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Regular Articles |
From the Departments of Ophthalmology,*
Neuroscience,
and Molecular Biology and
Genetics,
The Johns Hopkins University School
of Medicine, Baltimore, Maryland; and the National Cancer
Institute,§
National Institutes of Health,
Bethesda, Maryland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The PDGF family is made up of dimers of
the products from two genes, PDGF-A and PDGF-B,
resulting in three family members, PDGF-AA, PDGF-AB, and PDGF-BB. There
are two PDGF receptor (PDGFR) gene products, PDGFR
and PDGFRß,
that also form dimers (for review, see Hart and
Bowen-Pope9
). PDGFR binds both PDGF-A and -B, whereas
PDGFRß binds only PDGF-B. Differential expression of the receptors
results in differences in activity of the isoforms.
The retina provides a great model system to study the effects of growth factors and/or cellular interactions in the nervous system. PDGFs have been implicated in the differentiation of O2-A progenitor cells in the optic nerve into astrocytes that migrate into the retina and become localized in the nerve fiber layer and around blood vessels.10-13 Increased expression of PDGFs in retinal pigmented epithelial (RPE) cells after disruption of the RPE monolayer may be a mechanism by which defects are repaired, but may also contribute to scarring that underlies proliferative vitreoretinopathy, the major cause of failure of retinal reattachment surgery and disciform scarring, the end-stage of macular degeneration with choroidal neovascularization (NV).14 PDGFs produced by endothelial cells in retinal NV in diabetic retinopathy or retinopathy of prematurity may also contribute to glial and RPE cell recruitment into epiretinal scar tissue that can lead to retinal detachment.15,16 Therefore, neutralization of PDGFs may be a good strategy for preventing and treating these scarring processes. But to design the most specific treatments, it is necessary to know whether scarring is mediated by PDGF-A, PDGF-B, or both. It is also necessary to know what other effects PDGF-A and -B have in the retina, because these other effects are likely to be perturbed by neutralization of PDGFs. To investigate the effects of PDGF-A in the retina, we coupled the rhodopsin promoter to the coding region of the PDGF-A gene and generated transgenic mice that express PDGF-A in photoreceptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A full-length complementary DNA (cDNA) for human PDGF-A
was cloned into a plasmid containing the 2.2-kb
HindIII/NaeI fragment from the bovine rhodopsin
promoter.17
The plasmid also contained an intron and a
polyadenylic acid addition site derived from the mouse protamine gene
and a eukaryotic consensus ribosomal binding site. After
transformation, a clone with correct orientation was selected. DNA was
double CsCl purified and cut with EcoRI to provide a 3546-bp
fusion gene (Figure 1)
. The fusion gene
was purified, and transgenic mice were generated by established
techniques as previously described.17
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). Retinal Reverse Transcriptase-PCR
At appropriate time points, mice were sacrificed, eyes were
removed, and retinas were dissected. Retinal RNA was isolated using the
guanidine isothiocyanate method as described by Chomczynski and
Sacchi.18
Reverse transcription was carried out with
~0.5 µg of total RNA, reverse transcriptase (RT; SuperScript II,
Life Technologies, Inc., Gaithersburg, MD), and 5.0 µmol/L
oligodeoxythymidylate primer. Aliquots of the cDNAs were used
for PCR amplification with primers for the hPDGF-A/mP1
fusion gene that amplify across an intron-exon border, P3
(5'-AACACGAGCAGTGTCAAGTGCCAG-3') and P4
(5'-GATGTGGCGAGATGCTCTTGAAGTCTGGTA-3'; Figure 1
). The expected PCR
products for the hPDGF-A/mP1 fusion gene fragment from
genomic DNA and messenger RNA (mRNA) are 632 bp and 538 bp,
respectively. Titrations were performed to ensure that PCR reactions
were carried out in the linear range of amplification. Mouse S16
ribosomal protein primers (5'-CACTGCAAACGGGGAAATGG-3' and
5'-TGAGATGGACTGTCGGATGG-3') were used to provide an internal control
for the amount of template in the PCR reactions.
Northern Blot Analysis
RNA blot hybridization analysis was done as previously described,14 using 10 to 15 µg of total retinal RNA. The cDNA probe was the 1.1-kb EcoRI-HindIII fragment of hPDGF-A labeled with 32P by hexanucleotide random priming. The hybridization temperature was 65°C, and the membrane was washed twice for 60 minutes at room temperature in 2x standard saline citrate, 0.1% SDS, followed by a 15-minute wash at 58°C in 1x standard saline citrate, 0.1% SDS and a final 15-minute wash at 65°C in 0.5x standard saline citrate, 0.1% SDS.
Immunohistochemistry for PDGF
Transgene-positive and littermate control mice were sacrificed at various time points; their eyes were removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections (10 µm each) were cut and immunohistochemically stained as previously described14 with a 1:100 dilution of rabbit anti-hPDGF antibody (Genzyme, Cambridge, MA). Specificity of staining was assessed by substitution of nonimmune serum for primary antibody and by preabsorption of primary antibody with antigenic peptide.
Evaluation of the Retinal Phenotype of Transgenic Mice
At various time points, mice were sacrificed, and eyes were snap-frozen or briefly fixed in 4% paraformaldehyde and embedded in optical cutting temperature embedding media (OCT; Miles Diagnostics, Elkhart, IN) or paraffin. Frozen or paraffin sections were stained with hematoxylin and eosin (H&E), histochemically stained with biotinylated griffonia simplicifolia lectin B4 (GSA; Vector Laboratories, Burlingame, CA),19 or immunohistochemically stained with a 1:100 dilution of a rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; a gift of L. F. Eng, Palo Alto, CA)20 or a 1:100 dilution of a rabbit polyclonal antibody to human cellular retinaldehyde binding protein (a gift from J. Saari, Seattle, WA),21 a 1:50 dilution of a monoclonal anti-hFGF2 antibody (a gift from T. Reilly, Wilmington, DE)19,22 or a 1:20 dilution of a monoclonal antibody directed against proliferating cell nuclear antigen (a gift from P. Hall, Aberdeen, Scotland).14,23
Murine Model of Oxygen-Induced Ischemic Retinopathy
Ischemic retinopathy was produced in transgene-positive and littermate control mice by a method described by Smith et al.24 Seven-day-old mice and their mothers were placed in an airtight incubator and exposed to an atmosphere of 75 ± 3% oxygen for 5 days. Incubator temperature was maintained at 23 ± 2°C, and oxygen was measured every 8 hours with an oxygen analyzer. After 5 days, the mice were removed from the incubator and placed in room air for 5 days. After 5 days, the mice were killed, and their eyes were rapidly removed and frozen in OCT or fixed in 10% formalin and embedded in paraffin. To perform quantitative assessments, 10-µm serial sections were cut through the entire extent of each eye. The entire eye was sampled by staining sections roughly 50 to 60 µm apart, which provided 13 sections per eye for analysis. GSA-stained sections were examined with an Axioskop microscope (Zeiss, Thornwood, NY), and images were digitized using a 3 charge-coupled device color video camera (IK-TU40A, Toshiba, Tokyo, Japan) and a frame grabber. Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) was used to delineate lectin-stained cells on the surface of the retina, and their area was measured. The mean of the 13 measurements from each eye was used as a single experimental value.
Compound Transgenic Mice with Increased Expression of Both PDGF-A and Vascular Endothelial Growth Factor in the Retina
The production and screening of transgenic mice with overexpression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF-transgenic mice) have been previously described.25 Mice from the V-6 line that are heterozygous for the rhodopsin/VEGF transgene develop NV that originates from the deep capillary bed of the retina and grows into the subretinal space, where it gradually spreads and enlarges. V-6 rho/VEGF mice were mated with rho/PDGF-A2 or rho/PDGF-A3 mice and, at P21, the offspring were perfused with fluorescein-labeled dextran, and retinal NV was measured on retinal flat mounts as previously described.26 Briefly, mice were anesthetized, the descending aorta was clamped, the right atrium was cut, and the mice were perfused through the left ventricle with 1 ml of phosphate-buffered saline containing 50 mg/ml of fluorescein-labeled dextran (2 x 106 average molecular weight; Sigma Chemical Co., St. Louis, MO). The eyes were removed and fixed for 1 hour in 10% buffered formalin phosphate. The cornea and lens were removed, and then the entire retina was carefully dissected from the eyecup, radially cut from the edge of the retina to the equator in all four quadrants, and flat-mounted in Aquamount with photoreceptors facing upward. Flat mounts were examined by fluorescence microscopy and photographed with Ectachrome 400 film (Kodak, Rochester, NY), and slides were scanned with a QuickScan 35 scanner (Minolta, Osaka, Japan). Image files were imported into Adobe PhotoShop 4.0, labeled, and printed with a Fujix pictography 3000 printer (Fuji Photo Film Co., Tokyo, Japan). For quantitative assessments, retinal flat-mounts were examined by fluorescence microscopy at x400 magnification, which provides a narrow depth of field so that, when focusing on NV on the outer edge of the retina, the remainder of the retinal vessels are out of focus, allowing easy delineation of the NV. The outer edge of the retina, which corresponds to the subretinal space in vivo, is easily identified, and, therefore, there is standardization of focal plane from slide to slide. Images were digitized using a 3 charge-coupled device color video camera (IK-TU40A; Toshiba, Tokyo, Japan) and a frame grabber. Image-Pro Plus 3.0 software (Media Cybernetics, Silver Spring, MD) was used to delineate each of the lesions and calculate the number in each retina, the area of each lesion, and the total area of NV per retina. Measurements were repeated three times for each retina, and the mean was used for one experimental value; there was little variability among triplicate measurements.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six independent lines that incorporated the rhodopsin promoter/PDGF-A fusion gene were obtained (designated rho/PDGF-A1–6). The founders were backcrossed with C57BL/6J mice to establish transgenic lines; rho/PDGF-A6 did not reproduce and therefore only 5 lines were established. Mice that were heterozygous at the transgene locus were used in all analyses.
Expression of PDGF-A mRNA in the Retinas of Transgenic Mice
RT-PCR using total retinal RNA as template and primers specific
for transgene mRNA showed good expression in lines
rho/PDGF-A1, -A2, and -A3, moderate
expression in line 5, and weak expression in line 4 (Figure 2A)
. The time course of transgene mRNA
expression in the retina was assessed in lines 2 and 3 and showed that
in both lines mRNA was first detected at P3 and increased to a
steady-state level between P10 and P14 (Figure 2B)
.
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Only very rough quantitative comparisons can be made with
immunohistochemical staining, but it is safe to say that retinas of
adult mice from lines 1 and 3 showed a strong signal for PDGF-A,
whereas retinas from line 4 mice showed no signal above background
(Figure 3A)
. Lines 2 and 5 showed
staining above background, but less than that seen in lines 1 and 3.
The pattern of staining varied slightly among the lines, but in general
there was diffuse staining throughout the entire retina, suggesting
that PDGF-A is secreted by photoreceptors and spreads throughout the
inner retina. In lines 1 and 3, there was a distinct band of more
intense staining in the region of photoreceptor terminals (arrows),
suggesting that in these lines, secretion may occur primarily from the
synaptic terminals (Figures 3 and 4)
.
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Light microscopy of H&E-stained retinal sections of adult
rho/PDGF-A mice showed some irregularity and disruption of
the inner nuclear layer, which were caused by ectopic cells with
lightly staining cytoplasm in the periphery of the retina in lines 1–3
(Figure 3B)
. Lines 4 and 5, which have the lowest expression of PDGF-A,
showed no identifiable abnormalities. Staining for proliferating cell
nuclear antigen demonstrated a few positive nuclei on the surface of
the retina in transgene-negative mice, whereas transgene-positive mice
showed increased labeled nuclei in the inner retina (Figure 3C)
. All of
the rho/PDGF-A lines showed increased GFAP staining in the
inner nuclear layer, except line 4 (not shown). Figure 4
shows the time
course of PDGF-A protein and GFAP expression in line 3. PDGF-A
protein was first detectable in photoreceptors of transgenic mice on
P7, at which time transgenic mice appeared to have increased GFAP
staining in the nerve fiber layer compared with wild-type mice. At
later time points there was increased staining for PDGF-A and a
progressive increase in GFAP-reactive cells in the region corresponding
to the ectopic cells in the inner nuclear layer seen in H&E-stained
sections. Staining for GFAP was also done in line 2, and similar
changes were seen (not shown).
Normally, astrocytes are the only cells in the retina that express GFAP, but after various insults such as retinal detachment, light damage, or genetic photoreceptor degeneration, Muller glia become GFAP-positive.27,28 Staining for cellular retinaldehyde-binding protein (a marker for Muller cells29 ) showed no differences between rho/PDGF-A2 or -A3 transgene-positive mice and wild-type mice (not shown).
Astrocytes normally reside in the nerve fiber layer and surround
retinal blood vessels,30
and they have been demonstrated
to participate in the development of retinal blood
vessels31
and to affect their behavior, particularly for
induction of barrier characteristics.32
Staining with GSA,
an endothelial cell-selective marker, demonstrated that the
accumulation of GFAP-positive cells in the inner nuclear layer of
rho/PDGF-A transgene-positive mice was accompanied by an
accumulation of endothelial cells (Figure 5)
, demonstrating that the development of
the deep capillary beds was affected. The difference between
transgene-positive and -negative mice was most prominent at P14 and
became less noticeable at later time points.
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NV on the surface of the retina recruits cells to form epiretinal
membranes that exert traction on the retina, often resulting in
traction retinal detachments and severe loss of vision. Glial and RPE
cells are major components of vascularized epiretinal membranes in
diabetic retinopathy and other ischemic retinopathies. We hypothesized
that endothelial cell-derived PDGFs are likely to stimulate the
migration and proliferation of astrocytes and RPE cells and contribute
to their recruitment and growth in neovascular membranes. Therefore, we
anticipated that, in the oxygen-induced ischemic retinopathy model, the
excess PDGF-A and excess astrocytes in rho/PDGF-A mice would
result in increased NV compared with wild-type mice. Transgene-negative
mice with ischemic retinopathy developed extensive NV on the surface of
the retina, but, surprisingly, transgene-positive mice did not develop
any NV (Figure 6, A and B)
. A total of 10
transgene-positive mice with ischemic retinopathy were examined with
GSA lectin staining of serial sections, and none had any NV (Figure 6C)
.
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One possible explanation for the absence of NV in
rho/PDGF-A mice with ischemic retinopathy is
that the excess PDGF-A directly inhibits NV. Although this possibility
was considered unlikely, it was tested by crossing
rho/PDGF-A mice with rho/VEGF mice.
rho/VEGF mice express VEGF in photoreceptors and develop
intraretinal and subretinal NV.25
Double transgenic mice
that expressed both VEGF and PDGF-A in photoreceptors had more
neovascular lesions per retina than mice that expressed only VEGF, but
the area of individual lesions was significantly smaller, and therefore
the total area of NV per retina was not significantly different (Figure 7)
. These data indicate that PDGF-A does
not directly inhibit retinal NV.
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rho/PDGF-A and littermate control mice were placed in
75% oxygen at P7 and removed to room air at P12, which is the standard
protocol for oxygen-induced ischemic retinopathy. At sequential time
points after the onset of hyperoxia, mice were perfused with
fluorescein-labeled dextran, and retinal flat mounts were prepared. At
each time point, there was less capillary nonperfusion in the retinas
of rho/PDGF-A mice compared with wild-type mice (Figure 8)
. Measurements by image analysis
confirmed that rho/PDGF-A mice developed significantly less
nonperfused retina and had complete revascularization at P17, compared
with P21 in wild-type mice (Figure 8B)
.
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Hyperoxia results in capillary dropout in neonatal mice, because
it causes decreased expression of VEGF, and endothelial cells of newly
developed retinal vessels are dependent on VEGF for
survival.33
Exogenous VEGF rescues endothelial cells in
hyperoxic retinas and astrocytes produce VEGF.33
We
examined whether the decrease in hyperoxia-induced retinal capillary
dropout in rho/PDGF-A mice is caused by increased production
of VEGF by ectopic astrocytes, which overcomes the down-regulation by
hyperoxia. Northern blots demonstrated increased VEGF mRNA in the
retinas of P7 transgene-positive mice compared with transgene-negative
mice as predicted, but after either 4 hours or 3 days of hyperoxia,
VEGF mRNA levels were decreased to essentially undetectable levels in
both transgene-positive and -negative mice (Figure 9)
. Within hours of removing mice from
75% oxygen to the relative hypoxia of room air, there was a marked
increase in VEGF mRNA in the retinas of littermate controls, with only
a small increase in VEGF mRNA in the retinas of transgene-positive
mice. At later time points, there continued to be much greater VEGF
mRNA in the retinas of control mice compared with transgenic mice.
These data suggest that, despite increased VEGF expression by ectopic
astrocytes in the retinas of rho/PDGF-A mice under
normoxic conditions, hyperoxia effectively eliminates VEGF
expression in both transgenic and wild-type mice. Therefore, VEGF is
not responsible for retinal endothelial cell rescue from hyperoxic
damage in rho/PDGF-A mice. The increased expression of VEGF
mRNA after removal from 75% oxygen to room air in the retinas of
littermate controls compared with rho/PDGF-A mice is
consistent with the demonstration that rho/PDGF-A mice have
less retinal capillary dropout and therefore less retinal ischemia.
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Two factors other than VEGF that have survival-promoting activity
for endothelial cells are insulin-like growth factor-I (IGF-I) and
fibroblast growth factor 2 (FGF2). Northern blots with probes for IGF-I
showed that, after 5 days of hyperoxia followed by 1 hour in room air,
IGF-I mRNA was markedly increased, but there was no difference between
transgene-positive and -negative mice that would help to explain the
rescue of endothelial cells in transgenic mice. At P14 and P17 there
was somewhat greater IGF-I mRNA in the retinas of transgene-positive
mice, that could have contributed to the more rapid revascularization.
The differences for FGF2 mRNA were more striking, with a small increase
in transgene-positive mouse retinas at P12 and a large difference at
P14 (Figure 9)
. This experiment was repeated with mice from lines 2 and
3 and the differential increase in FGF2 mRNA at P12 and P14 in
transgenic mice compared with wild-type littermates was a consistent
finding. Immunohistochemistry demonstrated increased staining for FGF2
in the inner retina in the region of the ectopic glial cells of
transgenic mice (Figure 10)
. The first
time point examined was P10, which is 3 days after the onset of
hyperoxia, suggesting that hyperoxia somehow induces the production of
FGF2. These data are consistent with astrocyte-derived FGF2 acting as a
survival factor for retinal endothelial cells of hyperoxic/hypoxic
retinas of rho/PDGF-A mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important question is, why are rho/PDGF-A mice more resistant to hyperoxia-induced capillary dropout? The most likely explanation is that the additional astrocytes produce a factor(s) that promotes survival of vascular endothelial cells. Astrocytes are known to produce VEGF and exogenous VEGF rescues endothelial cells from hyperoxia-induced apoptosis.33 However, although rho/PDGF-A mice show prominent constitutive expression of VEGF, possibly due to increased numbers of astrocytes, hyperoxia causes decreased expression of VEGF in the retinas of rho/PDGF-A-transgenic mice to essentially undetectable levels, just as occurs in wild-type mice. Therefore, it is unlikely that VEGF is responsible for the decreased susceptibility to hyperoxia in rho/PDGF-A mice.
Two other survival factors for endothelial cells are IGF-I and FGF2. Northern blots showed that IGF-I was increased during the hypoxic period in both rho/PDGF-A and wild-type mice. There was no difference in IGF-I mRNA levels at P12, when there was a substantial difference in capillary nonperfusion, so IGF-I is unlikely to be responsible for the rescue of endothelial cells in rho/PDGF-A mice. However, it is interesting that IGF-I mRNA is increased in hypoxic retina, since it has been demonstrated to contribute to ischemic retinopathy34 and recently a hypoxia-inducible factor-1 response element has been demonstrated in the IGF-I promoter.35 At P14 and P17 there was slightly increased levels of IGF-I mRNA in transgenic mice, but the most dramatic difference during the critical period when enhanced survival of endothelial cells became manifested in transgenic mice was in FGF2 expression. This was confirmed to occur in mice from both lines 2 and 3, and immunohistochemistry showed that the increased staining for FGF2 occurred throughout the inner retina in the same sort of pattern of increased staining for GFAP. Increased production of FGF2 in ectopic astrocytes provides a good explanation for the decreased susceptibility to hyperoxia-induced capillary dropout in rho/PDGF-A mice. Astrocytes normally associate with retinal blood vessels and produce substances that promote barrier characteristics.32 Our findings in rho/PDGF-A mice suggest that another function of astrocytes in the retina is to promote endothelial cell survival and that FGF2 may be one mediator of this effect.
Previous studies have investigated the effect of increased expression
of PDGF-A in neurons of the inner retina or the lens. Fruttiger and
associates36
used the neuron-specific enolase (NSE)
promoter to drive expression of the short isoform of PDGF-A, beginning
before the day of birth in retinal ganglion cells and some cells of the
inner and outer nuclear layer of transgenic mice. These mice showed
numerous ectopic astrocytes that extended throughout the entire depth
of the retina, but the radial spread of astrocytes from the optic nerve
to the periphery was decreased. NV occurred in association with the
extensive astrocyte network in all retinal layers of posterior retina
and did not occur in the periphery of the retina, where there were no
astrocytes. Reneker and Overbeek37
used the
A-crystallin promoter to drive expression of PDGF-A, beginning
around embryonic day 12 in the lenses of transgenic mice. These mice
developed a several-layer-thick carpet of astrocytes on the surface of
the retina. NSE/PDGF-A36
and
A-crystallin/PDGF-A37
mice each have a larger number of
additional astrocytes and a more dramatic phenotype than
rho/PDGF-A mice. Astrocytes may be responsive to PDGF-A only
between P0 and P14,37,38
and therefore glial cell
proliferation may proceed for 2 weeks in NSE/PDGF-A and
A-crystallin/PDGF-A mice, compared with 1 week in
rho/PDGF-A mice, which would explain why the former two
types of transgenic mice show greater numbers of additional
astrocytes. The difference in location of ectopic astrocytes in
A-crystallin/PDGF-A mice compared with rho/PDGF-A mice is
probably explained by the location of PDGF-A expression. The production
of PDGF-A in the lens would provide a gradient favoring migration of
astrocytes to the retinal surface, whereas photoreceptor-derived PDGF-A
would provide a gradient favoring migration into the retina from the
retinal surface.
Unlike A-crystallin/PDGF-A or rho/PDGF-A mice, NSE/PDGF-A
mice show extensive NV in association with astrocyte proliferation.
Because astrocytes have been demonstrated to produce VEGF, it was
postulated that the NV was caused by excess astrocyte-derived VEGF from
the large number of ectopic astrocytes, although this was not
demonstrated. However, if this were the case, one would expect
prominent NV in A-crystallin/PDGF-A mice, and, although
there were a few GFAP-negative cells, which were postulated to be blood
vessel cells, there was no prominent NV. In addition to large numbers
of ectopic astrocytes, NSE/PDGF-A mice had areas of nonperfused
peripheral retina, because retinal blood vessel development did not
extend beyond the ectopic astrocytes located posteriorly. Nonperfused
retina is a source of VEGF, and this, combined with astrocyte-derived
VEGF, may be sufficient to cause NV, although the amount of VEGF
produced by ectopic astrocytes alone in A-crystallin/PDGF-A or
rho/PDGF-A mice is insufficient to cause NV.
Taken together, the findings in rho/PDGF-A,
A-crystallin/PDGF-A, and NSE/PDGF-A mice suggest that differences in
the temporal and/or spatial pattern of expression of PDGF-A in the eye
can result in large phenotypic differences in the retina. An inducible
promoter system combined with multiple retina-specific promoters would
provide a means for detailed analysis of the effects of alterations in
timing and location of PDGF-A expression. Such an analysis will be
needed to determine whether altered expression of PDGF-A in the retinas
of adult animals causes changes like those seen in proliferative
retinopathies. But even without such a detailed analysis, it appears
safe to conclude that alteration of PDGF-A expression in the eye during
development alters the number and location of astrocytes and
secondarily affects the development and survival of blood vessels.
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Footnotes |
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Supported by Public Health Service grants EY05951, EY12609, EY10017, EY09769, and core grant P30EY1765 from the National Eye Institute; a Juvenile Diabetes Foundation fellowship grant (to N. O.); a Lew R. Wasserman Merit Award (to P. A. C.); a career development award (to D. J. Z.); an unrestricted grant from Research to Prevent Blindness, Inc.; the Rebecca P. Moon, Charles M. Moon, Jr., and Dr. P. Thomas Manchester Research Fund; a grant from Mrs. Harry J. Duffey; a grant from Dr. and Mrs. William Lake; a grant from Project Insight; and a grant from the Association for Retinopathy of Prematurity and Related Diseases. P. A. C. is the George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience.
Current address for H. Yamada and E. Yamada: The Department of Ophthalmology, Kansai Medical University, Osaka, Japan.
Accepted for publication October 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit deleted patch mouse exhibits severe cardiovascular dysmorphogenesis. Teratology 1995, 51:351-366
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