 |
 Previous Article | Next Article 
The Journal of Neuroscience, June 15, 1998, 18(12):4511-4520
Early Onset Photoreceptor Abnormalities Induced by Targeted
Disruption of the Interphotoreceptor Retinoid-Binding Protein Gene
Gregory I.
Liou1,
Yijian
Fei1,
Neal S.
Peachey2,
Suraporn
Matragoon1,
Shuanghong
Wei3,
William S.
Blaner3,
Youxiang
Wang4,
Chengyu
Liu4,
Max E.
Gottesman4, and
Harris
Ripps5
1 Department of Ophthalmology, Medical College of
Georgia, Augusta, Georgia 30912, 2 Hines Veterans Affairs
Hospital, Hines, Illinois 60141, and Department of Neurology, Loyola
University Medical Center, Maywood, Illinois 60153, 3 Institute of Human Nutrition and 4 Institute
of Cancer Research, Columbia University, New York, New York 10032, and
5 Department of Ophthalmology and Visual Sciences,
University of Illinois, Chicago, Illinois 60612
 |
ABSTRACT |
Vision in all vertebrates is dependent on an exchange of retinoids
between the retinal pigment epithelium and the visual photoreceptors. It has been proposed that the interphotoreceptor retinoid-binding protein (IRBP) is essential for this intercellular exchange, and that
it serves to prevent the potentially cytotoxic effects of retinoids.
Although its precise function in vivo has yet to be defined, the early expression of IRBP suggests that it may also be
required for normal photoreceptor development. To further assess the
biological role of IRBP, we generated transgenic mice with targeted
disruption of the IRBP gene (IRBP / mice).
Specifically, homologous recombination was used to replace the first
exon and promoter region of the IRBP gene with a phosphoglycerate
kinase-promoted neomycin-resistant gene. Immunocytochemical and Western
blot analyses demonstrated the absence of IRBP expression in the
IRBP/ mice. As early as postnatal day 11, histological examination of the retinas of IRBP/
mice revealed a loss of photoreceptor nuclei and changes in the
structural integrity of the receptor outer segments. At 30 d of
age, the photoreceptor abnormalities in IRBP/ mice
were more severe, and electroretinographic recordings revealed a marked
loss in photic sensitivity. In contrast, no morphological or
electrophysiological changes were detected in age-matched
heterozygotes. These observations indicate that normal photoreceptor
development and function are highly dependent on the early expression
of IRBP, and that in the absence of IRBP there is a slowly progressive degeneration of retinal photoreceptors.
Key words:
homologous recombination; interphotoreceptor
retinoid-binding protein (IRBP); photoreceptor degeneration; retinal
development; vitamin A deficiency; Electroretinography (ERG)
| |
INTRODUCTION |
Vitamin A and its derivatives
(retinoids) play an essential role in the development and maintenance
of various tissues throughout the body. Because retinoids are typically
insoluble in aqueous media and are in many cases cytotoxic, they are
carried in the blood stream as well as intracellularly by a family of
retinoid-binding proteins. One of the more unique members of this
family is the interphotoreceptor retinoid-binding protein (IRBP), a
large glycoprotein (Mr ~140 kDa) synthesized by the
visual cells and extruded into the interphotoreceptor matrix (IPM) of
the vertebrate retina. IRBP is confined to the IPM by the permeability
barriers formed at the retinal pigment epithelium (RPE) (Cohen, 1965 )
and the outer limiting membrane (Bunt-Milam et al., 1985). This
localization, and the observation that the isomeric form of retinoid
bound to IRBP varies with light and dark adaptation (Liou et al., 1982; Adler et al., 1985; Saari et al., 1985), led to the suggestion that
IRBP plays a major role in the visual cycle, i.e., in the transport of
retinoids between the photoreceptor outer segments and the RPE during
the bleaching and regeneration of visual pigments (Lai et al., 1982;
Liou et al., 1982; Adler and Spencer, 1991; Pepperberg et al.,
1993).
Aside from its putative role in retinoid transport, it has been
suggested that IRBP may be essential for normal retinal development. Evidence that IRBP gene expression occurs early in photoreceptor differentiation (Carter-Dawson et al., 1986; Gonzalez-Fernandez and
Healy, 1990; Hauswirth et al., 1992; Liou et al., 1994) and that IRBP
binds fatty acids required for the structural integrity of
photoreceptor outer segments (Rodriguez et al., 1991; Chen et al.,
1993) lends credence to this hypothesis. Also consistent with this
notion is the observation that a reduction in IRBP may precede the loss
of photoreceptors seen in some animal models of hereditary retinal
degenerations (van Veen et al., 1988; Narfström et al., 1989;
Wiggert et al., 1994). In the abyssinian cat, for example, there is a
reduction in both IRBP message and protein before the onset of
photoreceptor cell death (Narfström et al., 1989; Wiggert et al.,
1994).
To assess further the role of IRBP in the development and maintenance
of retinal structure, we used homologous recombination (gene targeting)
to effectively disrupt the IRBP gene of transgenic mice. In the present
study, we report (1) the procedures for achieving the "knock-out"
of IRBP; (2) RT-PCR, Northern blot, Western blot, and
immunocytochemical evidence of the absence of IRBP expression in the
retinas of animals homozygous for the targeted allele; and (3)
histological and electrophysiological findings that reveal the
concomitant changes in retinal morphology and visual function.
| |
MATERIALS AND METHODS |
Construction of the targeting vector. The targeting
vector of 11.6 kb was composed of two IRBP genomic fragments isolated from a genomic library of mouse strain 129 (no substrain name was
designated; kindly provided by Dr. Hui Zheng, Merck Sharp & Dohme
Research Laboratories; originally from Dr. Alan Bradley, Baylor College
of Medicine), the bacterial neomycin resistant gene driven by a
phosphoglycerate kinase promoter (pgk-neo;
Colbere-Garapin et al., 1981; Adra et al., 1987), and the herpes
simplex virus thymidine kinase (hsv-tk) gene (Thomas and
Capecchi, 1987; Mansour et al., 1988). The 1.6 kb pgk-neo
gene was inversely placed downstream of an IRBP fragment spanning from
7200 to 1245, and upstream of another IRBP fragment spanning from
+3152 to +5049. The 1.9 kb fragment of the hsv-tk gene was
added to the upstream end of the vector. In the targeted allele, the
promoter region (from 1246 to +106), 81% of the translated sequence
of the IRBP gene (from +107 to +3151), and the hsv-tk gene
were deleted.
Generation of IRBP/ mice. Embryonic stem (ES)
cells of strain 129/Ola (Komang et al., 1995; kindly provided by Drs.
Peter Mombaerts and Richard Axel, Columbia University) were cocultured with STO-NEO and LIF (SNL) feeder cells (kindly provided by Dr. Hui
Zheng) and were transfected and cultured under the conditions of
positive selection for neomycin (G418) resistance (i.e., for the
presence of the expressed neomycin gene) and negative selection for
ganciclovir resistance (i.e., for the absence of hsv-tk gene expression). Appropriate targeting was confirmed by Southern analysis of genomic DNA using probes flanking the 5' and 3' ends of the targeting vector. The selected ES colonies were injected into the
blastocysts of strain C57BL/6J mice to generate chimeric founder mice.
Chimeric mice were bred with C57BL/6J mice to produce germ line-transmitted IRBP+/ mutants, which were identified by
Southern analysis. Mice that were heterozygous and homozygous for the
IRBP gene disruption were generated through inbreeding of germ
line-transmitted mutants; wild-type littermates were used as controls.
All animal procedures conformed to the Association for Research in
Vision and Ophthalmology and National Institutes of Health statements on the use of animals in ophthalmic and vision research.
PCR (Saiki, 1990) was used to identify the genotypes resulting from
this breeding protocol. The targeted (pgk-neo) allele was identified by amplification of the 530 bp fragment between 1273
of the IRBP gene and +592 of the pgk-neo gene.
The wild-type IRBP allele was identified by amplification of the 403 bp
fragment between +211 and +613 of the IRBP gene. IRBP gene expression
was analyzed by RT-PCR (Kawasaki, 1990). The cDNA of the IRBP gene was
identified by amplification of the same 403 bp fragment described above. Expression of the mouse  -actin gene was used as an internal control.
Northern blot analysis. To isolate total RNA, individual
retinas were homogenized in treated sand (Liou and Matragoon, 1992). RNA was isolated using the selective binding properties of silica gel-based membrane with the microspin technique RNeasy kit (Qiagen, Chatsworth, CA). Equal amounts (10 µg) of total RNA, determined by
absorbance at 260 nm, were electrophoresed in 1% agarose gel in
formaldehyde. RNA was blotted to Zeta-Probe GT blotting membrane (Bio-Rad, Richmond, CA) by capillary transfer and hybridized with probes for the mouse IRBP gene (a cDNA fragment extending from +3075 to
+3796), the pgk-neo gene, or the mouse -actin gene. Probes were labeled with [ -32P]dCTP with a Klenow
fragment of DNA polymerase I using random oligomers (Pharmacia,
Piscataway, NJ). Blotting, hybridization, and washing were all
performed according to the manufacturer's instructions.
RNase protection analysis (RPA). The protection assay has
been described previously (Liou et al., 1994). Briefly, sense and antisense RNA probes for the IRBP gene were synthesized using T3 or T7
polymerase on a cDNA template for the IRBP gene spanning from +3075 to
+3796 cloned in pBluescript. The coding strand probe will protect a 259 bp fragment of the noncoding strand IRBP transcript from +3333 to
+3075. The noncoding strand probe will protect a 214 bp fragment of the
coding strand IRBP transcript from +3583 to +3796. Linearized templates
were labeled with [-32P]UTP and purified as described
previously (Liou et al., 1994). One microgram of total RNA was
hybridized with each probe present in excess (500-800 pg or 4.0 × 105 cpm). Single-stranded RNA was removed with
RNase, purified, and subjected to electrophoresis on a denaturing 5%
polyacrylamide and urea gel. Gels were dried and exposed to x-ray film
with an intensifying screen.
Immunocytochemistry. Localization of IRBP was visualized by
immunocytochemical labeling with a monoclonal antibody (mAbH3-B5, kindly provided by Dr. Larry Donoso; Donoso et al., 1990) and two
polyclonal antibodies: a rabbit anti-monkey (Redmond et al., 1985) and
a goat anti-bovine (van Veen et al., 1988), both kindly provided by Dr.
Barbara Wiggert. Eyes were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, and then rinsed twice (30 min each) in cold buffer. After cryoprotection by overnight immersion in cold 30% sucrose, the tissue was mounted with Tissue-Tek OCT in a cryostat (20°C), sectioned at 8 µm, and picked up on gelatin-coated slides. The sections were washed in PBS at room temperature, and nonspecific binding sites were saturated by immersion in antiserum diluent (0.3% Triton X-100 and 0.02% sodium azide in
PBS) containing either 2.5% normal (nonimmune) serum or 3% nonfat dry
milk. The primary antibodies were applied to the sections in various
concentrations and allowed to incubate overnight at 4°C. The sections
were then exposed to an FITC-labeled secondary antibody for 1 hr at
room temperature, washed twice (10 min each) in PBS, coverslipped with
Vectashield, and photographed under incident light fluorescence.
Controls were prepared in the same manner, except that the primary
antibody was eliminated from the sequence; no fluorescent signal was
detected in control preparations, and they will not be considered
further.
Western immunoblot analysis. After removing the lens,
individual eyes were homogenized by an Omni International (Waterbury, CT) 2000 Polytron homogenizer in 500 µl of a buffer containing (in
mM): 10 HEPES, 1 EDTA, 500 NaCl, and 1 phenylmethylsulonylfluoride, pH 7.5, and centrifuged at 14,000 rpm in a
microcentrifuge at 4°C for 30 min. Ten micrograms of Protein,
determined by Bio-Rad Protein Assay (Bio-Rad, Richmond, CA) from the
supernatant fraction were analyzed by SDS-PAGE on 10% acrylamide gels
and electrophoretically transferred to a Trans-Blot transfer medium
polyvinylidene difluoride membrane (Bio-Rad). The antibodies used
included a monoclonal antibody for mouse -actin (Chemicon, Temecula,
CA) (Herman and Pollard, 1979) and two of the antibodies that were used
for immunocytochemistry, namely, the mouse mAb H3B5 directed against a
seven-amino acid sequence of the native molecule (Donoso et al., 1990)
and the polyclonal rabbit anti-monkey IRBP (Redmond et al., 1985).
Antibody-antigen reactions and their detection by enhanced
chemiluminescence (ECL; Amersham, Arlington Heights, IL) were performed
according to the manufacturer's instructions. A recombinant
Xenopus IRBP fusion protein of 37 kDa consisting of all but
the first eight amino acids of the corresponding fourth repeat of human
IRBP was used as a positive control in some of the Western analyses
(Baer et al., 1994; kindly provided by Dr. Federico Gonzalez-Fernandez, University of Virginia).
Histology and electroretinography. Structural studies were
conducted on mice at postnatal day 11 (P11), the age at which the mice
first opened their eyelids, and on 1-month-old animals. Enucleated eyes
were opened at the ora serrata and placed in 0.1 M
phosphate buffer, pH 7.4, containing 2% formaldehyde and 2.5%
glutaraldehyde, at 4°C. After overnight fixation, the anterior
segments were removed, and after three rinses in phosphate buffer, the
eyecups were post-fixed in 1% OsO4 in buffer for 90 min.
After dehydration through a graded ethanol series, the tissue was
infiltrated ultimately with a 1:1 mixture of Epon/Araldite, and 1 µm
sections were stained with azure II-methylene blue. Sections were cut
through the optic nerve head approximately along the horizontal
meridian of the eye, and micrographs were taken at ~300 µm from the
edge of the optic disk.
To determine whether the photoreceptors of IRBP/ mice
were capable of generating an electrical signal, we obtained recordings of the electroretinogram (ERG) from several 1-month-old mice of each
genotype; recordings from P11 mice were difficult to obtain, owing to
the size of the eye and partial lid closure. After an overnight period
of dark adaptation, the animals were anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg), and the ERG was recorded in response to
a high-intensity stimulus (0.3 log cd sec/m2). The
procedure has been described in detail in an earlier paper (Goto et
al., 1995).
| |
RESULTS |
Generation of IRBP/ mice
IRBP/ mice were generated by gene targeting in ES
cells. Figure 1A
illustrates the two wild-type IRBP alleles (top), the targeting vector (middle), and the result of homologous
recombination of the targeting vector with one of the wild-type alleles
(bottom). From 200 independent ES colonies that were G418-
and ganciclovir-resistant, a total of four colonies yielded the
predicted Southern hybridization pattern (Fig. 1B).
The variation in intensity of the bands identifying the wild-type and
the targeted alleles reflects differences in the amount of feeder cell
DNA. Of the four positive ES colonies, three were used for blastocyst
injection and generated chimeras that were 60-100% Agouti in coat
color.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 1.
Targeted disruption of the IRBP gene in ES cells.
A, Restriction maps of the mouse IRBP genomic locus, the
targeting vector, and the predicted structure of the targeted IRBP
gene. The locations of the 5' and 3' flanking probes used for Southern
analysis are indicated. B, BamHI;
Hc, HincII; X,
XhoI. B, Southern analysis of clones
positive for the targeting vector.
|
|
Figure 2A shows the PCR
identification of the genotypes of animals derived from inbreeding
heterozygous mice. The PCR-amplified fragments are of the expected
sizes and clearly distinguish the three genotypes. Expression of the
IRBP gene was determined by RT-PCR; as shown in Figure
2B, the 403 bp marker for the wild-type IRBP mRNA was
seen in wild-type mice (IRBP+/+), heterozygotes (IRBP+/) but not in homozygotes (IRBP/) in
which both IRBP alleles were disrupted.
View larger version (35K):
[in this window]
[in a new window]
|
Figure 2.
Identification of the genotype and IRBP gene
expression. A, PCR identification of the genotypes of
IRBP+/+, IRBP+/, and
IRBP/ mice; amplified sequences are indicated by
arrows. The 530 bp marker for the targeted
(pgk-neo) allele represents a region between
1273 of the IRBP gene and +592 of the pgk-neo gene.
For the wild-type allele, the 403 bp marker represents a region between
+211 and +613 of the IRBP gene. B, RT-PCR analysis of
wild-type IRBP gene expression. Mouse -actin was used as an internal
control.
|
|
Northern blot analysis
In the targeted IRBP allele, the IRBP gene segment from 1246 to
+3151 was deleted (Fig. 1). The remaining segment includes an upstream
conserved region between 1526 and 1245 (Liou et al., 1991), 24 bp
of exon 1, all of the introns, and exons 2-4. Expression of the IRBP
gene, the inserted pgk-neo gene, and the remaining segment
of the IRBP gene in IRBP+/ and IRBP/ mice was determined by Northern blot analysis. As shown in Figure
3A, a probe spanning from
+3075 to +3796 of the IRBP gene detected a retina-specific transcript
of 5.7 kb in IRBP+/ mice but not in IRBP/
mice. The same probe also detected a retina-specific transcript of 2.7 kb in both IRBP+/ and IRBP/ mice. A probe for pgk-neo detected a retina-specific transcript of 1.2 kb
in both IRBP+/ and IRBP/ mice. This result
indicates that although the upstream region of the IRBP gene including
its promoter was selectively deleted, the inserted pgk-neo
gene and the remaining exons of the IRBP gene continue to be
transcribed in the retina.
View larger version (40K):
[in this window]
[in a new window]
|
Figure 3.
Transcription analysis of the targeted IRBP gene.
A, Northern blot analysis of the total RNA isolated from
IRBP+/ and IRBP/ mice. Ten
micrograms of total RNA were run on a formaldehyde-agarose gel and
transferred to a Zeta Probe membrane. The top panel
depicts the structure of the wild-type and targeted IRBP alleles in
each genotype. The blot was hybridized with the following probes in
sequence: mouse IRBP cDNA, the pgk-neo
fragment, and a mouse -actin gene. R, Retina;
B, brain; K, kidney; L,
liver; S, spleen; T, testis;
 IRBP, the remaining segment of the IRBP gene.
B, RPA of total retinal RNA from IRBP+/+
and IRBP/ mice. One microgram of total RNA was
hybridized with 32P-labeled RNA probes from the coding
(sense) or noncoding (antisense) strand of the mouse IRBP
cDNA.
|
|
RNase protection analysis (RPA)
In the absence of the upstream promoter region and exon 1, the
retina-specific RNA transcript of 2.7 kb from the remaining IRBP gene
could be from either the upstream or downstream regions of the gene.
This cannot be determined by the DNA probe used in the Northern
analysis. Accordingly, we determined the orientation of this transcript
with RPA using sense and antisense RNA probes. Total RNA (1 µg) from
the retinas of IRBP/ mice was hybridized with labeled
probes, digested with RNase, and subjected to gel electrophoresis and
autoradiography. As shown in Figure 3B, only the antisense
RNA probe protected a band of bases from the total retinal RNA. This
result indicates that the 2.7 kb RNA that was transcribed from the
remaining IRBP gene was in the same orientation as the endogenous
gene.
Immunocytochemistry
The lack of IRBP gene expression in IRBP/ mice was
demonstrated by immunocytochemistry. Figure
4 shows IRBP immunoreactivity in sections
taken from 1-month-old IRBP+/+, IRBP+/, and
IRBP/ mice using a rabbit anti-monkey polyclonal
antibody (Redmond et al., 1985). The corresponding Nomarski images
demonstrate that the immunofluorescence was confined exclusively to the
IPM in IRBP+/+ and IRBP+/ animals; no
fluorescent signal was detected in IRBP/ mice. Similar
results (data not shown) were obtained with mAb H3B5 (Donoso et al.,
1990) and a goat anti-bovine polyclonal antibody (van Veen et al.,
1988). In addition, it is obvious that there is a thinning of the outer
nuclear layer and outer segment abnormalities in the
IRBP/ retina, which are more apparent in the
histological sections presented in Figure 6.
View larger version (144K):
[in this window]
[in a new window]
|
Figure 4.
IRBP immunocytochemistry. Retinal sections of
1-month-old IRBP+/+, IRBP+/, and
IRBP/ mice. The antigen is visualized with a 1:200
dilution of the primary (rabbit anti-monkey) polyclonal antibody
(Redmond et al., 1985) and FITC-labeled goat anti-rabbit IgG. The
corresponding Nomarski interference contrast images of the sections are
shown in the bottom panels. Scale bar, 20 µm.
|
|
Western blot analysis
The absence of IRBP in homozygous animals was confirmed by Western
blot analysis using monoclonal and polyclonal antibodies (Fig.
5). Figure 5A shows that a 144 kDa protein band was detected in IRBP+/+ and
IRBP+/ mice but not in IRBP/ animals.
Moreover, after normalization with mouse -actin, the amount of IRBP
expressed in heterozygous mice was approximately one-half of that in
normal littermates.
View larger version (35K):
[in this window]
[in a new window]
|
Figure 5.
Translation analysis of the targeted IRBP gene.
A, Western blot analysis of IRBP in
IRBP+/+, IRBP+/, and
IRBP/ mice. Ten micrograms of protein from the
supernatant fraction were analyzed. IRBP was identified with a
monoclonal antibody for IRBP (mAbH3B5, diluted 1:500; Donoso et al.,
1990). Internal standard, mouse -actin. Antibody-antigen reactions
and their detection are according to ECL (Amersham). B,
Western blot analysis of IRBP and a recombinant Xenopus
IRBP fusion protein of the fourth repeat (Baer et al., 1994). Antigen
detection is with a polyclonal rabbit anti-monkey antibody for IRBP
(diluted 1:100; Redmond et al., 1985).
|
|
In the targeted allele, 24 bp of exon 1 and exons 2-4 were spared. If
these exons were expressed and translated in-frame, the predicted
protein would consist of 230 amino acids, from position 1000 (Met) to
position 1231 (stop codon), and would have a molecular weight of ~23
kDa. Moreover, this protein would include ~75% of the fourth repeat
of IRBP, including all three conserved hydrophobic domains (Rajendran
et al., 1996). To test this possibility, we used a polyclonal antibody
(Redmond et al., 1985) that detects a recombinant Xenopus
IRBP fusion protein consisting of 298 amino acids, which correspond to
the fourth repeat of human IRBP, Xen IRBP.B1 (Baer et al., 1994). As
shown in Figure 5B, the polyclonal antibody detected IRBP of
the appropriate molecular weight (144 kDa) in IRBP+/+ but
not in IRBP/ mice. In addition, the antibody detected
the 37 kDa recombinant Xenopus fusion protein at levels as
low as 0.1 µg, whereas no signal was seen from IRBP+/+
mice (as expected) or from IRBP/ animals. These results
indicate that there was no protein that corresponded to the residual
portion of the IRBP gene in IRBP/ animals.
Histology and Electroretinography
Figure 6A shows
light micrographs of retinal sections from 11- and 30-d-old
IRBP+/+, IRBP+/, and IRBP/ mice.
No significant differences were seen between wild-type and
IRBP+/ mice in the two age groups. However,
IRBP/ mice in both age groups exhibited photoreceptor
abnormalities, but the changes were more severe in the older animals.
In the P11 mice, there was clear evidence of the loss of photoreceptor
cells and a thinning of the outer nuclear layer, i.e., a reduction in
thickness from ~10-11 cell nuclei (in IRBP+/+ mice) to
~6-8 cells deep in the IRBP/ mice. In addition, the
photoreceptor outer segments were poorly oriented and significantly
shorter than normal (~60% of the normal length). By contrast, there
were no detectable changes in the pigment epithelial cells or in the
more proximal layers of the IRBP/ retina; the cell
content of the inner nuclear and ganglion cell layers appeared normal,
and the thickness of the inner synaptic (plexiform) layer was
equivalent to that of wild-type animals. However, the loss of
photoreceptor cell perikarya and the shortened outer segments of the
remaining cells resulted in a significant thinning of the neural
retina.
View larger version (109K):
[in this window]
[in a new window]
|
Figure 6.
Retinal histology and electroretinography.
A, Light micrographs of the midperipheral retina from
IRBP+/+, IRBP+/, and
IRBP/ mice; the sections are from P11 (top
row) and P30 (bottom row) animals. Scale bar, 20 µm. B, Electroretinographic recordings from
1-month-old littermates. The ERG traces were in response to a
high-intensity (0.3 log cd sec/m2) flash stimulus
delivered to the dark-adapted retina; two successive traces obtained
for each mouse are superimposed.
|
|
At 1 month of age, retinal sections from IRBP/ mice
revealed a greater deterioration of retinal structure. The outer
nuclear layer showed a further reduction in thickness; the outer
segments were shorter and even more disoriented than at the P11 stage; and there were signs that many of the densely staining membraneous disks were lost or disrupted. The retinas of 1-month-old
IRBP+/ mice, on the other hand, still presented a normal
appearance.
Despite these structural abnormalities, the IRBP/ retina
retained the ability to generate a light-evoked electrical signal. As
shown in Figure 6B, the ERG response of the
1-month-old IRBP/ retina was significantly reduced in
amplitude compared with the recordings from either IRBP+/+
or IRBP+/ littermates. All components of the ERG waveform
were diminished, but it is particularly noteworthy that the ERG a-wave,
an index of photoreceptor activity (Penn and Hagins, 1969; Pugh and
Lamb, 1993), was retained in the 1-month-old IRBP/
mouse. Although the a-wave amplitude was less than half of that
recorded from wild-type and IRBP+/ mice, it is evident that even at this age the IRBP/ receptor outer segments
contain the light-sensitive pigments required to initiate the
electrical response.
| |
DISCUSSION |
The study of transgenic mice with targeted disruption of the IRBP
gene has made it possible to demonstrate the fundamental importance of
IRBP in preserving the structural and functional integrity of the
retina. The characterization of this in vivo model system is
still in its early stages, but the present results provide a clear
indication that IRBP/ mice exhibit a significant loss of
photoreceptors, as well as outer segment abnormalities, even before
their eyes have opened. Although it is highly unlikely that the damage
is related directly to photic exposure, the relatively transparent
eyelid of a newborn mouse does not preclude the possibility that the
photoreceptor abnormalities were exacerbated by light. Dark-rearing
experiments are currently being conducted to examine this
possibility.
In most other respects, the absence of IRBP appears to have had no
deleterious effects, and we could not detect any signs indicating that
the protein is essential for normal prenatal development. IRBP/ mice appeared to be healthy, and there were no
obvious cellular abnormalities in the postreceptoral layers of the
neural retina. Interestingly, the results obtained from
IRBP+/ mice suggest that IRBP may be produced in
overabundance in the normal retina. In the heterozygous animals, in
which only ~50% of the full complement of IRBP is present, the
photoreceptors have developed normally, and the electrical responses of
1-month-old mice are comparable to that of the normal. However, we have
yet to determine whether the decreased amounts of IRBP compromise other
aspects of photoreceptor function, e.g., the bleaching and regeneration cycle of rhodopsin, or lead to photoreceptor damage at later ages.
Although the IRBP promoter was deleted by the transgene, the inserted
pgk-neo gene and the remaining exons of the IRBP gene were
transcribed in the retina. This transcription may have been initiated
by the bidirectional activity of the pgk-neo promoter (Johnson and Friedman, 1990; Abeliovich et al., 1992). The explanation for the retina-specific nature of this transcription is not known but
may be related to the retina-specific hypomethylation of the mouse IRBP
allele (Liou et al., 1994). It may also reflect a retina-specific regulatory element within the remaining upstream conserved region of
the targeted IRBP gene (i.e., between 1526 and 1245; Liou et al.,
1991). Nevertheless, immunocytochemistry and Western blot analysis
indicated that these transcripts were not expressed. This failure is
likely to reflect the lack of a translation initiation codon at the
beginning of the last IRBP repeat. In eukaryotic cells, the efficient
use of AUG as a translation initiation codon is critically dependent on
the purines at positions 3 and +4 (the A in the AUG is designated +1;
Kozak, 1987; McBratney and Sarnow, 1996). The methionine encoded by the
sequence CCTATGC (+3167 to +3173) at the beginning of the
last IRBP repeat does not have purine at either 3 or +4.
Rhodopsin constitutes ~95% of the protein content of the rod outer
segment membranes (Smith et al., 1975; Krebs and Kühn, 1977). The
light-sensitive form requires the 11-cis isomer of vitamin A
aldehyde (11-cis retinal) to be linked to the apoprotein opsin. Because 11-cis retinal is not synthesized within the
rod photoreceptor, it must be transferred from its source in the RPE (Bernstein et al., 1987) through the IPM to the outer segment membranes. There is mounting evidence that IRBP is essential for the
removal of 11-cis retinal from the RPE (Flannery et al.,
1990; Okajima et al., 1990; Carlson and Bok, 1992) and for its delivery to the receptor outer segments (Okajima et al., 1990; Duffy et al.,
1993). Thus, in the absence of IRBP, the photoreceptors would suffer
essentially a local vitamin A deficiency.
Vitamin A deficiency is a well known clinical entity, and as Dowling
and Wald (1958, 1960) have shown in their classical studies of vitamin
A-deprived rats, animals reared on a vitamin A-deficient diet exhibit a
profound loss of visual sensitivity, widespread destruction of all
retinal cell layers, severe weight loss, and a broad range of defects
in other parts of the body. The pathology they describe is very
different from that encountered in the IRBP/ mouse; mice
lacking IRBP appeared to be healthy in all respects, and the only
abnormalities that we could detect were confined to the photoreceptor
layer.
However, it is important to recognize the fundamental difference in the
type of vitamin A deficiency that is seen under these different
experimental conditions. Vitamin A deficiency induced by dietary means
deprives all tissues of this essential substance, and despite the
enormous stores of retinol housed in the liver, prolonged deprivation
eventually leads to the extensive tissue damage reported by Dowling and
Wald (1958, 1960). On the other hand, the genetically induced depletion
of a retina-specific, extracellular binding protein such as IRBP has no
effect on the circulating levels of vitamin A within the blood stream.
Thus, the vitamin A requirements of the RPE, the cells of the inner retina, and virtually all tissues throughout the body are met. The fact
that IRBP is confined to the extracellular matrix of the subretinal
space and serves primarily to shuttle retinoids between the
photoreceptors and the RPE makes the photoreceptors the only cells
susceptible to damage in the IRBP/ mouse.
Despite the absence of IRBP, evidence from 1-month-old
IRBP/ mice indicates that light-evoked electrical
potentials can be elicited from the dark-adapted retina. Although the
histological abnormalities in the photoreceptor layers are more
extensive than in P11 IRBP/ mice, and the
electroretinographic responses are greatly reduced in amplitude, it is
evident that photopigments are present in visual cells of the
IRBP/ mice. As already indicated, the visual cells
require a source of 11-cis retinal to synthesize their
light-sensitive pigments, and it seems likely that some degree of
retinoid exchange between the RPE and photoreceptors has occurred in
the absence of IRBP. Whether this takes place entirely via aqueous
transfer through the IPM (Rando and Bangerter, 1982; Fex and
Johannesson, 1987; Ho et al., 1989) or whether another retinoid-binding
protein not normally expressed in the IPM is derived from other
cellular compartments that border the IPM (e.g., RPE and Müller
cells) has yet to be determined. Nevertheless, the early onset of
photoreceptor deterioration and the progressive loss of visual cells
are strong indications that whatever system is used, it is inadequate
to the task.
It is difficult to identify with certainty the cause of the
degenerative changes in the retinas of IRBP/ mice. The
observations that IRBP gene expression occurs well before the
appearance of opsin (Hauswirth et al., 1992; Liou et al., 1994) and
that IRBP binds fatty acids that are an essential component of the
photoreceptor outer segments (Rodriguez et al., 1991; Chen et al.,
1993) indicate that IRBP may be a sine qua non for normal
photoreceptor development. On this view, IRBP is essential to support
the normal synthesis of outer segment membranes, and its absence would
lead to outer segment malformation and ultimately to the death of
visual cells.
As mentioned earlier, it has been postulated that aberrant expression
of IRBP may be implicated in some genetically mediated retinal
degenerations seen in cat (Narfström et al., 1989; Wiggert et
al., 1994) and in mouse (van Veen et al., 1988). Abyssinian cats
homozygous for a slowly progressive form of hereditary rod and cone
degeneration show a 50% reduction in the levels of IRBP mRNA and
protein as early as 4 weeks of age, well before the onset of
significant changes in retinal structure or the appearance of ERG
abnormalities. The present findings are consistent with these
observations. The 1-month-old IRBP+/ mice we have studied show a similar reduction in IRBP levels and do not exhibit early signs
of photoreceptor abnormalities; as already indicated, we have yet to
determine whether pathological changes develop in older animals.
Alternatively, the reduced level of IRBP may be only one of several
factors contributing to the degenerative changes encountered in the
abyssinian cat (Applebury, 1992; Naash et al., 1996).
In double homozygous (rd/rd,rds/rds) mutant mice, on the
other hand, the loss of IRBP from the extracellular photoreceptor matrix is thought to result from an abnormality in the secretory mechanism responsible for extruding IRBP; i.e., the available IRBP is
retained within the photoreceptor cell soma (van Veen et al., 1988).
Whether the intracellular accumulation of IRBP is cytotoxic is not
known, but the profound structural changes seen in these animals at
very early ages are far greater than we have observed in 1-month-old
IRBP/ mice. Once again, the aberrant localization of
IRBP and its absence from the IPM are probably not the sole factors
contributing to the degenerative process. However, in both of these
animal models, the reduced levels of IRBP and the resultant
membranolytic effects of unbound retinoids may have contributed
to the course of the degeneration (Meeks et al., 1981). In any event,
these observations suggest that IRBP should be considered a potential
candidate gene for some forms of inherited retinal degeneration (Dryja,
1997).
| |
FOOTNOTES |
Received Jan. 27, 1998; revised March 26, 1998; accepted April 1, 1998.
This work was supported by National Institutes of Health Grants EY03829
(G.I.L.) and EY06516 (H.R.), the Department of Veterans Affairs, and
unrestricted awards from Research to Prevent Blindness, Inc. to the
Departments of Ophthalmology at the Medical College of Georgia and the
University of Illinois College of Medicine. We are grateful to Jane
Zakevicius for histology and immunocytochemistry, Cecilia Artacho for
providing animal care, and Brenda Sheppard for secretarial assistance.
We thank Drs. Hui Zheng and Howard Chen for helpful suggestions and
gifts of the mouse genomic library, plasmid clones, and SNL feeder
cells, Drs. Peter Mombaerts and Richard Axel for the ES cells, Dr.
Larry Donoso for the monoclonal antibody, Dr. Barbara Wiggert for the
polyclonal antibodies, and Dr. Federico Gonzalez-Fernandez for the
recombinant Xenopus IRBP fusion protein. We also thank
Dr. Keith Green for comments and suggestions on this manuscript.
Correspondence should be addressed to Dr. Gregory I. Liou, Department
of Ophthalmology, Medical College of Georgia, Augusta, GA 30912.
| |
REFERENCES |
-
Abeliovich A,
Gerber D,
Tanaka O,
Katsuki M,
Graybiel AM,
Tonegawa S
(1992)
On somatic recombination in the CNS of transgenic mice.
Science
257:404-408[Free Full Text].
-
Adler AJ,
Spencer SA
(1991)
Effect of light on endogenous ligands carried by interphotoreceptor retinoid-binding protein.
Exp Eye Res
53:337-346[ISI][Medline].
-
Adler AJ,
Evans CD,
Stafford WF
(1985)
Molecular properties of bovine interphotoreceptor retinoid-binding protein.
J Biol Chem
260:4850-4855[Abstract/Free Full Text].
-
Adra CN,
Boer PH,
McBurney MW
(1987)
Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter.
Gene
60:65-74[ISI][Medline].
-
Applebury ML
(1992)
Variations in retinal degeneration.
Curr Biol
2:113-115.
-
Baer CA,
Kittredge KL,
Klinger AL,
Briercheck DM,
Braiman M,
Gonzalez-Fernandez F
(1994)
Expression and characterization of the fourth repeat of Xenopus IRBP in E. coli.
Curr Eye Res
13:391-400[ISI][Medline].
-
Bernstein PS,
Law WC,
Rando RR
(1987)
Isomerization of all-trans-retinoids to 11-cis-retinoids in vitro.
Proc Natl Acad Sci USA
84:1849-1853[Abstract/Free Full Text].
-
Bunt-Milam AH,
Saari JC,
Klock IB,
Garwin GG
(1985)
Zonulae adherents pore size in the external limiting membrane of the rabbit retina.
Invest Ophthalmol Vis Sci
26:1477-1380.
-
Carlson A,
Bok D
(1992)
Promotion of the release of 11-cis retinal from cultured retinal pigment epithelium by interphotoreceptor retinoid-binding protein.
Biochemistry
31:9056-9062[Medline].
-
Carter-Dawson L,
Alvarez RA,
Fong S-L,
Liou GI,
Sperling HG,
Bridges CDB
(1986)
Rhodopsin, 11-cis vitamin A, and interstitial retinol-binding protein (IRBP) during retinal development in normal and rd mutant mice.
Dev Biol
116:431-438[ISI][Medline].
-
Chen Y,
Saari JC,
Noy N
(1993)
Interactions of all trans-retinol and long-chain fatty acids with IRBP.
Biochemistry
32:11311-11318[Medline].
-
Cohen AI
(1965)
A possible cytological basis for the "R" membrane in the vertebrate eye.
Nature
205:1222-1223.
-
Colbere-Garapin F,
Horodniceanu F,
Kourilsky P,
Garapin A
(1981)
A new dominant hybrid selective marker for higher eukaryotic cells.
J Mol Biol
150:1-14[ISI][Medline].
-
Donoso LA,
Rodrigues M,
Vrabec TR,
Sery TW,
Fong SL
(1990)
IRBP: preparation and characterization of site-specific monoclonal antibodies.
Curr Eye Res
9:357-362[ISI][Medline].
-
Dowling JE,
Wald G
(1958)
Vitamin A deficiency and night blindness.
Proc Natl Acad Sci USA
44:648-661[Free Full Text].
-
Dowling JE,
Wald G
(1960)
The biological function of vitamin A acid.
Proc Natl Acad Sci USA
46:587-608[Free Full Text].
-
Dryja TP
(1997)
Gene-based approach to human gene-phenotype correlations.
Proc Natl Acad Sci USA
94:12117-12121[Abstract/Free Full Text].
-
Duffy M,
Sun Y,
Wiggert B,
Duncan T,
Chader GJ,
Ripps H
(1993)
IRBP enhances rhodopsin regeneration in the experimentally detached retina.
Exp Eye Res
57:771-782[ISI][Medline].
-
Fex G,
Johannesson G
(1987)
Studies of the spontaneous transfer of retinol from the retinol:retinol-binding protein complex to unilamellar liposomes.
Biochim Biophys Acta
901:255-264[Medline].
-
Flannery JG,
O'Day W,
Pfeffer BA,
Horwitz J,
Bok D
(1990)
Uptake, processing and release of retinoids by cultured human retinal pigment epithelium.
Exp Eye Res
51:717-728[ISI][Medline].
-
Gonzalez-Fernandez F,
Healy JI
(1990)
Early expression of the gene for IRBP during photoreceptor differentiation suggests a critical role for the interphotoreceptor matrix in retinal development.
J Cell Biol
111:2775-2784[Abstract/Free Full Text].
-
Goto Y,
Peachey NS,
Ripps H,
Naash MI
(1995)
Functional abnormalities in transgenic mice expressing a mutant rhodopsin gene.
Invest Ophthalmol Vis Sci
36:62-71[Abstract/Free Full Text].
-
Hauswirth WW,
Langerijt AVD,
Timmers AM,
Adamus G,
Ulshafer RJ
(1992)
Early expression and localization of rhodopsin and IRBP in the developing fetal bovine retina.
Exp Eye Res
54:661-670[ISI][Medline].
-
Herman IM,
Pollard TD
(1979)
Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells.
J Cell Biol
80:509-520[Abstract/Free Full Text].
-
Ho MTP,
Massey JB,
Pownall HJ,
Anderson RE,
Hollyfield JG
(1989)
Mechanisms of vitamin A movement between rod outer segments, IRBP, and liposomes.
J Biol Chem
264:928-935[Abstract/Free Full Text].
-
Johnson P,
Friedmann T
(1990)
Limited bidirectional activity of two housekeeping gene promoters: human HPRT and PGK.
Gene
88:207-213[ISI][Medline].
-
Kawasaki ES
(1990)
Amplification of RNA.
In: PCR protocols: a guide to methods and applications (Innis MA,
Gelfand DH,
Sninsky JJ,
White TJ,
eds), pp 21-27. San Diego: Academic.
-
Komang H,
Fujiura Y,
Kawaguchi M,
Matsumoto S,
Hashimoto Y,
Obana S,
Mombaerts P,
Tonegawa S,
Yamamoto H,
Itohara S,
Nanno M,
Ishikawa H
(1995)
Homeostatic regulation of intestinal epithelia by intraepithelial T cells.
Proc Natl Acad Sci USA
92:6147-6151[Abstract/Free Full Text].
-
Kozak M
(1987)
An analysis of 5'-noncoding sequences upstream from 699 vertebrate messenger RNAs.
Nucleic Acids Res
15:8125-8148[Abstract/Free Full Text].
-
Krebs W,
Kühn H
(1977)
Structure of isolated bovine rod outer segment membranes.
Exp Eye Res
25:511-526[ISI][Medline].
-
Lai YL,
Wiggert B,
Liu YP,
Chader GJ
(1982)
Interphotoreceptor retinoid-binding proteins: possible transport vehicles between compartments of the retina.
Nature
298:848-849[Medline].
-
Liou GI,
Matragoon S
(1992)
A simple method to homogenize multiple tissue samples in small sizes without cross contamination.
Biotechniques
13:719[ISI][Medline].
-
Liou GI,
Bridges CDB,
Fong S-L,
Alvarez RA,
Gonzalez-Fernandez F
(1982)
Vitamin A transport between retina and pigment epithelium-an interstitial protein carrying endogenous retinol (IRBP).
Vision Res
22:1457-1467[ISI][Medline].
-
Liou GI,
Matragoon S,
Yang J,
Geng L,
Overbeek PA,
Ma D-P
(1991)
Retina-specific expression from the IRBP promoter in transgenic mice is conferred by 212 bp of the 5'-flanking region.
Biochem Biophys Res Commun
181:159-165[ISI][Medline].
-
Liou GI,
Wang M,
Matragoon S
(1994)
Timing of interphotoreceptor retinoid-binding protein (IRBP) gene expression and hypomethylation in developing mouse retina.
Dev Biol
161:345-356[ISI][Medline].
-
Mansour SL,
Thomas KR,
Capecchi MR
(1988)
Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to nonselectable genes.
Nature
336:348-352[Medline].
-
McBratney S,
Sarnow P
(1996)
Evidence for involvement of trans-acting factors in selection of the AUG start codon during eukaryotic translation initiation.
Mol Cell Biol
16:3523-3534[Abstract].
-
Meeks RG,
Zaharevitz D,
Chen RF
(1981)
Membrane effects of retinoids: possible correlation with toxicity.
Arch Biochem Biophys
207:141-147[ISI][Medline].
-
Naash MI,
Ripps H,
Li S,
Goto Y,
Peachey NS
(1996)
Polygenic disease and retinitis pigmentosa: albinism exacerbates photoreceptor degeneration induced by the expression of a mutant opsin in transgenic mice.
J Neurosci
16:7853-7858[Abstract/Free Full Text].
-
Narfström K,
Nilsson SE,
Wiggert B,
Lee L,
Chader GJ,
van Veen T
(1989)
Reduced level of interphotoreceptor retinoid-binding protein (IRBP), a possible cause for retinal degeneration in the abyssinian cat.
Cell Tissue Res
257:631-639[ISI][Medline].
-
Okajima T-IL,
Pepperberg DR,
Ripps H,
Wiggert B,
Chader GJ
(1990)
Interphotoreceptor retinoid-binding protein promotes rhodopsin regeneration in toad photoreceptors.
Proc Natl Acad Sci USA
87:6907-6911[Abstract/Free Full Text].
-
Penn RD,
Hagins WA
(1969)
Signal transmission along retinal rods and the origin of the electroretinographic a-wave.
Nature
223:201-205[Medline].
-
Pepperberg DR,
Okajima T-IL,
Wiggert B,
Ripps H,
Crouch RK,
Chader GJ
(1993)
Interphotoreceptor retinoid-binding protein (IRBP): molecular biology and physiological role in the visual cycle of rhodopsin.
Mol Neurobiol
7:61-84[ISI][Medline].
-
Pugh Jr EN,
Lamb TD
(1993)
Amplification and kinetics of the activation steps in phototransduction.
Biochim Biophys Acta
1141:111-149[Medline].
-
Rajendran RR,
van Niel EE,
Stenkamp DL,
Cunningham LL,
Raymond PA,
Gonzalez-Fernandez F
(1996)
Zebrafish IRBP: differential circadian expression among cone subtypes.
J Exp Biol
199:2775-2787[Abstract/Free Full Text].
-
Rando RR,
Bangerter FW
(1982)
The rapid intermembraneous transfer of retinoids.
Biochem Biophys Res Commun
104:430-436[ISI][Medline].
-
Redmond TM,
Wiggert B,
Robey FA,
Nguyen NY,
Lewis MS,
Lee L,
Chader GJ
(1985)
Isolation and characterization of monkey interphotoreceptor retinoid-binding protein-a unique extracellular matrix component of the retina.
Biochemistry
24:787-793[Medline].
-
Rodriguez de Turco EB,
Gordon WC,
Bazan NG
(1991)
Rapid and selective uptake, metabolism, and cellular distribution of docosahexaenoic acid among rod and cone photoreceptor cells in the frog retina.
Curr Eye Res
13:21-28.
-
Saari JC,
Teller DC,
Crabb JW,
Bredberg L
(1985)
Properties of an interphotoreceptor retinoid-binding protein from bovine retina.
J Biol Chem
260:195-201[Abstract/Free Full Text].
-
Saiki RK
(1990)
Amplification of genomic DNA.
In: PCR protocols: a guide to methods and applications (Innis MA,
Gelfand DH,
Sninsky JJ,
White TJ,
eds), pp 13-20. San Diego: Academic.
-
Smith Jr HG,
Stubbs GW,
Litman BJ
(1975)
The isolation and purification of osmotically intact discs from retinal rod outer segments.
Exp Eye Res
20:211-217[ISI][Medline].
-
Thomas KR,
Capecchi MR
(1987)
Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.
Cell
51:503-512[ISI][Medline].
-
van Veen T,
Ekstrom P,
Wiggert B,
Lee L,
Hirose Y,
Sanyal S,
Chader GJ
(1988)
A developmental study of interphotoreceptor retinoid-binding protein (IRBP) in single and double homozygous rd and rds mutant mouse retinae.
Exp Eye Res
47:291-305[ISI][Medline].
-
Wiggert B,
van Veen T,
Kutty G,
Lee L,
Nickerson J,
Si J,
Nilsson SEG,
Chader GJ,
Narfström K
(1994)
An early decrease in IRBP gene expression in abyssinian cats homozygous for hereditary rod-cone degeneration.
Cell Tissue Res
278:291-298[ISI][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18124511-10$05.00/0
This article has been cited by other articles:
|
|
|
|
 
L. M. Cortes, M. J. Mattapallil, P. B. Silver, L. A. Donoso, G. I. Liou, W. Zhu, C.-C. Chan, and R. R. Caspi
Repertoire Analysis and New Pathogenic Epitopes of IRBP in C57BL/6 (H-2b) and B10.RIII (H-2r) Mice
Invest. Ophthalmol. Vis. Sci.,
May 1, 2008;
49(5):
1946 - 1956.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Wenzel, J. von Lintig, V. Oberhauser, N. Tanimoto, C. Grimm, and M. W. Seeliger
RPE65 Is Essential for the Function of Cone Photoreceptors in NRL-Deficient Mice
Invest. Ophthalmol. Vis. Sci.,
February 1, 2007;
48(2):
534 - 542.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. DeVoss, Y. Hou, K. Johannes, W. Lu, G. I. Liou, J. Rinn, H. Chang, R. R. Caspi, L. Fong, and M. S. Anderson
Spontaneous autoimmunity prevented by thymic expression of a single self-antigen
J. Exp. Med.,
November 27, 2006;
203(12):
2727 - 2735.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. S. Grajewski, P. B. Silver, R. K. Agarwal, S.-B. Su, C.-C. Chan, G. I. Liou, and R. R. Caspi
Endogenous IRBP can be dispensable for generation of natural CD4+CD25+ regulatory T cells that protect from IRBP-induced retinal autoimmunity
J. Exp. Med.,
April 17, 2006;
203(4):
851 - 856.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Avichezer, R. S. Grajewski, C.-C. Chan, M. J. Mattapallil, P. B. Silver, J. A. Raber, G. I. Liou, B. Wiggert, G. M. Lewis, L. A. Donoso, et al.
An Immunologically Privileged Retinal Antigen Elicits Tolerance: Major Role for Central Selection Mechanisms
J. Exp. Med.,
December 1, 2003;
198(11):
1665 - 1676.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. C. Wendler, A. Schmoldt, G. R. Flentke, L. C. Case, L. Quadro, W. S. Blaner, J. Lough, and S. M. Smith
Increased Fibronectin Deposition in Embryonic Hearts of Retinol-Binding Protein-Null Mice
Circ. Res.,
May 2, 2003;
92(8):
920 - 928.
| |
Top
Abstract
Introduction
