Deletion of HIF-1α partially rescues the abnormal hyaloid vascular system in Cited2 conditional knockout mouse eyes.

Abstract

Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2) is a member of a new family of transcriptional modulators. Cited2 null embryos exhibit hyaloid hypercellularity consisting of aberrant vasculature in the eye. The purpose of the study is to address whether abnormal lenticular development is a primary defect of Cited2 deletion and whether deletion of hypoxia inducible factor (HIF)-1α or an HIF-1α target gene, vascular endothelial growth factor (VEGF), could rescue abnormal hyaloid vascular system (HVS) in Cited2 deficient adult eyes.
Le-Cre specific Cited2 knockout (Cited2(CKO)) mice with or without deletion of HIF-1α or VEGF were generated by standard Cre-Lox methods. Eyes collected from six-eight weeks old mice were characterized by Real Time PCR and immunohistological staining.
Cited2(CKO) mice had smaller lenses, abnormal lens stalk formation, and failed regression of the HVS in the adult eye. The eye phenotype had features similar to persistent hyperplastic primary vitreous (PHPV), a human congenital eye disorder leading to abnormal lenticular development. Deletion of HIF-1α or VEGF in Cited2 knockout eyes partially rescued the abnormal HVS but had no effect on the smaller lens and abnormal lens stalk differentiation. Intravitreal injection of Topotecan (TPT), a compound that inhibits HIF-1α expression, partially eliminated HVS defects in Cited2(CKO) lenses.
Abnormal HVS is a primary defect in Cited2 knockout mice, resulting in part from dysregulated functions of HIF-1 and VEGF. The Cited2(CKO) mouse line could be used as a novel disease model for PHPV and as an in vivo model for testing potential HIF-1 inhibitors.

Full text

Page 1

Deletion of HIF-1α partially rescues the abnormal hyaloid vascular
system in Cited2 conditional knockout mouse eyes
Tai-Qin Huang,1 Yiwei Wang,1 Quteba Ebrahem,2 Yu Chen,3 Cindy Cheng,1 Yong Qiu Doughman,4
Michiko Watanabe,4 Sally L. Dunwoodie,5 Yu-Chung Yang1
1Department of Biochemistry and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH;
2Department of Ophthalmology, Cole Eye Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, OH; 3Department of
Pharmacology, Rainbow Babies' and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, OH;
4Department of Pediatrics, Rainbow Babies' and Children's Hospital, Case Western Reserve University School of Medicine,
Cleveland, OH; 5Developmental and Stem Cell Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst NSW.
St. Vincent’s Clinical School University of New South Wales, Kensington, NSW, Australia
Purpose: Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2) is a
member of a new family of transcriptional modulators. Cited2 null embryos exhibit hyaloid hypercellularity consisting
of aberrant vasculature in the eye. The purpose of the study is to address whether abnormal lenticular development is a
primary defect of Cited2 deletion and whether deletion of hypoxia inducible factor (HIF)-1α or an HIF-1α target gene,
vascular endothelial growth factor (VEGF), could rescue abnormal hyaloid vascular system (HVS) in Cited2 deficient
adult eyes.
Methods: Le-Cre specific Cited2 knockout (Cited2CKO) mice with or without deletion of HIF-1α or VEGF were generated
by standard Cre-Lox methods. Eyes collected from six-eight weeks old mice were characterized by Real Time PCR and
immunohistological staining.
Results: Cited2CKO mice had smaller lenses, abnormal lens stalk formation, and failed regression of the HVS in the adult
eye. The eye phenotype had features similar to persistent hyperplastic primary vitreous (PHPV), a human congenital eye
disorder leading to abnormal lenticular development. Deletion of HIF-1α or VEGF in Cited2 knockout eyes partially
rescued the abnormal HVS but had no effect on the smaller lens and abnormal lens stalk differentiation. Intravitreal
injection of Topotecan (TPT), a compound that inhibits HIF-1α expression, partially eliminated HVS defects in
Cited2CKO lenses.
Conclusions: Abnormal HVS is a primary defect in Cited2 knockout mice, resulting in part from dysregulated functions
of HIF-1 and VEGF. The Cited2CKO mouse line could be used as a novel disease model for PHPV and as an in vivo model
for testing potential HIF-1 inhibitors.
The lens consists of the lens capsule, the lens epithelium,
and the lens fibers. The lens capsule is a collagen containing
basement membrane structure that completely surrounds the
lens. The lens epithelium is a simple cuboidal epithelium
between the lens capsule and the lens fibers in the anterior
portion of the lens. The lens fibers are long, thin, transparent
cells filled with crystalline proteins to ensure lens
transparency [1]. Lens development begins in the ectoderm
with formation of the lens placode. In the vertebrate eye, lens
development is accompanied by the growth of the surrounding
capillary network. This network is found within the anterior
papillary membrane (APM) on the anterior surface of the lens
and includes the tunica vasculosalentis (TVL) posterior to the
lens and provides nutrients to intraocular components. The
fetal vasculature normally regresses starting late in fetal life
and completes regression in the first two weeks after birth in
Correspondence to: Yu-Chung Yang Department of Biochemistry
and Cancer Center, Case Western Reserve University School of
Medicine, Cleveland, OH, 44106; Phone: (216) 368-6931; FAX:
(216) 368-3419; email: yxy36@cwru.edu
rodents [2]. After vascular regression, the lens becomes
transparent and resides in a hypoxic environment. However,
the role of hypoxia involved in lens development remains
elusive.
One of the major transcription factors governing hypoxic
responses is HIF-1, which is a heterodimeric protein
composed of HIF-1α and HIF-1β subunits [3]. HIF-1α is
degraded through a proteasome-mediated pathway under
normoxia but stabilized under hypoxia [4]. Stabilized
HIF-1α dimerizes with HIF-1β, binds to gene promoters
containing hypoxia-response elements (HREs) and recruits
transcriptional coactivators such as CREB-binding protein
(CBP), E1A binding protein p300 (p300), or steroid receptor
coactivator-1 (SRC-1) [5-7]. Although hypoxia has been
implicated in controlling normal homeostasis and
pathological conditions, the molecular mechanism of how
HIF-1α interacts with transcription cofactors to initiate target
gene expression is not clearly understood. As a coactivator,
CBP/p300 interacts with HIF-1α through its cysteine/
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132>
Received 20 February 2012 | Accepted 9 May 2012 | Published 11 May 2012
© 2012 Molecular Vision
1260

Page 2

histidine-rich 1 (CH1) domain to modulate HIF-1 target gene
expression [6].
Recently, we and others found that Cited2 (CBP/p300-
interacting transactivator, with glutamic acid (E) and aspartic
acid (D)-rich tail 2), a transcriptional modulator, is a negative
regulator for HIF-1 through its competitive binding with
HIF-1α to the CH1 domain of CBP/p300 [8,9]. It was first
demonstrated by Bhattacharya et al. [10] based on in vitro
transfection studies that a p300 CH1 mutant peptide, defective
in HIF-1α but not Cited2 binding, enhanced endogenous
HIF-1 function. This notion was further supported by the
similar nuclear magnetic resonance (NMR) structures of
HIF-1α/p300-CH1 and Cited2/p300-CH1 complexes and the
identification of the LPXL motif in both HIF-1α and Cited2
as the basis for the competition [8]. In our studies, we showed
that Cited2 is a negative regulator of HIF-1 in embryonic heart
[9,11] and eye [12] using Cited2 knockout mice. The
transcript levels of HIF-1 target genes such as
phosphoglycerate kinase 1 (PGK1), glucose transporter 1
(Glut1), and vascular endothelial growth factor (VEGF) were
highly expressed in the Cited2−/−embryonic heart [9].
Furthermore, embryonic heart defects could be rescued by
HIF-1α heterozygosity in Cited2−/−embryos [11]. Similarly,
deletion of HIF-1α in the lens specifically eliminated the
hypercellularity and aberrant structure of the hyaloid
vasculature in Cited2−/− embryonic eyes [12]. We therefore
demonstrated that Cited2 exerts a unique feedback regulatory
mechanism to limit excess HIF-1 activation and to maintain
normal tissue homeostasis.
VEGF is one of the HIF-1target genes involved in early
vascular development and angiogenesis. It functions by
binding to the transmembrane tyrosine kinase receptor
vascular endothelial growth factor receptor-1 (VEGFR1;
Flt-1) and VEGFR2 (Flk-1, KDR) on the cell surface. Deletion
of one allele of VEGF or disruption of VEGFR2 leads to
embryonic lethality [13]. VEGF is expressed in mouse lens
epithelial and fiber cells. Overexpression of VEGF in the
mouse lens induces microphthalmia, hypertrophy, and
persistence of the hyaloid vasculature [14]. Lenses lacking
VEGF are smaller in size with mild nuclear opacities that
regress with age [15]. Transgenic mice overexpressing stable
forms of HIF-1α in lens epithelial cells have smaller lenses at
birth and the tunica vasculosa lentis (TVL) do not form,
although the biologic consequences of HIF-1 overexpression
or hyperactivation on the hyaloid vasculature have not been
demonstrated [16].
Persistent hyperplastic primary vitreous (PHPV), also
known as persistent fetal vasculature, is a congenital
abnormality of the eye caused by the failure of regression of
the primary vitreous [17]. During embryogenesis of human
eye, nutrients are provided by a hyaloid artery between the
retina and crystalline lens, which is later replaced by the
developing retinal vasculature.
However, failure of regression of the primary vitreous
during third to ninth months of gestation causes PHPV [17,
18]. In most case, PHPV is sporadic and unilateral while
bilateral PHPV is rare [19]. The disease is complicated and
often associated with other ocular abnormalities. The
conditions that may mimic PHPV include microphthalmia,
congenital cataract, corneal opacity, uveal coloboma, and
retinal degeneration [17]. Although several genes, such as
protein 53 (p53) [20], alternative reading frame (Arf) [21,22],
norrie disease pseudoglioma (NDP) [23], and genes in the int
and wg (wingless; Wnt) [24] signaling pathway have been
implicated in the development of PHPV, the exact
mechanisms have not been resolved.
We previously reported lens stalk formation and hyaloid
hypercellularity in Cited2 knockout mouse embryos, most
likely through dysregulated HIF-1 function. To address
whether the phenotype is a primary defect in the lens, we
generated tissue-specific Cited2 knockout mice. Since
deletion of HIF-1α partially rescues hyaloid hypercellularity
and aberrant vasculature in Cited2 knockout embryos, we also
tested the role of HIF-1 and its target gene VEGF in lens
development by generating compound mice in which Cited2
and HIF-1α or VEGF were deleted in the lens. Based on the
fact that Cited2 is a negative regulator of HIF-1, we explored
the possibility that the Cited2CKO mouse could be an in vivo
model for testing potential HIF-1 inhibitors that may be useful
for therapies in several clinical settings.
METHODS
Animals: Cited2flox/flox (Cited2fl/fl) mice (Cited2tm1.1Dunw [25]);
with C57BL6 genetic background were mated with Le-Cre+/
−mice [26] to generate Cited2fl/fl;Le-Cre- and Cited2fl/fl;Le-Cre
+ (referred to as Cited2CKO) mice. To generate Le-Cre specific
Cited2 and HIF-1α knockout mice, Cited2fl/fl;HIF-1αfl/+ mice
were mated with Cited2fl/fl;HIF-1αfl/fl;Le-Cre+ mice. Mice
with deletion of Cited2 and VEGF in the lens were generated
by mating Cited2fl/fl;VEGFfl/+ mice with Cited2fl/fl;VEGFfl/
+;Le-Cre+/−. Primers for genotyping were: Cited2-flox:
antisense (a), 5′-CTG CTG CTG CTG GTG ATG AT-3′ and
sense(s), 5′-GTC TCA GCG TCT GCT CGT TT-3′; Le-Cre:
(a), 5′-GCA TTA CCG GTC GAT GCA ACG AGT GAT
GAG-3′ and (s), 5′-GAG TGA ACG AAC CTG GTC GAA
ATC AGT GCG-3′; HIF-1α-flox: (a), 5′-ATA TGC TCT TAT
GAA GGG GCC TAT GGA GGC-3′ and (s), 5′-GAT CTT
TCC GAG GAC CTG GAT TCA ATT CCC-3′; VEGF-flox:
(a), 5′-ACA CAG GAC GGC TTG AAG AT-3′ and (S), 5′-
CTA CTG CCG TCC GAT TGA GA-3′.
Histology: Eyes were removed from euthanized mice and
fixed in 10% formalin at 4 0C for 24 h. Transverse 7 μm
paraffin embedded sections were collected, hematoxylin-
eosin (H&E) stained and examined by light microscopy.
Immunofluorescence staining: Eyes were removed and
mounted in optimal cutting temperature (OCT) immediately
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132> © 2012 Molecular Vision
1261

Page 3

following sacrifice of the animal. Five μm frozen sections
were fixed in 4% paraformaldehyde/PBS for 10 min and
blocked with 10% normal goat serum (NGS) and 0.1% Triton
X-100 in PBS for 30 min at room temperature. Sections were
incubated with primary antibodies in a humidified chamber
overnight at 4 0C. Immunostaining was performed by applying
antibodies against Cited2 (Santa Cruz, Santa Cruz, CA or R&
D, Minneapolis, MN), α-smooth muscle actin (α-SMA;
Sigma, St. Louis, MO), F4/80 (AbDSerotec, Raleigh, NC),
and CD31 (BD PharMingen, San Jose, CA) and detected using
Cy3-conjugated anti-mouse or anti-rat secondary antibody.
Digital images were collected using a Leica DMLB
fluorescence microscope (Buffalo Grove, IL).
Pimonidazole hydrochloride (PIM) staining: Six-week old
mice were treated with PIM (20 mg/kg bodyweight;
HypoxyprobeTM-1; Chemicon, Billerica, MA) by intravenous
(i.v.) injection. Two hours after injection, mice were
sacrificed and the eyes were immediately embedded in OCT.
Five μm thick sections were used for immunostaining with
FITC-conjugated Mab1 antibody (Hypoxyprobe, Inc.
Burlington, MA).
Real Time PCR: Total RNA was extracted from lens using the
RNeasy Kit (Qiagen, Valencia, CA) and reverse transcribed
into cDNA using SuperScript First-Strand Synthesis System
for RT–PCR Kit (Invitrogen, Grand Island, NY). Real-Time
PCR was performed by using primers for Cited2: sense (s) 5′-
CGC ATC ATC ACC AGC AGC AG-3′ and antisense (a) 5′-
CGC TCG TGG CAT TCA TGT TG-3′; HIF-1α: (s) 5′-GGA
CGA TGA ACA TCA AGT CAG-3′ and (a) 5′-GGA ATG
GGT TCA CAA ATC AGC-3′; VEGF: (s) 5′-ATC TTC AAG
CCG TCC TGT GT-3′ and (a) 5′-CTG CAT GGT GAT GTT
GCT CT-3′; 18S: (s) 5′-CGT CTG CCC TAT CAA CTT
TCG-3′ and (a)5′-CCT TGG ATG TGG TAG CCG TT-3′.
Real-Time PCR was performed using iQTM SYBR Green
Supermix PCR kit and iCycler machine (Bio-Rad, Hercules,
CA). Cycling conditions were 95 °C for 10 min, and 40 cycles
of 95 °C for 15 s and 60 °C for 30 s. A melting curve analysis
of products was performed routinely following the
amplification to test the specificity of the PCR products. 18S
was used as an internal control for normalization. After
normalization with 18S, average was calculated for each
group and control in each group was set as one.
Intravitreal injection: Six- to eight-week old Cited2fl/fl;Le-
Cre- and Cited2CKO mice received intravitreal injection of
Topotecan (TPT; Sigma) using a 33-gauge needle. TPT was
dissolved in dimethyl sulfoxide (DMSO) and diluted with
saline to 20 μg/μl and 40 μg/μl and 2.5 μl was injected into
each eye. In each mouse, the left eye was treated with TPT in
saline; the right eye was treated with saline containing the
same portion of DMSO to minimize the effect of DMSO used
to dissolve TPT. Right eye was used as a negative vehicle
control. After injection, mouse eyes were covered with
Gentak ointment (Akorn, Inc. Lake Forest, IL) to prevent
inflammation. After 3 weeks, injected mice were euthanized
and eyes were collected for histological analysis.
RESULTS
Cited2 is important in HVS formation and regression in the
lens: To test whether the lens abnormalities in Cited2 total
knockout embryos are primary defects, Le-Cre was used to
excise the Cited2 gene in the mouse lens. Le-Cre is expressed
in the surface ectoderm from embryonic day (E) 9.5 and in
surface ectoderm derived structures including the lens,
cornea, conjunctiva, and the eye lid. As expected, Cited2
mRNA expression level was significantly decreased in six-
week old Cited2CKO mouse lens compared to control
littermates, demonstrating the high deletion efficiency of Le-
Cre. Since Cited2 is a negative regulator of HIF-1 and
VEGF is one of HIF-1 target genes, we also examined the
expression of HIF-1α and VEGF mRNAs. A modest increase
of HIF-1α and a substantial increase of VEGF transcripts were
observed in Cited2CKO mouse lenses compared to those of
control littermates (Figurer 1A). Cited2CKO mice showed
corneal opacity and the eyes were smaller than control
littermates at six weeks of age. These mice also had lens stalks
due to failed separation of the lens from the cornea and
defective anterior chamber formation. In addition, abnormal
retrolental tissue was observed in Cited2CKO mouse eyes and
lenses from these mice lost their transparency (Figure 1B).
Immunostaining showed that Cited2 protein was mainly
expressed in the nuclei of lens epithelial cells in control mouse
lenses (Figure 1C). Compared with strong expression of
Cited2 in control littermate lenses, Cited2CKO mice showed a
very weak signal in the disorganized lens epithelial cells. To
address cell types involved
immunotstaining was performed with antibodies against α-
SMA for pericytes, F4/80 for macrophages, and CD31 for
endothelial cells. In Cited2CKO mouse eyes, positive signals
were detected for these proteins in abnormal HVS of the lens
(Figure 1D). These results indicate that Cited2 deficiency is
the primary defect during lens HVS regression.
HIF-1α controls HVS formation and regression mediated by
Cited2: To address the mechanism of Cited2 deletion-induced
abnormal hyaloid vasculature, our study focused on HIF-1
and its target gene, VEGF. Deletion of Cited2 and HIF-1α by
Le-Cre resulted in lower expression levels of HIF-1α in
Cited2fl/fl;HIF-1αfl/+ ;Le-Cre+ and Cited2fl/fl;HIF-1αfl/fl;Le-Cre
+ mice compared to control mice (Figure 2A). The VEGF
expression level was high in Cited2fl/fl;Le-Cre+ mouse lens but
decreased to the normal level in Cited2fl/fl;HIF-1αfl/fl;Le-Cre+
mouse lens (Figure 2B). These results indicate that the
deletion efficiency of Cited2 and HIF-1α by Le-Cre was
sufficient to study phenotypes rescued by deletion of
HIF-1α. Eyes from Cited2fl/fl;HIF-1αfl/fl;Le-Cre+ showed
corneal and lens opacity with a smaller size than control
littermates at six weeks of age (Figure 2C). These mice
showed lens stalk formation and abnormal retrolental tissues.
in abnormal HVS,
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132>© 2012 Molecular Vision
1262

Page 4

Figure 1. Le-Cre mediated deletion of Cited2 and morphological changes in Cited2CKO mice. A: Expression of Cited2 was decreased in
Cited2CKO mice compared to control (n=3, *p<0.05). Modest increase of HIF-1α and substantial increase of VEGF were observed in
Cited2CKO mice. B: Under a dissecting microscope, eyes collected from Cited2CKO mice showed smaller sizes and cornea opacity (upper panel).
H&E staining showed smaller lens, lens stalk (LS) formation at the anterior side of the lens, and hyaloid hypercellularity and aberrant
vasculature at the posterior side of the lens in Cited2CKO mice (lower panel). The insets represent higher magnification of the areas indicated
by arrows. C: Immunostaining with Cited2 antibody showed decreased expression of Cited2 (magenta) in Cited2CKO mouse lens.
Counterstaining with DAPI (blue) indicated that Cited2 localized in the nucleus. H&E pictures were taken at 5× magnification to show the
entire eye structure. DAPI pictures were taken at 10× using adjacent sections. Red box indicates the area shown in the Cited2/DAPI
counterstained pictures (20×). Scale bar in each picture indicates 100 μm. D: Immunostaining with α-SMA, F4/80, and CD31 (red) showed
composition of the hyaloid vascular system. Pictures were taken at 20× to show the red-boxed area. Scale bar in each picture indicates 100
μm. A↔P: anterior and posterior orientation of the eye.
1263
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132>© 2012 Molecular Vision

Page 5

Interestingly, abnormal retrolental tissues disappeared in
Cited2fl/fl;HIF-1α fl/fl;Le-Cre+ mouse eyes even though lens
stalks persisted. This is consistent with our previous result that
deletion of HIF-1α in Cited2 knockout embryonic lens
specifically eliminates abnormal retrolental tissues without
affecting the corneal-lenticular stalk phenotype [12].
Immunostaining for Cited2 showed decreased Cited2 protein
expression in Cited2fl/fl;HIF-1αfl/fl;Le-Cre+ mouse lens
compared to controls (Figure 2D). Most Cited2fl/fl;HIF-1αfl/
+;Le-Cre+ mice expressed α-SMA, CD31, and F4/80 in the
posterior lens indicating the presence of the pericytes,
endothelial cells, and macrophage cell types (Figure 2E).
To detect the level of local hypoxia in the absence of
Cited2 or both Cited2 and HIF-1α, we injected mice with
pimonidazole (PIM) [27], a chemical that interacts with
macromolecules under hypoxia, and sacrificed mice 2 h later.
By immunostaining, we observed strong PIM signals in
Cited2CKO mouse lens compared to the control mouse lens and
the lens from the negative control mouse injected with saline.
Deletion of HIF-1α in Cited2CKO mice (Cited2fl/fl;HIF-1αfl/
fl;Le-Cre+) showed a weaker PIM signal compared to the
Cited2fl/fl;Le-Cre+ mouse lens (Figure 3). This is consistent
with our previous finding that, unlike wild type embryos,
Cited2-deficient embryos remained highly hypoxic at 15.5
days post coitum (d.p.c.) in defective cardiac regions and both
the severe hypoxia and cardiovascular defects were absent
when the gene dosage of HIF-1α was reduced in Cited2-
deficient embryos [11].
Figure 2. Le-Cre mediated deletion of Cited2 and HIF-1α and phenotypic rescue by HIF-1α deletion. A: The expression levels of Cited2 and
HIF-1α were decreased in Cited2fl/fl;HIFfl/fl;Le-Cre+ mice compared to the control (n=3, *p<0.05). B: Increased VEGF in Cited2CKO resumed
to the normal level after HIF-1α was deleted in Cited2CKO mice. C: Under dissecting microscope, eyes collected from Cited2fl/fl;HIFfl/fl;Le-
Cre+ mice showed smaller sizes and cornea opacity (upper Panel). H&E staining showed smaller lens and lens stalk formation at the anterior
part of the lens in Cited2fl/fl;HIFfl/+;Le-Cre+ and Cited2fl/fl;HIFfl/fl;Le-Cre+ mice. The insets represent higher magnification of the areas indicated
by arrows. D: Expression level of Cited2 (magenta) was low in Cited2fl/fl;HIFfl/fl;Le-Cre+ mice compared to the control . H&E pictures were
taken at 5× magnification to show the entire eye structure. DAPI pictures were taken at 10× using adjacent sections. Red box indicates the
area shown in the counterstained pictures (20×). Scale bar in each picture indicates 100 μm. E: α-SMA, F4/80, and CD31 protein expression
indicated the cell types in the hyaloid vascular system. Pictures were taken at 20× to show the red-boxed area. Scale bar in each picture indicates
100 μm. A↔P: anterior and posterior orientation of the eye.
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132>© 2012 Molecular Vision
1264

Page 6

To compare the rescuing efficiency of HIF-1α deficiency,
six-week old Cited2CKO and Cited2fl/fl;HIF-1αfl/+;Le-Cre+ and
Cited2fl/fl;HIF-1αfl/fl;Le-Cre+ mice were analyzed and number
of mice in each group with abnormal HVS was determined
(Table 1). All Cited2CKO mice (100%) showed abnormal HVS
formation in the lens. When one allele of HIF-1α was deleted
in Cited2CKO mice (Cited2fl/fl;HIF-1αfl/+;Le-Cre+), 71.4% of the
mice showed abnormal HVS. Interestingly, when both alleles
of HIF-1α were deleted in Cited2CKO mice (Cited2fl/
fl;HIF-1αfl/fl;Le-Cre+), the number of mice with abnormal HVS
decreased to 27.3%.
Deletion of VEGF partially rescues the HVS defects in
Cited2 deficient lens: We previously observed increased
mRNA expression of VEGF, a HIF-1 target gene, in Cited2
deficient lens [12]. To answer whether VEGF plays a role in
the formation of abnormal hyaloid vasculature induced by
Cited2 deficiency, we generated Cited2fl/fl;VEGFfl/fl;Le-Cre+
mice. Relative mRNA expression levels of Cited2 (Figure 4A)
and VEGF (Figure 4B) were decreased in Cited2fl/fl;VEGFfl/
fl;Le-Cre+ mouse lens compared to controls. Most of Cited2fl/
fl;VEGFfl/+;Le-Cre+ and Cited2fl/fl;VEGFfl/fl;Le-Cre+ mice
showed a similar phenotype with the Cited2CKO mice,
including smaller eyes, lens stalk formation, lens opacity, and
abnormal hyaloid formation (Figure 4C). Abnormal hyaloid
vasculature was observed in 100% (4/4) Cited2fl/fl;VEGFfl/
+;Le-Cre+ and 70% (7/10) Cited2fl/fl;VEGFfl/fl;Le-Cre+ mouse
lenses (Figure 4C; Table 1), although the rescuing efficiency
was not as high as with HIF-1α deletion. These results suggest
that deletion of VEGF partially rescues the phenotype induced
by Cited2 deletion but that other HIF-1 downstream genes
besides VEGF may be involved.
Cited2CKO mouse can be used as a mouse model for testing
HIF-1α inhibitors: Since deletion of HIF-1α significantly
rescued Cited2 deficiency induced abnormal HVS in mouse
lens, we tested whether the Cited2CKO mouse could be used as
a mouse model for testing potential HIF-1 inhibitors. Six- to
eight-week old wild type or Cited2CKO mice received once
intravitreal injection 50 μg or 100 μg of Topotecan (TPT), a
compound that inhibits the synthesis of HIF-1α, into the left
eye [28]. Saline was injected into the right eye as a negative
Figure 3. PIM staining for the hypoxic region. Eyes from different genotypes were stained with antibodies against PIM. PIM signal (Green)
was strong in Cited2CKO compared to the control. Eyes collected from Cited2fl/fl;HIFfl/fl;Le-Cre+ showed a weaker signal compared to
Cited2CKO mice. Saline injection was used as a negative control.
TABLE 1. NUMBER OF MICE WITH ABERRANT HVS FORMATION.
Genotypes
Cited2fl/fl
Cited2fl/fl;HIF-1αfl/+/Cited2fl/fl;HIF-1αfl/fl
Cited2cko
Cited2fl/fl;HIF-1αfl/+; LeCre+
Cited2fl/fl;HIF-1αfl/fl; LeCre+
Cited2fl/fl;VEGFfl/+; LeCre+
Cited2fl/fl;VEGFfl/fl; LeCre+
Age (weeks)
6
6
6
6
6
6
6
Lens stalk
0/8 (0%)
0/15 (0%)
7/7 (100%)
5/5 (100%)
11/11 (100%)
4/4 (100%)
10/10 (100%)
Abnormal HVS
0/8 (0%)
0/15 (0%)
7/7 (100%)
5/7 (71.4%)
3/11 (27.3%)
4/4 (100%)
7/10 (70%)
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132>© 2012 Molecular Vision
1265

Page 7

control. Three weeks after injection, eyes were collected and
analyzed by standard histological methods. 20% (1/5) of mice
with 50 μg of TPT injection showed less severe HVS and 25%
(2/8) of mice with 100 μg of TPT injection showed improved
HVS compared to saline injected right eyes (Figure 5). In
addition, TPT injection did not affect lens stalk formation in
the Cited2CKO mouse. These results suggest that TPT injection
partially eliminated abnormal HVS in the posterior lens in
Cited2CKO mouse.
Figure 4. Le-Cre mediated deletion of Cited2 and VEGF and phenotypic rescue by VEGF deletion. The expression level of Cited2 was decreased
in Cited2fl/fl;VEGFfl/fl;Le-Cre+ mouse lens compared to the control (n=3, *p<0.05). B: Relative VEGF mRNA expression was decreased in
Cited2fl/fl;VEGFfl/fl;Le-Cre+ mouse lens compared to Cited2CKO. C: Morphological changes were similar between Cited2CKO and Cited2fl/
fl;VEGFfl/fl;Le-Cre+ by gross examination or under dissecting microscope (upper panel). The insets represent higher magnification of the areas
indicated by arrows.
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132>© 2012 Molecular Vision
1266

Page 8

DISCUSSION
Cited2 deficient embryos exhibit several developmental
defects and die in mid- to late gestation [9,12,29-35].
Although Cited2 knockout
abnormalities [12], the mechanisms attributed to abnormal
HVS was not fully addressed. Our results in this study clearly
demonstrate that the lens abnormalities observed in Cited2
null embryos are primary defects of Cited2 deficiency in part
through the deregulation of the HIF-1 pathway.
embryos showed lens
Previous studies have shown that intraocular injection of
PIM at different stages of development detected adduct
formation in all the stages examined, indicating that the lens
exists in a chronically hypoxic state throughout embryonic
development [27]. Our results showed that the lens from
Cited2CKO mouse exists in a highly hypoxic environment
compared to the normal lens. After deletion of HIF-1α in
Cited2CKO mice, the hypoxic levels of the lens reached normal
levels. It indicated that Le-Cre specific deletion of Cited2
affects the oxygen tension in the lens, which is consistent with
our finding in the Cited2 knockout embryonic heart [11] and
further support our hypothesis that Cited2 is a negative
regulator of HIF-1.
The transcriptional regulator HIF-1 is the master
mediator in the process of oxygen sensing and homeostasis.
Under hypoxia, cell proliferation is inhibited by stabilized
HIF-1 in cultured cells. Based on the fact that the lens is a
hypoxic organ during chick development and HIF-1α is highly
expressed in lens epithelial cells at mid-gestation in mouse
embryos, we tested the hypothesis that Cited2 deficiency
induced abnormal HVS in the lens is mediated by HIF-1α. In
Cited2CKO mice, deletion of HIF-1α partially rescued
abnormal HVS in Cited2CKO mice. The results are consistent
with our previous data that HIF-1α homozygous deletion in
the lens eliminates the aberrant HVS formation in Cited2
knockout embryonic eyes [12].
The potential role of VEGF in mediating the effect of a
dysregulated Cited2-HIF-1 genetic interaction was suggested
by previous studies in which we showed increased VEGF
expression in Cited2 deficient hearts and HIF-1α
haploinsufficiency not only decreased VEGF expression but
also rescued the heart defect in Cited2 deficient embryos
[11]. In the Cited2 deficient embryonic lens, we also observed
increased VEGF mRNA level. VEGF is one of the HIF-1
target genes that contributes to the formation of the
vasculature in wounded tissues and tumors. Murine VEGF
exists in three isoforms of 120, 164, and 188 amino acids
[36]. Overexpression of specific VEGF isoforms results in
different vascular patterning phenotypes: VEGF120
transgenics show several vascular patterning defects in
retinopathy of prematurity; VEGF164–188 transgenics show
features consistent with persistent hyperplastic primary
vitreous (PHPV) [37,38]. Although Cited2CKO mice showed
higher expression levels of VEGF with aberrant formation of
Figure 5. Intravitreal injection of a HIF-1α inhibitor in Cited2CKO mice. Six-eight weeks old mice were received intravitreal injection of
Topotecan (TPT). Fifty or 100 μg of TPT was injected into the left eye of Cited2CKO mouse. Saline was injected into right eye as a negative
control. Representative pictures are shown for 50 μg and 100 μg of TPT injection.
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132>© 2012 Molecular Vision
1267

Page 9

hyaloid vasculature in adult lens compared to normal mouse
lens, it is not clear which isoform(s) are overexpressed and
responsible for the vascular phenotype. Although the
abnormalities were only partially corrected with deletion of
VEGF in Cited2 conditional knockout lens, it is consistent
with a previous study by Shui et al. [16] that VEGF secreted
by lens cells may stimulate the formation of the fetal
vasculature, but regression of these vessels is not likely to be
caused by a reduction in VEGF production by the lens. It is
also possible that HIF-1 signaling could crosstalk with other
regulators, such as TGFβ family cytokines. TGFβ2 has been
shown to be an anti-angiogenic factor required for proper HVS
remodeling during development since TGFβ2 knockout mice
display aberrant HVS formation, which is similar to the HVS
phenotype in Cited2 knockout eyes and can be rescued by
overexpression of TGFβ1. It is possible that decreased
expression of negative growth factors inhibiting angiogenesis,
such as TGFβ, may also contribute to the HVS phenotypes
observed in Cited2CKO mice. Consistent with such a notion,
we have previously shown that Cited2 interacts with Smads
to modulate TGFβ signaling [39,40]. A more thorough
expression profile analysis is necessary to identify possible
cytokines and associated pathways other than VEGF that are
involved.
Failed regression of HVS in Cited2CKO lens also
associates with smaller lens, lens opacity, and vitreous
abnormality. These characteristics are similar to the
pathological features of PHPV. Interestingly, PHPV has been
diagnosed in a patient inflicted with von Hippel-Lindau
disease, in which HIF-1 is overexpressed, suggesting a
potential involvement of deregulated HIF-1 signaling in the
pathogenesis of PHPV [41,42]. Cited2 induced abnormal
HVS is also similar to the phenotype in Arf−/−mice. P19Arf may
directly or indirectly alter pericyte biology through repression
of VEGF to destabilize the underlying vessels [22].
Interestingly, several studies have suggested a possible link
between Arf and Cited2 in other cell types, although a formal
connection in the ocular system has not been established
[43,44].
In this study, the Cited2-HIF1α pathway in lens
development was further confirmed by intravitreal injection
of a HIF-1α inhibitor in Cited2CKO mice. Topotecan (TPT), a
drug that inhibits the synthesis of HIF-1α proteins, has been
used in clinical trials in treating ovarian cancer and lung
cancer. Periocular TPT in fibrin sealant can achieve volume
reduction of small and recurrent retinoblastoma sufficient to
allow successful focal therapy [45]. Our finding that
intraocular injection of TPT eliminates aberrant vasculature
in Cited2CKO mice not only supports the hypothesis that the
HIF-1 pathway is involved in abnormal HVS formation but
also provides a novel mouse model for testing HIF-1
inhibitors. The major advantage of using this mouse line is
that one does not have to generate tumor-bearing mice, which
is time-consuming. In addition, only a small amount of
inhibitor is needed for intravitreal injection, which is
convenient for screening before a drug candidate is fully
developed or synthesized. It is clear that TPT, like many HIF-1
inhibitors, is not a specific inhibitor. Since TPT did not affect
lens stalk formation, it is likely that TPT did not induce general
apoptosis in the injected lens, although more studies and the
use of more specific HIF-1 inhibitors as they become available
will be necessary to further validate our mouse model.
In conclusion, abnormal HVS formation in Cited2CKO
mice shows that the phenotype is a primary defect in the lens.
In addition, deletion of HIF-1α or VEGF partially rescues the
defect and HIF-1 inhibitor eliminates aberrant vasculature in
Cited2CKO mice. Based on these findings, Cited2CKO mouse
line could be used as a novel disease model for PHPV and for
testing of potential HIF-1 inhibitors.
ACKNOWLEDGMENTS
We thank Dr. Napoleone Ferrara at Genentech for providing
VEGF-flox mice, Dr. Randy Johnson at UCSD for HIF-1α-
flox mice and Dr. David Beebe at Washington University for
providing Le-Cre:HIF-1α-flox mice and help with the
manuscript. This work was support in part by NIHR21
EY018424 (to Y.C.Y.).
REFERENCES
1.Graw J. Genetics of crystallins: cataract and beyond. Exp Eye
Res 2009; 88:173-89. [PMID: 19007775]
Eiden LE, Latker C, Zatz M. Retinal toxicity of
methylazoxymethanol acetate is developmentally specific.
Brain Res 1981; 227:425-8. [PMID: 7260649]
Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible
factor 1 is a basic-helix-loop-helix-PAS heterodimer
regulated by cellular O2 tension. Proc Natl Acad Sci USA
1995; 92:5510-4. [PMID: 7539918]
Salceda S, Caro J. Hypoxia-inducible factor 1alpha
(HIF-1alpha) protein is rapidly degraded by the ubiquitin-
proteasome system under normoxic conditions. Its
stabilization by hypoxia depends on redox-induced changes.
J Biol Chem 1997; 272:22642-7. [PMID: 9278421]
Ebert BL, Bunn HF. Regulation of transcription by hypoxia
requires a multiprotein complex that includes hypoxia-
inducible factor 1, an adjacent transcription factor, and p300/
CREB binding protein. Mol Cell Biol 1998; 18:4089-96.
[PMID: 9632793]
Arany Z, Huang LE, Eckner R, Bhattacharya S, Jiang C,
Goldberg MA, Bunn HF, Livingston DM. An essential role
for p300/CBP in the cellular response to hypoxia. Proc Natl
Acad Sci USA 1996; 93:12969-73. [PMID: 8917528]
Carrero P, Okamoto K, Coumailleau P, O'Brien S, Tanaka H,
Poellinger L. Redox-regulated
transcriptional coactivators CREB-binding protein and
SRC-1 to hypoxia-inducible factor 1alpha. Mol Cell Biol
2000; 20:402-15. [PMID: 10594042]
Freedman SJ, Sun ZY, Kung AL, France DS, Wagner G, Eck
MJ. Structural basis for negative regulation of hypoxia-
inducible factor-1alpha by CITED2. Nat Struct Biol 2003;
10:504-12. [PMID: 12778114]
2.
3.
4.
5.
6.
7.
recruitment of the
8.
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132> © 2012 Molecular Vision
1268

Page 10

9.Yin Z, Haynie J, Yang X, Han B, Kiatchoosakun S, Restivo J,
Yuan S, Prabhakar NR, Herrup K, Conlon RA, Hoit BD,
Watanabe M, Yang YC. The essential role of Cited2, a
negative regulator for HIF-1alpha, in heart development and
neurulation. Proc Natl Acad Sci USA 2002; 99:10488-93.
[PMID: 12149478]
10. Bhattacharya S, Michels CL, Leung MK, Arany ZP, Kung AL,
Livingston DM. Functional role of p35srj, a novel p300/CBP
binding protein, during transactivation by HIF-1. Genes Dev
1999; 13:64-75. [PMID: 9887100]
11. Xu B, Doughman Y, Turakhia M, Jiang W, Landsettle CE,
Agani FH, Semenza GL, Watanabe M, Yang YC. Partial
rescue of defects in Cited2-deficient embryos by HIF-1alpha
heterozygosity. Dev Biol 2007; 301:130-40. [PMID:
17022961]
12. Chen Y, Doughman YQ, Gu S, Jarrell A, Aota S, Cvekl A,
Watanabe M, Dunwoodie SL, Johnson RS, van Heyningen V,
Kleinjan DA, Beebe DC, Yang YC. Cited2 is required for the
proper formation of the hyaloid vasculature and for lens
morphogenesis. Development 2008; 135:2939-48. [PMID:
18653562]
13. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea
KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous
embryonic lethality induced by targeted inactivation of the
VEGF gene. Nature 1996; 380:439-42. [PMID: 8602242]
14. Rutland CS, Mitchell CA, Nasir M, Konerding MA, Drexler
HC. Microphthalmia, persistent hyperplastic hyaloid
vasculature and lens anomalies following overexpression of
VEGF-A188 from the alphaA-crystallin promoter. Mol Vis
2007; 13:47-56. [PMID: 17277743]
15. Garcia CM, Shui YB, Kamath M, DeVillar J, Johnson RS,
Gerber HP, Ferrara N, Robinson ML, Beebe DC. The function
of VEGF-A in lens development: formation of the hyaloid
capillary network and protection against transient nuclear
cataracts. Exp Eye Res 2009; 88:270-6. [PMID: 18782574]
16. Shui YB, Arbeit JM, Johnson RS, Beebe DC. HIF-1: an age-
dependent regulator of lens cell proliferation. Invest
Ophthalmol Vis Sci 2008; 49:4961-70. [PMID: 18586877]
17. Reese AB. Persistent hyperplastic primary vitreous. Am J
Ophthalmol 1955; 40:317-31. [PMID: 13248898]
18. 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. [PMID: 9372715]
19. Jain TP. Bilateral persistent hyperplastic primary vitreous.
Indian J Ophthalmol 2009; 57:53-4. [PMID: 19075412]
20. Reichel MB, Ali RR, D'Esposito F, Clarke AR, Luthert PJ,
Bhattacharya SS, Hunt DM. High frequency of persistent
hyperplastic primary vitreous and cataracts in p53-deficient
mice. Cell Death Differ 1998; 5:156-62. [PMID: 10200460]
21. Thornton JD, Swanson DJ, Mary MN, Pei D, Martin AC,
Pounds S, Goldowitz D, Skapek SX. Persistent hyperplastic
primary vitreous due to somatic mosaic deletion of the arf
tumor suppressor. Invest Ophthalmol Vis Sci 2007;
48:491-9. [PMID: 17251441]
22. Martin AC, Thornton JD, Liu J, Wang X, Zuo J, Jablonski MM,
Chaum E, Zindy F, Skapek SX. Pathogenesis of persistent
hyperplastic primary vitreous in mice lacking the arf tumor
suppressor gene. Invest Ophthalmol Vis Sci 2004;
45:3387-96. [PMID: 15452040]
23. Ohlmann AV, Adamek E, Ohlmann A, Lutjen-Drecoll E. Norrie
gene product is necessary for regression of hyaloid vessels.
Invest Ophthalmol Vis Sci 2004; 45:2384-90. [PMID:
15223821]
24. Saint-Geniez M, D'Amore PA. Development and pathology of
the hyaloid, choroidal and retinal vasculature. Int J Dev Biol
2004; 48:1045-58. [PMID: 15558494]
25. Preis JI, Wise N, Solloway MJ, Harvey RP, Sparrow DB,
Dunwoodie SL. Generation of conditional Cited2 null alleles.
Genesis 2006; 44:579-83. [PMID: 17133411]
26. Ashery-Padan R, Marquardt T, Zhou X, Gruss P. Pax6 activity
in the lens primordium is required for lens formation and for
correct placement of a single retina in the eye. Genes Dev
2000; 14:2701-11. [PMID: 11069887]
27. Bassnett S, McNulty R. The effect of elevated intraocular
oxygen on organelle degradation in the embryonic chicken
lens. J Exp Biol 2003; 206:4353-61. [PMID: 14581604]
28. Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville
EA, Shoemaker RH, Melillo G. Identification of small
molecule inhibitors of
transcriptional activation pathway. Cancer Res 2002;
62:4316-24. [PMID: 12154035]
29. Bamforth SD, Braganca J, Eloranta JJ, Murdoch JN, Marques
FI, Kranc KR, Farza H, Henderson DJ, Hurst HC,
Bhattacharya S. Cardiac malformations, adrenal agenesis,
neural crest defects and exencephaly in mice lacking Cited2,
a new Tfap2 co-activator. Nat Genet 2001; 29:469-74.
[PMID: 11694877]
30. Bamforth SD, Braganca J, Farthing CR, Schneider JE,
Broadbent C, Michell AC, Clarke K, Neubauer S, Norris D,
Brown NA, Anderson RH, Bhattacharya S. Cited2 controls
left-right patterning and heart development through a Nodal-
Pitx2c pathway. Nat Genet 2004; 36:1189-96. [PMID:
15475956]
31. Withington SL, Scott AN, Saunders DN, Lopes Floro K, Preis
JI, Michalicek J, Maclean K, Sparrow DB, Barbera JP,
Dunwoodie SL. Loss of Cited2 affects trophoblast formation
and vascularization of the mouse placenta. Dev Biol 2006;
294:67-82. [PMID: 16579983]
32. Xu B, Qu X, Gu S, Doughman YQ, Watanabe M, Dunwoodie
SL, Yang YC. Cited2 is required for fetal lung maturation.
Dev Biol 2008; 317:95-105. [PMID: 18358466]
33. Qu X, Lam E, Doughman YQ, Chen Y, Chou YT, Lam M,
Turakhia M, Dunwoodie SL, Watanabe M, Xu B, Duncan SA,
Yang YC. Cited2, a coactivator of HNF4alpha, is essential for
liver development. EMBO J 2007; 26:4445-56. [PMID:
17932483]
34. Buaas FW, Val P, Swain A. The transcription co-factor CITED2
functions during sex determination and early gonad
development. Hum Mol Genet 2009; 18:2989-3001. [PMID:
19457926]
35. Chen Y, Haviernik P, Bunting KD, Yang YC. Cited2 is required
for normal hematopoiesis in the murine fetal liver. Blood
2007; 110:2889-98. [PMID: 17644732]
36. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and
biological properties of the vascular endothelial growth factor
family of proteins. Endocr Rev 1992; 13:18-32. [PMID:
1372863]
hypoxia-inducible factor 1
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132> © 2012 Molecular Vision
1269

Page 11

37. Stalmans I, Ng YS, Rohan R, Fruttiger M, Bouche A, Yuce A,
Fujisawa H, Hermans B, Shani M, Jansen S, Hicklin D,
Anderson DJ, Gardiner T, Hammes HP, Moons L, Dewerchin
M, Collen D, Carmeliet P, D'Amore PA. Arteriolar and
venular patterning in retinas of mice selectively expressing
VEGF isoforms. J Clin Invest 2002; 109:327-36. [PMID:
11827992]
38. Mitchell CA, Rutland CS, Walker M, Nasir M, Foss AJ, Stewart
C, Gerhardt H, Konerding MA, Risau W, Drexler HC. Unique
vascular phenotypes following over-expression of individual
VEGFA isoforms from the developing lens. Angiogenesis
2006; 9:209-24. [PMID: 17109192]
39. Chou YT, Wang H, Chen Y, Danielpour D, Yang YC. Cited2
modulates TGF-beta-mediated upregulation of MMP9.
Oncogene 2006; 25:5547-60. [PMID: 16619037]
40. Lopes Floro K, Artap ST, Preis JI, Fatkin D, Chapman G,
Furtado MB, Harvey RP, Hamada H, Sparrow DB,
Dunwoodie SL. Loss of Cited2 causes congenital heart
disease by perturbing left-right patterning of the body axis.
Hum Mol Genet 2011; 20:1097-110. [PMID: 21224256]
41. Kurihara T, Kubota Y, Ozawa Y, Takubo K, Noda K, Simon
MC, Johnson RS, Suematsu M, Tsubota K, Ishida S, Goda N,
Suda T, Okano H. von Hippel-Lindau protein regulates
transition from the fetal to the adult circulatory system in
retina. Development 2010; 137:1563-71. [PMID: 20388654]
42. Kurihara T, Westenskow PD, Krohne TU, Aguilar E, Johnson
RS, Friedlander M. Astrocyte pVHL and HIF-alpha isoforms
are required for embryonic-to-adult vascular transition in the
eye. J Cell Biol 2011; 195:689-701. [PMID: 22084310]
43. Kranc KR, Schepers H, Rodrigues NP, Bamforth S, Villadsen
E, Ferry H, Bouriez-Jones T, Sigvardsson M, Bhattacharya S,
Jacobsen SE, Enver T. Cited2 is an essential regulator of adult
hematopoietic stem cells. Cell Stem Cell 2009; 5:659-65.
[PMID: 19951693]
44. Kranc KR, Bamforth SD, Braganca J, Norbury C, van Lohuizen
M, Bhattacharya S. Transcriptional coactivator Cited2
induces Bmi1 and Mel18 and controls fibroblast proliferation
via Ink4a/ARF. Mol Cell Biol 2003; 23:7658-66. [PMID:
14560011]
45. Mallipatna AC, Dimaras H, Chan HSL, Heon E, Gallie BL.
Periocular Topotecan for Intraocular Retinoblastoma. Arch
Ophthalmol 2011; 129:738-45. [PMID: 21670340]
Molecular Vision 2012; 18:1260-1270 <http://www.molvis.org/molvis/v18/a132>© 2012 Molecular Vision
Articles are provided courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China.
The print version of this article was created on 9 May 2012. This reflects all typographical corrections and errata to the article
through that date. Details of any changes may be found in the online version of the article.
1270