesis in humans.

24

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