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First published online 12 April 2006
doi: 10.1242/dev.02361
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1 School of Optometry and Vision Science Program, University of California at
Berkeley, Berkeley, CA 94720, USA.
2 UC Berkeley/UCSF Joint Bioengineering Graduate Program, University of
California at Berkeley, Berkeley, CA 94720, USA.
3 Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037,
USA.
4 Department of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta,
GA 30310, USA.
* Author for correspondence (e-mail: xgong{at}berkeley.edu)
Accepted 15 March 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 connexin (Cx46 or Gja8) and
8 connexin (Cx50 or Gja8), subunits of lens gap junction
channels, cause a variety of cataracts via unknown mechanisms. We identified a
dominant cataractous mouse line (L1), caused by a missense 8
connexin mutation that resulted in the expression of 8-S50P mutant
proteins. Histology studies showed that primary lens fiber cells failed to
fully elongate in heterozygous 8S50P/+ embryonic lenses, but
not in homozygous 8S50P/S50P, 8-/- and
3-/- 8-/- mutant embryonic lenses. We
hypothesized that 8-S50P mutant subunits interacted with wild-type
3 or 8, or with both subunits to affect fiber cell formation. We
found that the combination of mutant 8-S50P and wild-type 8
subunits specifically inhibited the elongation of primary fiber cells, while
the combination of 8-S50P and wild-type 3 subunits disrupted the
formation of secondary fiber cells. Thus, this work provides the first in vivo
evidence that distinct mechanisms, modulated by diverse gap junctions, control
the formation of primary and secondary fiber cells during lens development.
This explains why and how different connexin mutations lead to a variety of
cataracts. The principle of this explanation can also be applied to mutations
of other connexin isoforms that cause different diseases in other organs.
Key words: Cataract, Connexin, Gap junction, Lens fiber cell
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Piatigorsky, 1981). A
mechanistic understanding of fiber cell elongation in vivo has relied on
speculations based on in vitro studies of cultured lens epithelial cells.
Moreover, mechanisms that differentially regulate lens primary and secondary
fiber cell formation are unknown.
Lens fiber cells are coupled by intercellular gap junction channels formed
by 3 (connexin 46) and 8 (connexin 50) connexin subunits to
maintain the homeostasis required for lens transparency
(Goodenough, 1992;
Mathias et al., 1997).
Mutations in the 3 (Gja3) and 8 (Gja8)
connexin genes are one of the common causes for inherited cataracts in humans
and mice. Connexin proteins have four transmembrane domains with three
intracellular regions (the N terminus, a cytoplasmic loop and the C terminus)
and two extracellular loops (E1 and E2)
(Yeager and Nicholson, 2000).
Six connexin subunits oligomerize to form one connexon (hemichannel). A gap
junction channel is formed by the docking of extracellular loops of two
opposing connexons in the plasma membrane. Hundreds of gap junction channels
come together to form gap junctions that are morphologically defined as
specialized punctate `plaques' of cell-to-cell contacts. These channels with
small pores provide pathways for the direct exchange of small molecules
between adjacent cells (Fleishman et al.,
2004; Unger et al.,
1999). Co-expression of two types of connexin subunits in cells
will allow the formation of homomeric connexons (consisting of one type of
subunit), heteromeric connexons (consisting of two types of subunits),
homotypic channels (the docking of two identical connexons) and heterotypic
channels (the docking of two different types of connexons)
(Kumar and Gilula, 1996).
Studies of 3-/- knockout mice have suggested that
3 connexin is essential for maintaining lens transparency
(Gong et al., 1997), while the
analyses of 8-/- knockout mice and knock-in
3(50KI46/50KI46) mice have revealed that 8 connexin is important
for lens growth (Martinez-Wittinghan et
al., 2003; Martinez-Wittinghan
et al., 2004; Rong et al.,
2002; White et al.,
1998). Although 8-/- lenses show reduced
epithelial proliferation and delayed fiber cell maturation, the mechanism for
how the loss of 8 connexin leads to smaller lenses is unknown
(Rong et al., 2002;
Sellitto et al., 2004). The
interaction between 3 and 8 subunits has been suggested by their
colocalization in the fiber cells of different vertebrate lenses and the
biochemical isolation of heteromeric connexons from the lens
(Gong et al., 1997;
Jiang and Goodenough, 1996;
Konig and Zampighi, 1995;
Lo et al., 1996). The 3
and 8 connexin subunits are able to form heteromeric and heterotypic
channels in paired Xenopus oocytes and cultured cells in vitro
(Hopperstad et al., 2000;
White et al., 1994). Thus,
diverse gap junctions formed by 3 and 8 subunits need to be
further investigated in vivo.
The roles of diverse gap junctions have never been elucidated during lens
development due to a lack of an appropriate experimental model in vivo. We
hypothesize that dominant cataracts are caused by altered intercellular
communication mediated by diverse gap junction channels consisting of mutant
and wild-type connexin subunits in the lens. In this work, we have found that
the combination of mutant 8-S50P subunits (a mutation in the
extracellular loop 1) and wild-type 8 subunits specifically inhibits
the elongation of embryonic lens fiber cells, while the combination of mutant
8-S50P and wild-type 3 subunits disrupts the differentiation and
elongation of postnatal lens fibers. This work reveals that diverse gap
junctions mediate distinct mechanisms to control the formation of lens primary
and secondary fiber cells, and this also explains why and how a variety of
cataracts can result from perturbations of different types of gap junctions
during lens development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The dominant L1 mutation, in the C57BL/6J
(B6) strain background, was outcrossed with wild-type C3H/Hej mice to produce
affected G1 hybrid mice that were further crossed with wild-type C3H/Hej mice
to produce second generation (G2) mice. The G2 mice were phenotyped with a
slit lamp and genotyped with genomic DNA for a genome-wide linkage analysis by
using a total of 59 microsatellite markers
(Du et al., 2004). Based on the
chromosomal location, causative gene candidates were identified from the Mouse
Genome Database at the National Center for Biotechnology Information (NCBI)
website. PCR fragments of the 8 connexin gene were amplified from
mutant genomic DNA isolated from homozygous mutant mice, as previously
described (Chang et al., 2002),
and followed by DNA sequencing analysis.
Examination of lens phenotypes
Mouse pupils were dilated by using an eye drop containing 1% phenylephrine
and 1% atropine before lens clarity was examined using a slit lamp. The
cataract was directly imaged in living animals by a slit lamp (Nikon-FS3)
using a camera and Kodak elite2 200ASA color slide films. Fresh lenses,
dissected from enucleated eyeballs of wild-type and mutant mice, were imaged
under a Leica MZ16 dissecting scope using a digital camera.
Generation of compound mutant mice
L1 heterozygous 8S50P/+ 3+/+
mice were bred with 8-/- 3-/- double
knockout mice to produce the 8S50P/- 3+/-
and 8+/- 3+/- mutant mice. Both
L1 homozygous 8S50P/S50P 3+/+ and
double mutant 8S50P/S50P 3-/- mice were
generated from the intercross of 8S50P/-
3+/- mice. Double mutant 8S50P/S50P
3-/- mice were bred with 3-/- knockout
mice to produce 8S50P/+ 3-/- mice.
Previously described PCR methods were used to distinguish 3 or 8
knockout alleles from wild-type or mutant 8 alleles
(Chang et al., 2002).
Histology, thin-section TEM and immunohistochemistry
Enucleated eyeballs were fixed in a solution containing 2% glutaraldehyde
and 2.5% formaldehyde in 0.1 M cacodylate buffer (pH 7.2) at room temperature
for at least 5 days and were postfixed in 1% aqueous OsO4, stained
en bloc with 2% aqueous uranyl acetate and then dehydrated through graded
acetone. Samples were embedded in Epon resin (Ted Pella, Redding, CA).
Sections (1 µm) were collected on glass slides and stained with Toluidine
Blue. Bright-field images were acquired via a Zeiss Axiovert 200 light
microscope with a digital camera. Thin sections (80 nm) were cut with a
diamond knife, stained with 5% uranyl acetate followed by Reynold's lead
citrate before examination under a JEOL JEM-1200EX electron microscope (JEOL,
Tokyo, Japan).
A previously described method was used to prepare lens frozen sections for
immunohistochemical analysis with different antibodies
(Gong et al., 1997). The
following reagents were used for immunostaining embryonic lens frozen
sections: a rabbit polyclonal antibody against the C-terminal region of
8 connexin (generously provided by Dr M. J. Wolosin at Mount Sinai
School of Medicine, New York), rhodamine phalloidin (Molecular Probe) for
detecting F-actin and DAPI (Vector Laboratories) for labeling cell nuclei.
Fluorescent images were collected under a Zeiss Axiovert 200 fluorescent
microscope with an Axiocam camera or a Leica laser confocal microscope with a
high resolution digital camera.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8-S50P point mutation causes dominant cataracts in L1 mutant mice8 connexin) gene
(Fig. 1C).
It is known that 8 connexin mutations cause cataracts in both humans
and mice. Therefore, we performed DNA sequencing analysis using PCR fragments
amplified from the genomic DNA of L1 homozygous mice. We found a
missense mutation (T
C) of the Gja8 gene, resulting in the
replacement of the serine residue at codon 50 by a proline residue (S50P) in
the extracellular loop 1 (E1-loop) of the 8 connexin protein
(Fig. 1D). Thus, the dominant
cataracts in the L1 mouse line are caused by the 8-S50P
mutation. The genotypes for L1 heterozygous and homozygous mice are
labeled as 8S50P/+ 3+/+ and
8S50P/S50P 3+/+, respectively. The
wild-type, homozygous 8 knockout and 8/3 double knockout
mice are labeled as 8+/+ 3+/+,
8-/- 3+/+ and 8-/-
3-/-.
We further confirmed that the 8S50P/S50P
3+/+ mice developed small and ruptured cataractous lenses
similar to the 8S50P/+ 3+/+ mice at
weaning age (Fig. 2A,B).
Histological data showed severely disorganized fiber cells, vacuole formation
and posterior capsule rupture in both 8S50P/+
3+/+ and 8S50P/S50P 3+/+
lenses (Fig. 2C,D). Thus,
secondary fiber cell formation was severely disrupted in both
8S50P/+ 3+/+ and
8S50P/S50P 3+/+ postnatal lenses.
Primary fiber cell elongation is inhibited only in the 8S50P/+ 3+/+ lenses
As altered fiber cells were obviously observed in the neonatal lenses of
both 8S50P/+ 3+/+ and
8S50P/S50P 3+/+ mice (data not shown), we
carried out histological studies of their embryonic lenses. At 15.5 days
post-conception (E15.5), both 8+/+ 3+/+
and 8-/- 3+/+ embryonic lenses showed no
space between the primary fibers and overlying anterior epithelium
(Fig. 3A,B). Unexpectedly, a
large cystic lumen between posterior primary fiber cells and anterior
epithelium was observed in the E15.5 8S50P/+
3+/+ embryonic lenses with 100% penetrance
(Fig. 3C), but not in the E15.5
8S50P/S50P 3+/+ embryonic lenses
(Fig. 3D). Histology data
further verified that at earlier stages (E13.5), posterior primary fiber cells
did not reach the anterior epithelium in the 8S50P/+
3+/+ lenses, while primary fiber cells in wild-type lenses
elongated and obliterated the lumen of the lens vesicle at the same embryonic
stage (data not shown).
|
8S50P/+ 3+/+ and
8S50P/S50P 3+/+ lenses
(Fig. 2), the elongation of
primary fiber cells is significantly perturbed only in
8S50P/+ 3+/+ embryonic lenses, not in
8S50P/S50P 3+/+ and 8-/-
3+/+ lenses during embryonic development. We hypothesize
that 8-S50P is a gain-of-function mutant subunit, as neither a
loss-of-function of 8 connexin in 8-/-
3+/+ lenses nor a normal function of 8 connexin in
wild-type 8+/+ 3+/+ lenses inhibits
primary fiber cell elongation. Moreover, normal elongation of primary fiber
cells in 8S50P/S50P 3+/+ lenses suggests
that an interaction between mutant 8-S50P subunits and endogenous
wild-type 8 subunits is probably required for the suppression of
primary fiber cell elongation in 8S50P/+
3+/+ embryonic lenses.
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8-S50P mutation alters the
intercellular communication in lens fiber cells by interacting with endogenous
wild-type 8 and/or 3 subunits to form mutant gap junctions. To
test this hypothesis, we examined the presence of gap junctions in
8S50P/+ 3+/+ embryonic lenses. A
representative immunostaining image showed typical punctate fluorescent spots
of 8 connexin in an E15.5 wild-type lens frozen section detected by an
anti-8 antibody (Fig.
3E). Similar fluorescent signals were also detected in posterior
fiber cells of E15.5 8S50P/+ 3+/+ lens
sections (Fig. 3F,G).
Transmission electron microscope (TEM) analysis further confirmed the presence
of bona fide gap junctions in 8S50P/+ 3+/+
lenses (Fig. 3H). Therefore, we
investigated the subunit composition of mutant gap junctions that perturb the
formation of primary and secondary fiber cells in 8-S50P mutant lenses
by a genetic approach.
The combination of wild-type 8 and mutant 8-S50P subunits inhibits the elongation of lens primary fibers
In order to determine the subunit composition of mutant gap junction
channels that mediate the inhibition of primary fiber cell elongation in
8S50P/+ 3+/+ embryonic lenses, we replaced
wild-type 3 and 8 alleles with the null alleles. By breeding the
8S50P/+ 3+/+ mutant mice with the
3-/- knockout and 8-/- knockout mice, we
have generated two different compound mutant mice: 8S50P/+
3-/- mice that lack wild-type 3 connexin and
8S50P/- 3+/+ mice that lack wild-type
8 connexin. Histological data showed a cystic lumen only in
8S50P/+ 3-/- embryonic lenses
(Fig. 4A), but not in
8S50P/- 3+/+ lenses
(Fig. 4B). Gap junctions were
also detected in the fiber cells of these compound mutant embryonic lenses by
immunohistochemistry and TEM (data not shown). These results suggest that
8-S50P subunits interact with endogenous wild-type 8 subunits to
inhibit primary fiber cell elongation, while the presence of endogenous
wild-type 3 subunits does not affect primary fiber formation. Thus, it
is possible that 8-S50P and wild-type 8 subunits form mutant gap
junction channels that modulate a unique mechanism essential for the
elongation of lens primary fiber cells.
|
3 and mutant 8-S50P subunits disrupts the formation of postnatal secondary fibers8S50P/+
3+/+ and 8S50P/S50P 3+/+
mice developed whole cataracts at weaning age
(Fig. 2), we further examined
the postnatal lens phenotypes of 8S50P/+
3-/- and 8S50P/- 3+/+
mice. Surprisingly, the 8S50P/+ 3-/- mice
developed a nuclear cataract rather than a whole cataract. A lens from a P21
8S50P/+ 3-/- mouse revealed a nuclear
cataract with a transparent cortex (Fig.
4E). Histological data showed degenerated nuclear fibers but
normal peripheral cortical fibers in the P14 8S50P/+
3-/- lens (Fig.
4C). By contrast, 8S50P/- 3+/+
mice developed microphthalmia with whole cataracts similar to the
8S50P/+ 3+/+ and
8S50P/S50P 3+/+ mice. A lens from a P21
8S50P/- 3+/+ mouse was small and ruptured
with a cataract (Fig. 4F).
Disrupted secondary fiber cells, enlarged vacuole-like extracellular spaces
and posterior capsule rupture were observed in the P14
8S50P/- 3+/+ lens
(Fig. 4D).
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3
and mutant 8-S50P subunits disrupts the formation of postnatal lens
secondary fibers, while the presence of endogenous wild-type 8 connexin
does not affect secondary fiber formation. These results also provide a
molecular explanation for why the L1 heterozygous
(8S50P/+ 3+/+) and L1 homozygous
(8S50P/S50P 3+/+) mice developed similar
phenotypes, such as ruptured lenses and microphthalmia, at the weaning age.
Thus, it is possible that 8-S50P and wild-type 3 subunits form
mutant gap junction channels that modulate a different mechanism for
regulating proper formation of secondary fibers in postnatal lenses.
Mutant 8-S50P subunits alone have no effect on the formation of lens primary or secondary fiber cells
We have also generated 8S50P/- 3-/-
mutant mice that lack both endogenous wild-type 8 and 3
connexins and compared their lens phenotypes with those of double knockout
8-/- 3-/- mice. Histological data revealed
that both 8S50P/- 3-/- and
8-/- 3-/- embryonic lenses had normal
elongation of primary fiber cells, and both mutant mice developed large
nuclear cataracts at the age of three weeks
(Fig. 5A). A histological
section of a P14 8S50P/- 3-/- lens
displayed normal secondary fiber cells in lens periphery but degenerated inner
mature fiber cells (Fig. 5B).
Lens phenotypes of different types of 8-S50P mutant mice are summarized
in Table 1. In summary, these
data suggest that mutant 8-S50P subunits probably have no function in
vivo and that the interactions between these mutant subunits and endogenous
wild-type 8 or 3 connexins perturb primary fiber cell elongation
or secondary fiber cell formation, respectively.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 (Cx43), 3
(Cx46) and 8 (Cx50) subunits have been suggested to provide a
sophisticated regulatory network to coordinate lens growth and to maintain
lens transparency throughout life
(Goodenough, 1992). Lens fiber
cells are coupled by gap junction channels consisting of 3 and 8
connexin subunits. This work demonstrates that the combination of
8-S50P and endogenous wild-type 8 subunits specifically inhibits
the elongation of primary fiber cells in embryonic lenses, while the
combination of 8-S50P and endogenous wild-type 3 subunits
disrupts the proper formation of secondary fiber cells in postnatal lenses.
These results suggest that gap junctions formed by 8-S50P and wild-type
8 subunits alter a unique mechanism required for the elongation of lens
primary fiber cells, while gap junctions formed by 8-S50P and wild-type
3 subunits perturb a separate and distinct mechanism needed for proper
formation of secondary fiber cells in postnatal lenses. Thus, this work
provides the first in vivo evidence for a working model that diverse gap
junction communications modulate different signaling mechanisms for primary
fiber cell elongation or secondary fiber cell formation during lens
development (Fig. 6).
This work provides an in vivo model to understand a fundamental mechanism
for why and how diverse gap junction channels are used in almost all organs
supported by the fact that two or more types of connexin subunits are commonly
co-expressed in cells (Goodenough et al.,
1996; Kumar and Gilula,
1996). It is generally accepted that diverse gap junctions provide
a broad spectrum of pathways to ensure the homeostasis needed for various
cellular functions in vivo. However, studies to define the roles of these
diverse gap junctions have previously been hindered by an inability to
distinguish gap junction channels formed by different types of subunits from
the channels formed by one type of subunits in vivo. Previous results from
studies of loss-of-function knockout mice suggest that 3 connexin is
essential for maintaining lens transparency, while 8 connexin is
required for lens growth. Functional differences between channels consisting
of mixed 8 and 3 subunits and channels consisting of either
8 or 3 subunits in vivo are unclear
(Martinez-Wittinghan et al.,
2003). Studies of the 8-G22R mutation (a mutation in the
N-terminal domain) have demonstrated that severe lens phenotypes are partly
caused by the mutant gap junction channels consisting of both mutant
8-G22R and endogenous wild-type 3 subunits
(Chang et al., 2002). The
8-G22R mutant probably acts as a dominant-negative inhibitor to perturb
gap junction communication by oligomerizing with wild-type 8 and/or
3 subunits. This predication is also supported by the fact that the
elongation of primary fiber cells is normal in both 8-G22R heterozygous
and homozygous embryonic lenses (C.-h.X, D.C. and X.G., unpublished), and in
8-/- 3-/- lenses without gap junction
channels. Therefore, based on the result that a combination of 8-S50P
and wild-type 8 subunits inhibits the elongation of primary fiber
cells, we propose that 8-S50P mutant proteins are gain-of-function
subunits that perturb the intercellular gap junction communication or possibly
hemichannels by interacting with wild-type 8 subunits. A
gain-of-function approach has never been used to investigate the roles of
intercellular gap junction communications in vivo. The downstream signaling
mechanisms modulated by diverse intercellular gap junctions formed by
8-S50P and 8 or 3 connexins in the lens are unknown.
Thus, the 8-S50P mutation provides a useful experimental model with
which to further investigate the mechanisms that uniquely regulate the
elongation of primary fibers and the formation of secondary fiber cells during
lens development.
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3 mutants (F32L, P59L, N63S, P187L, S380fs and N188T)
and five 8 mutants (R23T, E48K, P88S, I247M and V64G) have been linked
to dominant cataracts in humans (Bennett et
al., 2004; Berry et al.,
1999; Jiang et al.,
2003; Li et al.,
2004; Mackay et al.,
1999; Polyakov et al.,
2001; Rees et al.,
2000; Shiels et al.,
1998; Willoughby et al.,
2003; Zheng et al.,
2005). In addition, three 8 mutants (G22R, D47A and V64A)
cause different dominant cataracts in mice
(Chang et al., 2002;
Graw et al., 2001;
Steele et al., 1998).
Previously reported mutations of 8 connexin are illustrated in a
topological model of 8 protein (Fig.
7). These mutations cause a variety of dominant cataracts in
humans and mice. A previous study of human 8-P88S mutant (located in
the second transmembrane domain) suggests a hypothesis that the dominant
cataract is caused by cellular defects triggered by mistrafficking and
intracellular accumulation of toxic aggregates composed of mutant connexin
proteins (Berthoud et al.,
2003). However, this hypothesis cannot explain the cataracts
caused by the 8-S50P mutation and the 8-G22R mutation. Data from
our current study of the 8-S50P mutation and our previous study of the
8-G22R mutation reveal that different types of cataracts result from a
perturbation of intercellular gap junction communication by the specific
interactions between mutant subunits and endogenous wild-type subunits during
lens development. Therefore, our current work demonstrates a new mechanistic
explanation for why and how different connexin mutations lead to a variety of
cataracts. Moreover, this new mechanism explains how mutations of other
connexin isoforms cause different diseases in other organs, such as a recent
finding that the Cx43-G60S mutation in extracellular loop 1 causes
oculodentodigital dysplasia (Flenniken et
al., 2005). The 1 connexin (Cx43 or Gja1) is
predominantly expressed in lens epithelial cells. We have not characterized
the properties of epithelial cells in different 8-S50P mutant mice. It
would be an interesting area to explore in future studies.
Fiber cell elongation is a morphological hallmark of lens fiber cell
differentiation. The mechanism for fiber cell elongation has never been
investigated in vivo due to a lack of proper experimental systems. Current
understanding of fiber cell elongation is based on the results obtained from
elongation studies of cultured lens epithelial cells in vitro
(Beebe et al., 1982;
Parmelee and Beebe, 1988) and
some descriptive in vivo information
(Bassnett, 2005;
Kuszak et al., 2004). Several
transcriptional factors have been reported to be essential for the formation
of lens primary fiber cells (Kim et al.,
1999; Ogino and Yasuda,
2000; Wigle et al.,
1999). However, mechanistic differences between lens primary and
secondary fiber formation are unknown. No previous results have ever
demonstrated the involvement of 8 connexin or gap junction channels in
the regulation of elongation or formation of lens primary fiber cells. This
work provides molecular evidence for regulatory differences between lens
primary and secondary fiber cells.
The 8-S50P mutant subunits alone are nonfunctional in the lens. The
mutant subunits must rely on the presence of wild-type 8 subunits to
inhibit the elongation of primary fiber cells. However, the combination of
8-S50P and wild-type 8 subunits does not disrupt the formation
of secondary fiber cells in postnatal lenses. As neither
8-/- knockout nor 8-/-
3-/- double knockout inhibits the elongation of lens primary
fibers, a complete blockage of fiber-to-fiber coupling is not responsible for
the inhibition of primary fiber cell elongation. Normal fiber-to-fiber
coupling does not inhibit primary fiber cell elongation in wild-type lenses.
We hypothesize that gap junction channels formed by 8-S50P and
wild-type 8 subunits gain their mutant function by increasing channel
permeability to facilitate the fiber-to-fiber transport or by transmitting a
unique signal that inhibits the elongation of primary fiber cells. Future
experiments will be needed to evaluate this hypothesis or an alternative
hypothesis that the interaction of 8-S50P and wild-type 8
subunits perturbs a non-junction signaling event to inhibit the elongation of
primary fiber cells.
The molecular basis for the proper formation of differentiating secondary
fiber cells and the maturation of fiber cells remains largely unknown despite
substantial descriptive information that have been published in the last few
decades. This work demonstrates that 8-S50P must rely on the presence
of endogenous wild-type 3 to disrupt the formation of secondary fiber
cells in postnatal lenses. A previous study of the 8-G22R mutation also
revealed the importance of the interaction between mutant 8 and
wild-type 3 connexins in regulating the formation of secondary fiber
cells (Chang et al., 2002).
These results suggest that the intercellular gap junctions formed by 8
and 3 connexin subunits modulate the formation of secondary fiber cells
in the lens periphery.
In summary, this work reveals that the 8-S50P mutation causes lens
cataracts via distinct mechanisms, which are different from those causing
cataracts in the 8-/- knockout and 8-G22R mutant
mice. More importantly, this study clearly demonstrates that connexin point
mutations such as 8-S50P can be used as useful experimental models to
investigate fundamental mechanisms during lens development and
cataractogenesis in vivo.
|
ACKNOWLEDGMENTS |
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|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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