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Persistent Hyperplastic Primary Vitreous in Transgenic
Mice Expressing IE180 of the Pseudorabies Virus
Satoshi Taharaguchi,1,2,3 Kazuhiko Yoshida,3,4 Yukiko Tomioka,1 Saori Yoshino,1,5
Toshimitsu Uede,6 and Etsuro Ono1
PURPOSE. Pseudorabies virus (PRV), a representative member of
the ␣-herpesvirus family, causes nervous symptoms and ocular
lesions, such as keratoconjunctivitis and retinal degeneration
in piglets. The immediate-early protein IE180 of the PRV is
known to be essential, not only in viral gene expression, but
also in the cellular gene expression in host cells. The purpose
of this study was to examine the effect of IE180 on the development of the mouse eye, by using transgenic technology.
METHODS. Transgenic mice expressing IE180 were generated
and their eyes analyzed by histology, immunocytochemistry,
and the bromodeoxyuridine cell proliferation assay.
RESULTS. A fibrovascular retrolental tissue analogous to persistent hyperplastic primary vitreous (PHPV) in humans was
observed in a transgenic mouse line expressing IE180. The
gross anatomy of the eye showed white pupils. Analysis of
hematoxylin and eosin–stained sections revealed that the
retrolental tissue adhered to the neuroretina, the inner nuclear and ganglion cell layers were disorganized, and rosettelike arrangements of dysplastic photoreceptor cells were
present. Bromodeoxyuridine-positive cells were detected in
the retrolental tissues of postnatal day (P)1, P7, and P14
mice. The retrolental mass in the P7 transgenic mouse was
composed of melanocytes and endothelial cells, which were
detected by a cocktail of antibodies against endoglin, CD31,
and VEGF receptor-2.
CONCLUSIONS. The observation that the eye disease in transgenic
mice is similar to that in PHPV in humans raises the possibility
that expression of the immediate-early gene of ␣-herpesviruses
From the 1Laboratory of Animal Experiment for Disease Model,
Institute of Genetic Medicine, the 4Department of Ophthalmology,
School of Medicine, and the 6Division of Molecular Immunology, Research Section of Molecular Pathogenesis, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; the 2Laboratory of Veterinary Microbiology, Department of Veterinary Medicine, Faculty of
Agriculture, Kagoshima University, Kagoshima, Japan; and 5Gene
Techno Science, Sapporo, Japan.
3
Contributed equally to the work and therefore should be considered equivalent authors.
Supported by Grants-in-Aid for Scientific Research (B)(2) and
(C)(2), and Encouragement of Young Scientists (A) from The Ministry
of Education, Culture, Sports, Science and Technology, Japan.
Submitted for publication June 24, 2004; revised December 2,
2004; accepted January 10, 2005.
Disclosure: S. Taharaguchi, None; K. Yoshida, None; Y.
Tomioka, None; S. Yoshino, Gene Techno Science (E); T. Uede,
None; E. Ono, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Etsuro Ono, Laboratory of Animal Experimental Models of Disease, Institute for Genetic Medicine, Hokkaido
University, Sapporo 060-0815, Japan; [email protected].
may contribute to PHPV. (Invest Ophthalmol Vis Sci. 2005;46:
1551–1556) DOI:10.1167/iovs.04-0743
P
ersistent hyperplastic primary vitreous (PHPV) is thought
to be a congenital anomaly in which the normal regression
of the primary vitreous body and hyaloid vasculature does not
occur.1 The primary vitreous is a part of the embryonic vasculature of the eye and supplies nutrients to the developing lens
and retina during early gestation.2 It is composed of the hyaloid
artery, the vasa hyaloidea propria, and the tunica vasculosa
lentis.3 PHPV, first identified by Reese,1 is a congenital malformation of the primary vitreous that is characterized by a retrolental white plaque of fibrovascular tissue. Subsequently,
Pruett and Schepens4 classified malformations involving a retrolental mass as anterior PHPV, as described by Reese,1 and
malformations involving a congenital retinal fold or ablatio
falciformis congenita as posterior PHPV. A variety of clinical
findings are associated with PHPV, including microphthalmos,
glaucoma due to closure of the chamber angle, shallowing of
the anterior chamber, corneal opacity, cataract, uveal
coloboma, and retinal degeneration. Most cases of PHPV have
no known cause, are unilateral, and are not associated with
diseases in other tissues of the body. They are therefore best
considered to be idiopathic isolated sporadic congenital malformation syndromes localized to the eye.5
Pseudorabies virus (PRV) is classified into the genus Varicellovirus of the subfamily Alphaherpesvirinae.6 PRV causes
not only nervous symptoms, such as unbalanced walking, trembling, staggering, and convulsions, but also ocular lesions, such
as keratoconjunctivitis7,8 and retinal degeneration9 in piglets.
PRV invades and spreads within the trigeminal pathway (the
nasal mucosa, the trigeminal ganglion, the pons/medulla, and
the cerebellum/thalamus) of neonatal pigs.10,11
PRV expresses a single IE protein (IE180), with a molecular
weight of 180 kDa, for continuous transcription of late genes
and shutting off the synthesis of its own RNA.12–14 In addition,
IE180 is known to be a strong transactivator of several promoters, including other viral and cellular genes,12,14 –16 and binds
to specific sites on class II gene promoters17 and singlestranded DNA.18 Taken together, these findings suggest the
possibility that IE180 is not only essential in viral gene expression, but also participates in gene expression in the host cells.
To investigate whether IE180 affects the murine’s normal development, we have generated transgenic mouse lines expressing IE180 and have shown that IE180 affects the cascade of
gene expression for development of the murine cerebellum.19
IE180 is a multifunctional transcription factor that activates
transcription from a variety of promoters and represses transcription from its own promoter.12–16 We have reported that
transcription factors were involved in development of the
eye.20 –24
In the current study, a transgenic mouse line expressing
IE180 showed an abnormality analogous to PHPV in humans in
their eyes. The transgenic mice showing PHPV described in
Investigative Ophthalmology & Visual Science, May 2005, Vol. 46, No. 5
Copyright © Association for Research in Vision and Ophthalmology
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FIGURE 1. Eyes of TgIE110 and littermate mice showing the white
pupil observed in the transgenic eye.
this study are the first demonstration that PHPV can be caused
by expression of the immediate-early gene of ␣-herpesviruses.
MATERIALS
AND
were detected by PCR analysis using specific primers, as described
previously.19 The transgene copy number was estimated by comparing
the band intensity of transgenic mouse DNA with that of control DNA
by Southern blot analysis. The DNA samples (10 ␮g) were digested
with EcoRI, fractionated on 0.8% agarose gels, and transferred to
membranes (Hybond N⫹; Amersham Pharmacia Biotech, Piscataway,
NJ) by capillary blotting. Digoxigenin (DIG)-labeled DNA probes for
detection of the transgene were derived from pTet/IE180 using the
specific primers and a PCR DIG probe synthesis kit (Roche Diagnostics,
Indianapolis, IN). Hybridization and detection of the transgene were
performed as described previously.26 All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research. Briefly, all mice were maintained in the animal facility
at our institute and treated according to the Laboratory Animal Control
Guidelines of our institute, which conform to those of the National
Institutes of Health-American Association of Laboratory Animal
Control.
METHODS
Generation of Transgenic Mice
Analysis of the Transgene Expression
19
Transgenic mice were generated as described previously. Briefly,
approximately 500 copies of the 7.2-kb HindIII transgene fragment and
the 3.3-kb XhoI-NotI fragment containing the Tet promoter, the tetracycline transactivator (tTA) gene, and the SV40 intron/polyA signal
from pTet-tTAk (Invitrogen, Carlsbad, CA) were comicroinjected into
the pronuclei of fertilized B6C3F1 (C57BL/6 X C3H/He) mouse embryos. Genomic DNA was isolated from mouse tail.25 Both transgenes
Transgenic mice (3 weeks old) were killed by decapitation, and tissue
samples were immediately removed and frozen in liquid nitrogen.
Total cellular RNA was isolated from various tissues of the transgenic
mice and RT-PCR analyses were performed as described previously.19
To analyze expression level of IE180 gene in transgenic eyes at postnatal day (P)1, P7 and P14, we performed quantitative RT-PCR assays,
as described previously.19
FIGURE 2. (a) Southern blot analysis
of the introduced transgene. Copy
numbers of control DNA derived
from pTet/IE180 are shown at the
top of the four right lanes. Arrow:
position of the 4.8-kb EcoRI fragment
containing the IE180 gene. The copy
number of the transgene integrated
in the genomic DNA is shown below.
(b) Expression of the IE180 gene in
various tissues of TgIE110. Total
RNAs prepared from the tissues
shown were analyzed by RT-PCR.
Shown is a representative result of
Southern blot analysis using RT-PCR
products from the various tissues.
␤-Actin was the control. ⫹, Positive
control product synthesized from the
transgene DNA fragment by PCR. (c)
Quantitative RT-PCR analysis of the
transgene expression in the transgenic eyes at P1, P7, and P14.
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IOVS, May 2005, Vol. 46, No. 5
PHPV in Transgenic Mice Expressing PRV IE180
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compound (Sakura Finetek, Inc., Torrance, CA), and snap frozen.
Frozen histologic sections (20 ␮m) were cut with a cryostat (Reichert,
Inc., Buffalo, NY). Endothelial cells were identified by a cocktail of
antibodies against endoglin (CD105), CD 31, and VEGF receptor-2
(flk-1; BD-PharMingen, San Diego, CA), a strategy that has been used to
maximize labeling of all vascular endothelial cells, both immature and
mature.32,33
RESULTS
Characterization of Transgenic Mouse Lines
To assess neuropathogenic potentials of pseudorabies virus
IE180, transgenic mice expressing the immediate-early gene
were generated. In the course of breeding of transgenic mice,
we observed that one of the transgenic mouse lines (TgIE110)
showed abnormalities in their eyes. White pupils were observed in 11 of 12 transgenic mice, but not in 12 of their
nontransgenic littermates (Fig. 1). TgIE110 mice gave rise to
FIGURE 3. Abnormal retrolental tissue observed in the eyes of
TgIE110 mice. Photomicrographs of sections of eyes from TgIE110
mice at 12 weeks of age. (c, e) Detail of (a); (d, f) detail of (b). Hyaloid
arteries were present in the tissue (a–c, e, f, arrows). The retrolental
tissue adhered to the neuroretina (b, d, arrowhead), (e, ✽) lens. Lens
sections were stained with hematoxylin and eosin. Scale bar: (a, b) 330
␮m; (c, d) 60 ␮m; (e, f) 20 ␮m.
Treadmill Test
A treadmill apparatus (Rotarod; Ugo Basile, Camerio, Italy) was used
for measuring the mice’s fore- and hindlimb motor coordination and
balance.27 Mice (8 weeks old) were placed on the treadmill at a
constant speed (20 rpm/min) for a maximum of 300 seconds, and the
time until they fell off the treadmill within this period was recorded.
Mice underwent three trials per day, by which time a steady baseline
level of performance was attained.
Histological Procedures
Various tissue samples including eyes of decapitated animals (12 weeks
old and P1, P7, and P14) were excised and immersion-fixed in 4%
paraformaldehyde (PFA) solution for 24 hours at room temperature,
embedded in paraffin, and cut into 4-␮m-thick slices with a microtome.
For bromodeoxyuridine (BrdU) labeling, mice were injected with
BrdU peritoneally at a dosage of 30 mg/kg body weight 30 minutes
before sampling. Anti-BrdU staining was performed as described previously.28 –31 Briefly, the sections were immersed in pepsin solution
(0.4 mg/mL) in 0.1 M HCl at 30°C for 1 minute and then in 2 M HCl at
40°C for 1 hour. After a wash in PBS, the slides were incubated with
normal goat serum and then with the anti-BrdU antibody (dilution,
1:1000; BD Biosciences, Franklin Lakes, NJ). Binding of the primary
antibody was localized by fluorescence microscopy using FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories,
West Grove, PA) at a dilution of 1:200. The slides were examined by
laser scanning confocal microscopy (MRC-1024; Bio-Rad, Hercules, CA;
and LSM 510; Carl Zeiss Meditec, Dublin, CA).
Tissue samples from mice at P7, after fixation, were immersed in
20% sucrose in PBS at 4°C, embedded in optimal temperature cutting
FIGURE 4. Abnormal retrolental tissues observed in the eyes of neonatal TgIE110 mice. Hematoxylin and eosin staining (a–f) and immunodetection of BrdU (g, h) of the nontransgenic (a, c, e, g) and
TgIE110 (b, d, f, h) mice at postnatal day 1. The retrolental tissue was
present in the TgIE110 mouse (b, arrow and arrowhead). Melanocytes were present in the tissue (f, arrows). BrdU-positive cells were
detected in the retrolental tissue of the TgIE110 mouse (h), but not in
the hyaloid vessels in the nontransgenic mouse (c, e, arrowhead).
Scale bar: (a, b) 400 ␮m; (c, d) 100 ␮m; (e–h) 40 ␮m.
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Taharaguchi et al.
FIGURE 5. Immunodetection of BrdU-positive cells in the retrolental
tissue of the TgIE110 mice. Hematoxylin and eosin staining (a, c, e, g)
and immunodetection of BrdU (b, d, f, h) of the nontransgenic (a, b,
e, f) and TgIE110 (c, d, g, h) mice of P7 (a–d) and P14 (e–h). Scale bar:
100 ␮m.
offspring in crosses with C57BL/6 mice and transmitted the
introduced gene in Mendelian fashion. Southern blot analysis
performed on genomic DNA from the transgenic mouse
showed that 35 copies of the transgene were integrated (Fig.
2a). The transgene expression in various tissues at 3 weeks of
age was assessed by RT-PCR. The expected 296-bp PCR product of IE180 mRNA was detected in all the tested tissues,
including the eye (Fig. 2b). The expression level of the transgene varied among the tested tissues, with expression consistently high in cerebrum and cerebellum. However, the expression levels in cerebella of TgIE110 mice were rather low
compared with TgIE96 mice showing severe cerebellar symptoms (approximately 5% of that of TgIE96 mice), although
there was no difference between both lines in the tissue specificity of the transgene expression and in the expression levels
in eyes (data not shown). In the tTA gene expression, a similar
expression pattern was observed (data not shown). To examine whether IE180 expression level changed in early postnatal
stages, quantitative RT-PCR analysis using transgenic eyes was
performed. There was no difference in the levels of IE mRNA
accumulation among the eyes at postnatal day (P)1, P7, and
P14 (Fig. 2c). Because we have reported that four transgenic
mouse lines expressing IE180 showed motor discoordination,19 motor coordination and balance of TgIE110 mice were
measured on a treadmill (Rotarod; Ugo Basile). TgIE110 mice
maintained constant balance on the treadmill, similar to their
nontransgenic littermates (data not shown). Histopathological
analyses of the cerebella of several TgIE110 mice were performed. Failure of layer formation and/or reduction in size, as
reported previously,19 was not observed in their cerebella
(data not shown).
IOVS, May 2005, Vol. 46, No. 5
ganglion cell layer (GCL) were disorganized, and rosettelike
arrangements of dysplastic photoreceptor cells were observed
(Fig. 3c).
On P1, hyaloid vessels were present in the vitreous of
nontransgenic littermates (Figs. 4c, 4e, arrowhead), as described previously.34 In contrast, a retrolental mass was
present in the vitreous of TgIE110 eyes on P1 (Figs. 4b, 4d, 4f).
Retrolental tissue was observed in both eyes of all TgIE110
mice (3/3), but not in nontransgenic littermates (0/4). In the
retrolental tissue on the vitreous of TgIE110 mice, melanocytes
were present (Fig. 4f, arrows). Whereas retinal folds were
observed in TgIE110 eyes, the rest of the neuroretinas of
TgIE110 mice were morphologically similar to those of nontransgenic littermates. We examined the incorporation of
BrdU, which was incorporated only into cells that were in the
S-phase, and a portion of these reflected proliferative activity.
When TgIE110 mice and nontransgenic littermates were injected with BrdU 30 minutes before death, BrdU-positive cells
were detected in the retrolental tissue of the TgIE110 (Fig. 4h),
but not in the hyaloid vessels in the nontransgenic littermates
(Fig. 4g). The BrdU-positive retrolental mass was also present
in the TgIE110 mice at P7 and P14, as shown in Figure 5.
Careful examination revealed that the retrolental mass was
composed of melanocytes and endothelial cells, detected by a
cocktail of antibodies against endoglin (CD105), CD 31, and
VEGF receptor-2 (flk-1; Fig. 6).
DISCUSSION
A retrolental mass presented in the vitreous of TgIE110 eyes on
postnatal day 1 (P1; Figs. 4b, 4d, 4f). Retrolental tissue was
observed in both eyes of all TgIE110 mice (3/3), but not in
nontransgenic littermates (0/4). The phenotype was completely penetrant in the TgIE110 line. More than half of the
adult animals showed a folding of the retina adjacent to the
hyaloid artery. In these areas, the retrolental tissue adhered to
the neuroretina (Figs. 3b, 3d, 3f), the INL and GCL were
disorganized, and rosettelike arrangements of dysplastic photoreceptor cells were observed. The remainder of the neuroretina of TgIE110 mice was morphologically similar to that of
nontransgenic littermates, suggesting that the progressive nature of the neuroretina was secondary to the progressive attachment of the retrolental tissue to these structures.
Ocular abnormalities found in transgenic mice expressing
PRV IE180 were similar to those in patients with PHPV, with
Histopathological Analyses of Transgenic Eyes
In control mice at 12 week of age, there was no evidence of
hyaloid vasculature and primary vitreous (data not shown). In
contrast to the vitreous in littermates, fibrovascular retrolental
tissue was detected in TgIE110 mice. Many melanocytes were
present in the mesenchymal tissue on the vitreous (Figs. 3e,
3f). The manifestation of retrolental tissue in TgIE110 mice was
variable. Some animals had only a thin, tapering stalk of persistent hyaloid artery (Figs. 3a, 3c, 3e), which just reached the
lens where it spread into a funnel-shaped retrolental mass.
However, in more than half of the adult animals, a folding of
the retina adjacent to the hyaloid artery was observed (Fig. 3b).
In these areas, the retrolental tissue adhered to the neuroretina
(Figs. 3b, 3d, arrowhead), the inner nuclear layer (INL) and the
FIGURE 6. Immunodetection of endothelium cells in the retrolental
tissue of the TgIE110 mice. (a) Detail in the retrolental tissues in the
eye 7 days after birth. (b) Immunodetection of endothelium cells in
the retrolental tissue. A serial section was processed by immunohistochemistry to identify the vascular endothelium (combined
CD31⫹CD105⫹flk-1). Scale bars: 20 ␮m.
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IOVS, May 2005, Vol. 46, No. 5
the following pathologic characteristics: (1) the presence of
retrolental tissue, which can contain melanocytes; (2) the attachment of the retrolental tissue to the inner neuroretina; (3)
retrolental tissue-induced traction on the neuroretina, which
causes neuroretinal detachment from the retina pigment epithelium; and (4) cellular disorganization and other dysplastic
changes in the neuroretina.5,35
The vessels in the retrolental mass of TgIE110 mice included endothelial cells that were detected by a cocktail of
antibodies against endoglin (CD105), CD 31, and VEGF receptor-2 (flk-1; Fig. 6). When the transgenic mice were injected
with BrdU 30 minutes before death, BrdU-positive cells were
detected in the retrolental tissue of TgIE110 mice but were not
detected in the hyaloid vessels in the wild-type mice. These
findings suggest that IE180 is involved in cell proliferation
during the development of the vitreous. The mesenchymal
tissue of the vitreous of TgIE110 mice contained scattered
melanocytes. It has been reported that the primary vitreous
develops from melanocytes.36 The pathologic observation in
TgIE110 mice indicates that the mesenchymal retrolental tissues were caused, at least in part, by the abnormal migration of
excessive cells derived from the neural crest cells, because
melanocytes and pericytes in the craniofacial structures including eye and brain have been shown to be of neural crest
origin.37– 40
IE180 is a multifunctional transcription factor that activates
transcription from a variety of promoters and represses transcription from its own promoter. It is therefore thought that
IE180 affects the serially and precisely regulated gene expression in murine eye development, resulting in disruption of
ocular morphogenesis. The observation that the eye disease in
transgenic mice is similar to that in PHPV in humans raises the
possibility that expression of the immediate early gene of
␣-herpesviruses may contribute to this disease, which is usually
unilateral and sporadic.5,35
PHPV in Transgenic Mice Expressing PRV IE180
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References
1. Reese AB. Persistent hyperplastic primary vitreous. Am J Ophthalmol. 1955;40:317–331.
2. Kaste SC, Jenkins JJ III, Meyer D, Fontanesi J, Pratt CB. Persistent
hyperplastic primary vitreous of the eye: imaging findings with
pathologic correlation. AJR Am J Roentgenol. 1994;162:437– 440.
3. Wells RG, Miro P, Brummond R. Color-flow Doppler sonography of
persistent hyperplastic primary vitreous. J Ultrasound Med. 1991;
10:405– 407.
4. Pruett RC, Schepens CL. Posterior hyperplastic primary vitreous.
Am J Ophthalmol. 1970;69:534 –543.
5. Goldberg MF. Persistent fetal vasculature (PFV): an integrated
interpretation of signs and symptoms associated with persistent
hyperplastic primary vitreous (PHPV). LIV Edward Jackson Memorial Lecture. Am J Ophthalmol. 1997;124:587– 626.
6. Kit S. Pseudorabies virus. Webster RG, Granoff A, eds. Encyclopedia of Virology. London: Academic Press; 1994.
7. Schneider WJ. Viral pathogenesis during experimental pseudorabies keratoconjunctivitis in the pig. J Infect Dis. 1973;128:598 –
604.
8. Schneider WJ, Howarth JA. Clinical course and histopathologic
features of pseudorabies virus-induced keratoconjunctivitis in pigs.
Am J Vet Res. 1973;34:393– 401.
9. Howarth JA, De Paoli A. An enzootic of pseuodorabies in swain in
California. J Am Vet Med Assoc. 1968;152:1114 –1118.
10. Kritas SK, Pensaert MB. Role of gp63 and gIII of Aujeszky’s disease
virus in the invasion of the olfactory nervous pathway in neonatal
pigs. Acta Vet Hung. 1994;42:309 –316.
11. Kritas SK, Pensaert MB, Mettenleiter TC. Invasion and spread of
single glycoprotein deleted mutants of Aujeszky’s disease virus
25.
26.
27.
28.
29.
30.
31.
32.
33.
1555
(ADV) in the trigeminal nervous pathway of pigs after intranasal
inoculation. Vet Microbiol. 1994;40:323–334.
Green MR, Treisman R, Maniatis T. Transcriptional activation of
cloned human beta-globin genes by viral immediate-early gene
products. Cell. 1983;35:137–148.
Ihara S, Feldman L, Watanabe S, Ben-Porat T. Characterization of
the immediate-early functions of pseudorabies virus. Virology.
1983;131:437– 454.
Wu L, Berk AJ. Transcriptional activation by the pseudorabies virus
immediate early protein requires the TATA box element in the
adenovirus 2 E1B promoter. Virology. 1988;167:318 –322.
Wong ML, Ho TY, Huang JH, Hsiang CY, Chang TJ. Stimulation of
type I DNA topoisomerase gene expression by pseudorabies virus.
Arch Virol. 1997;142:2099 –2105.
Yuan R, Bohan C, Shiao FC, et al. Activation of HIV LTR-directed
expression: analysis with pseudorabies virus immediate early gene.
Virology. 1989;172:92–99.
Cromlish WA, Abmayr SM, Workman JL, Horikoshi M, Roeder RG.
Transcriptionally active immediate-early protein of pseudorabies
virus binds to specific sites on class II gene promoters. J Virol.
1989;63:1869 –1876.
Chlan CA, Coulter C, Feldman LT. Binding of the pseudorabies
virus immediate-early protein to single-stranded DNA. J Virol.
1987;61:1855–1860.
Taharaguchi S, Kon Y, Yoshino S, Ono E. Impaired development of
the cerebellum in transgenic mice expressing the immediate-early
protein IE180 of pseudorabies virus. Virology. 2003;307:243–254.
Sakai M, Serria MS, Ikeda H, et al. Regulation of c-maf gene expression by Pax6 in cultured cells. Nucleic Acids Res. 2001;29:1228 –
1237.
Sakai MIJ, Yoshida K, Ogata A, et al. Rat maf related genes: specific
expression in chondrocytes, lens and spinal cord. Oncogene.
1997;14:745–750.
Yoshida K, Hu Y, Karin M. I␬B kinase alpha is essential for development of the mammalian cornea and conjunctiva. Invest Ophthalmol Vis Sci. 2000;41:3665–3669.
Yoshida K, Imaki J, Koyama Y, et al. Differential expression of
maf-1 and maf-2 genes in the developing rat lens. Invest Ophthalmol Vis Sci. 1997;38:2679 –2683.
Yoshida K, Kim JI, Imaki J, et al. Proliferation in the posterior
region of the lens of c-maf⫺/⫺ mice. Curr Eye Res. 2001;23:116 –
119.
Hogan BF, Constantin F, Kacy E. Manipulating the Mouse Embryo. A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1986:153–163.
Ono E, Tasaki T, Kobayashi T, et al. Resistance to pseudorabies
virus infection in transgenic mice expressing the chimeric transgene that represses the immediate-early gene transcription. Virology. 1999;262:72–78.
Jones BJ, Roberts DJ. A rotarod suitable for quantitative measurements of motor incoordination in naive mice. Naunyn Schmiedebergs Arch Exp Pathol Pharmakol. 1968;259:211.
Dean PN, Dolbeare F, Gratzner H, Rice GC, Gray JW. Cell-cycle
analysis using a monoclonal antibody to BrdUrd. Cell Tissue Kinet.
1984;17:427– 436.
Dolbeare F, Gratzner H, Pallavicini MG, Gray JW. Flow cytometric
measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci USA. 1983;80:5573–5577.
Nowakowski RS, Lewin SB, Miller MW. Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and
the DNA-synthetic phase for an anatomically defined population.
J Neurocytol. 1989;18:311–318.
Yoshiki A, Hanazono M, Oda S, et al. Developmental analysis of the
eye lens obsolescence (Elo) gene in the mouse: cell proliferation
and Elo gene expression in the aggregation chimera. Development. 1991;113:1293–1304.
Chang YS, di Tomaso E, McDonald DM, et al. Mosaic blood vessels
in tumors: frequency of cancer cells in contact with flowing blood.
Proc Natl Acad Sci USA. 2000;97:14608 –14613.
Ozerdem U, Stallcup WB. Early contribution of pericytes to angiogenic sprouting and tube formation. Angiogenesis. 2003;6:241–
249.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933231/ on 05/08/2017
1556
Taharaguchi et al.
IOVS, May 2005, Vol. 46, No. 5
34. Ito M, Yoshioka M. Regression of the hyaloid vessels and pupillary
membrane of the mouse. Anat Embryol (Berl). 1999;200:403–
411.
35. Haddad R, Font RL, Reeser F. Persistent hyperplastic primary
vitreous: a clinicopathologic study of 62 cases and review of the
literature. Surv Ophthalmol. 1978;23:123–134.
36. Matsuo T. The genes involved in the morphogenesis of the eye.
Jpn J Ophthalmol. 1993;37:215–251.
37. Beauchamp GR, Knepper PA. Role of the neural crest in anterior
segment development and disease. J Pediatr Ophthalmol Strabismus. 1984;21:209 –214.
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38. Noden DM. Periocular mesenchyme: neural crest and mesodermal interactions. In: Tasman W, Jaeger EA, eds. Foundations of Clinical Ophthalmology. Philadelphia: JB Lippincott;
1991:1–23.
39. Etchevers HC, Vincent C, Le Douarin NM, Couly GF. The cephalic
neural crest provides pericytes and smooth muscle cells to all
blood vessels of the face and forebrain. Development. 2001;128:
1059 –1068.
40. Korn J, Christ B, Kurz H. Neuroectodermal origin of brain pericytes and vascular smooth muscle cells. J Comp Neurol. 2002;442:
78 – 88.
M
Erratum in: “The Inhibitory Interaction between Human Corneal and Conjunctival Sensory
Channels” by Feng and Simpson (Invest Ophthalmol Vis Sci. 2005;46:1251–1255).
In the published Figure 1, the lower right-hand labels were switched. The correct figure and
labels are shown below.
FIGURE 1. Corneal and conjunctival mechanical transducer functions. The conjunctival scaling curves are
lower than the corneal one. The paired conjunctival curve is even lower than the unpaired one. The
separation of the two conjunctival curves is more apparent at high than at low stimulus intensity.
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