539 Epigenetics, Stem Cells and Ocular Development
Thursday, May 5, 2011, 11:15 AM - 1:00 PM
Hall B/C Poster Session
Program #/Board # Range: 5978-6024/A127-A173
Organizing Section: Biochemistry/Molecular Biology
Program Number: 5978 Poster Board Number: A127
Presentation Time: 11:15 AM - 1:00 PM
Characterization of Ocular Diseases Prevalent Amongst Southwestern Native
American Patients
Robert B. Avery1A, Lawrence C. Tafoya1B. AOphthalmology, Surgery, BSchool of
Medicine, 1University of New Mexico, Albuquerque, NM.
Purpose: To determine the prevalence of various eye diseases afflicting a group of
Southwestern Native American patients over a seven-year period. To classify
common disorders in an underserved population that is largely unrepresented in the
current literature.
Methods: We performed a retrospective review of clinical records at the University
of new Mexico Hospital eye Clinic. This yielded a population of 324 adult Native
American patients which were treated in our clinic from 2003-2010. We then
compared this Native American population to control groups of 3333 Hispanic
patients and 2495 Caucasian patients matched for age, sex, and insurance carrier
type.
Results: Overall results showed Native American patients had increased rates of
glaucoma (32% of patients) and more severe diabetic complications including
proliferative retinopathy (44% of diabetics) and diabetic cataracts (14% of
diabetics). Native American patients also had increased levels of other preventable
conditions, such as mature cataract formation, pseudoexfoliation, and trauma.
Native American patients were 3.5 to 6 times more likely to have a traumaassociated diagnosis than their Hispanic and Caucasian counterparts. In addition,
Native American patients were, on average, significantly younger at the time of
trauma in comparison to the other ethnic groups surveyed.
Conclusions: The Southwestern Native American patient population is at an
increased risk for many devastating, yet avoidable, eye conditions compared to
other ethnic groups in the region. It is hoped that this information will aid
Ophthalmologists in anticipating the unique and highly treatable eye care needs of
Native American patients.
Commercial Relationships: Robert B. Avery, None; Lawrence C. Tafoya,
None
Support: None
Program Number: 5979 Poster Board Number: A128
Presentation Time: 11:15 AM - 1:00 PM
DNA Methylation Patterns During Retinal Development In Mice
Verity F. Oliver, Kieron Torres, Mariam S. Assadian, Ray A. Enke, Shannath L.
Merbs. Ophthalmology, Johns Hopkins University School of Medicine, Baltimore,
MD.
Purpose: The retina is a highly organized tissue with specialized cells types that
develop from a common retinal progenitor cell type. Our previous studies have
shown that in the adult mouse, genes that are preferentially expressed in
photoreceptors (PRs) from the outer nuclear layer (ONL) are unmethylated in PRs
but are methylated in non-expressing, non-PR cells from the inner nuclear layer
(INL). To understand when this differential DNA methylation pattern is established
and to begin to understand its significance to retinal development, we investigated
the methylation status of PR-specific genes in the developing mouse retina.
Methods: PRs (~97% rods) from the ONL and non-PR cells from the INL were
isolated by laser capture microdissection from wild-type retina of mice aged E11.5,
E17.5, P0 and P6 and were compared to adult whole retina, brain, kidney and
testes. S-cones (which comprise only 1% of the PRs in the wild type ONL), were
isolated from adult Nrl-/- mice ONL (~98% S-cones). Genomic DNA was isolated
from each cell type and modified by bisulfite treatment. 200-500 bp regions within
1000 bp of the TSS of Rho (rod-specific), Rbp3 (rod/ cone-specific) and Opn1sw
(cone-specific) were sequenced.
Results: At least 3 different DNA methylation patterns of PR-specific genes were
observed: one specific for non-retinal tissues and non-PR retinal cells
(methylation), another specific for expressing PR cells (hypomethylation), and a
third pattern that did not simply correlate with expression (regional
hypomethylation). This regional hypomethylation was seen close to the TSS of
Opn1sw (cone-specific) in wild-type ONL (~97% rods) and in the promoter of Rho
(rod-specific) in Nrl-/- ONL (~98% S-cones). Hypomethylation of the promoter
region of Rho and Rbp3 was also seen in developing cells not yet expressing these
genes.
Conclusions: We hypothesize that the regional hypomethylation of Rho and
Opn1sw is characteristic of PR precursor cells and once cell fate is determined,
these early methylation patterns become completely unmethylated in expressing
PRs but persist in the non-expressing PRs. Additionally, the regional
hypomethylation near the TSS of Rbp3 and Rho that did not correlate with
expression at time points as early as E11.5 likely represent differential methylation
that leads to lineage determination by regulating gene expression in the developing
retina.
Commercial Relationships: Verity F. Oliver, None; Kieron Torres,
None; Mariam S. Assadian, None; Ray A. Enke, None; Shannath L. Merbs,
None
Support: NIH R21EY018703
Program Number: 5980 Poster Board Number: A129
Presentation Time: 11:15 AM - 1:00 PM
DNA Methylation Is Associated With Altered Gene Expression in AMD
Allan A. Hunter, III1, Anna K. Hunter2, Alyssa Cwanger1, Paul A. Spechler1, Zhe
Zhang1, Ed deZoeten2, Joshua L. Dunaief1. 1Ophthalmology, F.M. Kirby Center for
Molecular Ophthalmology, Scheie Eye Institute, Philadelphia, PA; 2Children’s
Hospital of Philadelphia Research Institute, Philadelphia, PA.
Purpose: Previous studies indicate that AMD disease risk is influenced by several
susceptibility loci. To assess the potential contribution of epigenetic regulation to
AMD pathogenesis, we evaluated DNA methylation, a tissue specific genetic
modulation that affects gene expression.
Methods: Using the Infinium HumanMethylation 27 Illumina platform, we
performed DNA bisulfite sequencing to compare the methylation status between
patients with AMD vs. age-matched controls. DNA was extracted from frozen
retinal pigment epithelium (RPE) and neurosensory retina (NSR) from 21 postmortem human eyes. Bisulfite conversion of genomic DNA was done using EZ
DNA Methylation kit (Zymo Research). RNA was isolated with the RNeasy Mini
Kit (Qiagen Inc). TaqMan gene expression assays were used for qPCR analysis.
Results: The Illumina platform determines the methylation status of over 27,578
CpG dinucleotides spanning 14,495 genes. CpG measurements were represented as
β values outputted by BeadStudio. CpG measurements of all samples were further
normalized by QSPLINE method using autosomal sites. 67 sites having p<0.1 and
β difference more than 0.02 were selected. We compared these results to gene
expression profiles in the same tissues determined by the Affymetrix exon
microarray. The data set includes 23,536 unique Entrez genes. Of the 67 genetic
loci identified by the Illumina platform, 45 genes had expression changes recorded
through the expression array with fold-change > 25% and p < 0.05. These exon
expression results were confirmed by qPCR. Compared to controls,
hypomethylation was noted in 8 genes with elevated expression levels, while
hypermethylation was found in 11 genes with decreased expression. Of these 19
genes identified, some had gene function considered important in AMD
pathogenesis, e.g. oxidative stress and inflammation modulation.
Conclusions: These data suggests that DNA methylation of cis regulatory CpG
islands may play a role in regulation of genes that contribute to AMD pathogenesis.
Commercial Relationships: Allan A. Hunter, III, None; Anna K. Hunter,
None; Alyssa Cwanger, None; Paul A. Spechler, None; Zhe Zhang, None; Ed
deZoeten, None; Joshua L. Dunaief, None
Support: Allan Hunter's NIH K Award 0904, F.M. Kirby Foundation
Program Number: 5981 Poster Board Number: A130
Presentation Time: 11:15 AM - 1:00 PM
Diabetic Cataract: Endoplasmic Reticulum-Stress/Epigenetic Suppression of
Nrf2-Keap1 Pathway for Many Antioxidant Enzymes
Palsamy Periyasamy1, Elanchezhian Rajan1, Christian J. Madson1, David W.
Lynch1, Masahiko Ayaki2, Jiro Usukura3, Toshimichi Shinohara4. 1Ophthalmology
and Visual Sciences, Univ of Neb Med Center, Omaha, NE; 2Saitama National
Hospital, Wako City, Japan; 3Department of Materials Physics and Engineering,
Adv. Sci. and Technol., Nagoya, Japan; 4Ophthalmol and Vis Sci, Univ of
Nebraska Medical Center, Omaha, NE.
Purpose: Early onset of age-related cataract (ARC) is often found in diabetic
patients, whose glucose concentration fluctuates from hyper- to hypoglycemia.
Endoplasmic reticulum stress responds to these glucose concentrations and
activates protective signals through translational and transcriptional regulations,
which is termed as unfolded protein response (UPR). There are increasing
evidences available on the aging and cancer studies, which indicate that
hypermethylation in the CpG islands of NF-E2-related factor 2 (Nrf2 gene), and its
negative regulator, Kelch-like ECH-associated protein 1 (Keap1 gene), suppresses
the expression of many antioxidant genes. During oxidative stress, Nrf2 escapes
from Keap1-mediated repression and activates expression of antioxidant responsive
element-dependent genes to preserve cellular redox homeostasis. Here, we aimed to
investigate whether hypo- and hyperglycemia induces the UPR and also the
antioxidant defense system failure during prolonged exposure to hypo- and
hyperglycemia.
Methods: Human lens epithelial cells (LECs) and rat lenses were cultured in
different glucose concentrations in DMEM at 1% oxygen. H2-DCFH-DA and EthD
staining were used to detect ROS and cell death, respectively. Protein blot analyses
were performed with Abs specific to UPR proteins. MS-PCR and DNA sequencing
were used to identify the methylated DNA.
Results: Activation of UPR in 0, 50, 100 mM glucose after 3 hrs was observed and
was increased with incubation time, along with higher levels of ROS and cell death.
Nrf2 and Keap1 were also upregulated in 0, 50, and 100 mM glucose but prolonged
exposure decreased the levels of Nrf2 and Keap1. Fluctuations of glucose
concentrations from 100 mM to 1 mM did not enhance the activation of the UPR.
UPR specific proteins were predominantly localized in LECs of the rat lenses
treated with no or high levels of glucose. Bisulfite DNA sequencing showed that
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
the CpG island of Nrf2 and Keap1 promoters had methylated cytosines in more
than 30 capsulotomy specimens of ARCs.
Conclusions: No or high glucose induces UPR and generates ROS. Eventually
Nrf2 and Keap1 are suppressed by epigenetic modifications which ultimately lower
the transcription of antioxidant genes and induce lens oxidation and cell death in
LECs, perhaps in ARCs.
Commercial Relationships: Palsamy Periyasamy, None; Elanchezhian Rajan,
None; Christian J. Madson, None; David W. Lynch, None; Masahiko Ayaki,
None; Jiro Usukura, None; Toshimichi Shinohara, None
Support: RPB and EY018172
Program Number: 5982 Poster Board Number: A131
Presentation Time: 11:15 AM - 1:00 PM
The Role of Dnmt1 in Retinal Differentiation
Krushangi Patel1, Igor Nasonkin1, Jerome Roger1, Kevin Lazo1, Dustin
Hambright1, Rudolf Jaenisch2, Milan Jamrich3, Anand Swaroop1. 1NeurobiolNeurodegen & Repair Lab, NEI, Bethesda, MD; 2Dept. of Biol, Massachusetts Inst.
of Technology, Whitehead Inst. for Biomed. Research, Cambridge, MA; 3Dept. of
Molec. and Human Genetics and Molec. & Cell. Bio., Baylor College of Medicine,
Houston, TX.
Purpose: Methylation is generally associated with repression of gene expression
(Razin 1991). Conditional ablation of exons 4 and 5 in Dnmt1 2lox mouse model is
expected to cause demethylation (Jackson-Grusby et al. 2001) and as a result
derepression of the genetic program in the developing neural retina (nr) leading to
premature gene expression resulting in aberrant maturation of nr cells including
photoreceptors (PRs). The goal of this project is to delineate the role of DNA
Methyltransferase-1 (Dnmt1) in retinal development.
Methods: We generated Dnmt12lox, Six3Cre mice to study the effect of early
demethylation on nr development. Using histology and immunohistochemistry
(IHC), we examined the changes in nr development in mutant mice at several
stages of differentiation: P0.5, P10.5, 3wk, 6 wk, and 3-4 months.
Results: We observed the early and severe degeneration of the central retina in the
mutant retinas by postnatal day P10.5, while peripheral retinal changes were only
subtle. Pathology was observed in the peripheral retina only by 3-4 months. There
is further indication of uneven changes in mutants along the dorso-vental nr axis.
We also detected a significant loss of cones in mutant retina, as evidenced by Cone
Arrestin (CAR) and Peanut Agglutinin Lectin (PNA) IHC.
Conclusions: DNMT1-mediated DNA methylation and DNMT1 function are
crucial for retinal development and cell survival.
Commercial Relationships: Krushangi Patel, None; Igor Nasonkin,
None; Jerome Roger, None; Kevin Lazo, None; Dustin Hambright,
None; Rudolf Jaenisch, None; Milan Jamrich, None; Anand Swaroop, None
Support: None
Program Number: 5983 Poster Board Number: A132
Presentation Time: 11:15 AM - 1:00 PM
Characterizing the Role of Epigenetic Mechanisms in Apoptotic Retinal Cells
Ray A. Enke, Karl J. Wahlin, Verity F. Oliver, Mariam S. Assadian, Kieron Torres,
Donald J. Zack, Shannath L. Merbs. Ophthalmology, Johns Hopkins University
School of Medicine, Baltimore, MD.
Purpose: Retinal cells undergo programmed cell death (PCD) during normal
development as well as in response to injury and disease. We hypothesize that
epigenetic mechanisms may contribute to apoptosis of retinal neurons in the
developing and diseased retina. The role of epigenetic modifications, such as DNA
methylation, in public health and medicine has primarily concentrated on the
malignant transformation of cells. A more recent focus has been the possible
contribution of epigenetic changes to tissue development and tissue-specific
diseases. To determine a correlation between epigenetic mechanisms and PCD in
the retina, we used immunohistochemical (IHC) analysis of the epigenetic
modification 5-methyl cytosine (5-meC) in experimental models of retinal disease
and development.
Methods: Retinal cross sections were prepared from chicken at different time
points during development. Retinal sections were also prepared from adult mouse
models of retinal disease, the rd1 model of retinal degeneration and the DBA/2J
model of glaucoma. Eye cups from adult animals or heads from embryonic animals
were fixed in 4% PFA and cryoprotected. Retinal cross sections were cut and used
for IHC analysis using an antibody against 5-meC as well as markers of PCD.
Results: TUNEL positive neurons in the degenerating retina of adult rd1 mice
demonstrate enhanced staining for 5-meC. This was also observed in a
developmental time course of chicken retinas. Although cleaved caspase-3
(cCaspase-3) positive retinal neurons were present at similar periods of
development there was little overlap with 5-meC positive cells suggesting that they
are expressed at different stages of PCD. We are currently evaluating the 5-meC
IHC staining of the DBA/2J mouse glaucoma model to determine if there is a
similar correlation in retinal ganglion cells undergoing apoptosis, and these results
will also be presented.
Conclusions: The genomes of retinal neurons in the late stages of PCD
demonstrate DNA hypermethylation by IHC. This finding suggests that, in
vertebrates, epigenetic mechanisms may play a role in apoptosis of neurons during
retinal development and retinal degeneration. Further investigation is needed to
determine if other canonical epigenetic marks correlate with hypermethylation in
apoptotic neural retina.
Commercial Relationships: Ray A. Enke, None; Karl J. Wahlin, None; Verity
F. Oliver, None; Mariam S. Assadian, None; Kieron Torres, None; Donald J.
Zack, None; Shannath L. Merbs, None
Support: NIH R21EY018703
Program Number: 5984 Poster Board Number: A133
Presentation Time: 11:15 AM - 1:00 PM
Retina-specific Dna Methylation
Mariam S. Assadian, Verity F. Oliver, Kieron Torres, Ray A. Enke, Shannath L.
Merbs. Ophthalmology, Johns Hopkins University School of Medicine, Baltimore,
MD.
Purpose: DNA methylation is known to play a critical role in the differentiation of
cells and tissues, and changes in DNA methylation have been shown to be
important in the development of some diseases. We have previously demonstrated
that certain retina-specific genes, including Rho and Rbp3, are differentially
hypomethylated in the retina when compared to non-expressing tissues in the
mouse. We believe that a genome-wide DNA methylation map of the retina will be
invaluable as we search to understand the role of DNA methylation in retinal
development and disease.
Methods: Whole retinas were extruded from 3 C57B/6J mice at 2 months of age.
Mouse cortex was isolated as non-retinal control tissue. Genomic DNA was
isolated and fragmented to an average of 300 bp. The fragmented DNA was divided
in to 2 fractions: input DNA and a second fraction that was enriched for methylated
DNA using MethylCollector (ActiveMotif). Enrichment was confirmed by QPCR
of Rho, which is relatively unmethylated in the retina and methylated in the brain.
The input DNA and methylated fractions were subjected to whole genome
amplification (WGA), and maintenance of the enrichment was confirmed by
QPCR, prior to being labeled and hybridized to mouse CHARM arrays
(Comprehensive High-throughput Analysis of Relative Methylation, NimbleGen).
Results: Multiple techniques for WGA were compared to insure that we were
using the technique that best maintained our methylated DNA enrichment. The
enriched and labeled DNAs are currently being evaluated in our ongoing, genomewide analysis using the CHARM array. Retina-specific DNA methylation sites
(when compared to brain) will be compared to known tissue-differentially
methylated regions that have previously been identified by CHARM in other
tissues.
Conclusions: Our pangenomic characterization of retina-specific DNA patterns
provides an important resource for future studies to investigate the relationship
between DNA methylation and retinal development and disease.
Commercial Relationships: Mariam S. Assadian, None; Verity F. Oliver,
None; Kieron Torres, None; Ray A. Enke, None; Shannath L. Merbs, None
Support: NIH R21EY018703
Program Number: 5985 Poster Board Number: A134
Presentation Time: 11:15 AM - 1:00 PM
Isolation Of Cell Layer-specific DNA From Human Eyes With Glaucoma Or
AMD
Kieron Torres, Mariam S. Assadian, Verity F. Oliver, Ray A. Enke, Shannath L.
Merbs. Ophthalmology, Johns Hopkins University School of Medicine, Baltimore,
MD.
Purpose: Little is known about the relationship between DNA methylation
patterns, retinal gene expression, and retinal disease. In the US alone, almost 4
million individuals currently have either primary open angle glaucoma (POAG) or
age-related macular degeneration (AMD). With both diseases, gene expression
changes in the retina have been observed. One modulator of gene expression is
DNA methylation. We hypothesize that alterations in DNA methylation,
accompany and may actually precede the gene expression changes seen with the
onset of POAG and AMD and are working towards a genome-wide map of the
DNA methylation changes in the human retina associated with these two diseases.
High quality, cell layer-specific DNA from diseased and control eyes is required
for our genomic analysis.
Methods: Adult human eyes, enucleated within 6hrs after autopsy and transported
on ice within 24hrs after enucleation, are obtained from NDRI. Eyes are from
individuals with AMD, POAG, or without history of either disease. The optic nerve
is cut sharply with a razor blade, placed in 4% paraformaldehyde, and embedded in
plastic. The cornea is removed. Four radial cuts are made in the quadrants between
rectus muscles and are extended to the equator of the globe. A high-resolution
photograph is taken of the macula and optic nerve. The eye is put through a 6.2525% sucrose gradient on ice over 2hrs. A 6mm trephine is used to isolate the
macula and optic nerve head in separate calottes. The calottes are flash frozen in a
2:1 mixture of 25% sucrose:OCT and cryosectioned.
Results: Cryosections through the optic nerve head, as well as plastic sections
though the optic nerve, are used to confirm axonal loss in the POAG samples and
exclude axonal loss in control eyes. Gross examination of the macular photographs
and cryosections of the macular calottes are used to confirm or exclude the
diagnosis of AMD. LCM of cryosections on PEN membrane slides is used to
collect ganglion cells from POAG eyes and photoreceptors and RPE cells from
AMD eyes, as well as all 3 cell layers from control eyes. DNA and RNA are
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
isolated with AllPrep (Qiagen). Pyrosequencing of the rhodopsin gene, and QPCR
of ganglion cell-, photoreceptor cell- and RPE-specific genes, have demonstrated
our ability to isolate quality, cell layer-specific DNA and RNA.
Conclusions: Using cryosectioning and microdissection of a 6mm macular calotte,
we are able to obtain quality, cell layer-specific DNA and RNA from the ganglion
cell, photoreceptor and RPE cell layers. The DNA will be used for a pangenomic
epigenetic analysis to identify DNA methylation changes associated with AMD and
POAG.
Commercial Relationships: Kieron Torres, None; Mariam S. Assadian,
None; Verity F. Oliver, None; Ray A. Enke, None; Shannath L. Merbs, None
Support: NIH Grant R01EY020406
Program Number: 5986 Poster Board Number: A135
Presentation Time: 11:15 AM - 1:00 PM
DNA Demethylation Increases Expression of Pluripotent and Retinal Ganglion
Cell-Specific Genes in ImM10 Müller Glia-Derived Sphere Cultures
Tihomira D. Petkova, Deborah C. Otteson. College of Optometry, University of
Houston, Houston, TX.
Purpose: In the mammalian retina, Müller glia have limited stem cell properties
and the mechanisms that restrict their potential to proliferate and differentiate into
retinal ganglion cells (RGCs) are poorly understood. The purpose of this study was
to determine if key pluripotency and developmental genes are transcriptionally
silenced by DNA methylation in Müller-derived retinal stem cells.
Methods: Genomic DNA and RNA were isolated from untreated or 5-Azadeoxycytidine (AzadC) (1 and 10µM) treated, conditionally immortalized mouse
Müller glia (ImM10) cultured as spheres in serum-free medium with 20 ng/ml EGF
and FGF2. DNA was bisulfite converted (ZymoGold), CpG islands were PCR
amplified, subcloned and sequenced. Gene expression was analyzed by quantitative
RT-PCR. Statistical analyses used the Mann-Whitney U-test for methylation
analysis and Student's T-tests for expression analysis.
Results: In Müller-derived spheres Atoh7 was hypermethylated (66%) and Atoh7
mRNA expression was not detected. AzadC treatment decreased methylation of
Atoh7 to 26% (p<0.0001) and increased mRNA expression by at least 97-fold
(p<0.0001). AzadC treatment also increased expression of several pluripotency
genes: Pou5f1 (from no expression to 35.3Ct, p<0.0001), Nanog2 (from 34.7 to
32.6 Ct, 2.7-fold, p=0.0003) and Sox2 (from 23.5 to 22.5 Ct, 1.7-fold, p=0.0007)
and retinal ganglion cell specific genes: Isl1 (from 35.6 to 29.6 Ct, 26.8-fold,
p<0.0001), Pou4f3 (from 37.5 to 29.8 Ct, 110-fold; p<0.0001). Pou4f2 mRNA was
not detected in control or AzadC-treated ImM10 spheres.
Conclusions: The inverse relationship between DNA methylation and mRNA
expression of Atoh7 in Müller glia-derived sphere cultures supports a role for DNA
methylation in regulating Atoh7 gene expression in vitro. Up-regulation of
pluripotent and RGC developmental genes following DNA demethylation suggests
that epigenetic mechanisms could limit the proliferative and neurogenic potential of
Müller glia. Reversing epigenetic silencing of neuronal and pluripotency genes
offers a potential strategy for increasing neurogenesis from Müller-derived stem
cells and enhancing retinal regeneration in mammals.
Commercial Relationships: Tihomira D. Petkova, None; Deborah C. Otteson,
None
Support: Fight for Sight Summer Fellowship, Borish Ezell Fellowship (TDP);
Glaucoma Research Foundation Shaffer Grant (DCO); NIH NEI P30EY07551
(core UHCO).
Program Number: 5987 Poster Board Number: A136
Presentation Time: 11:15 AM - 1:00 PM
Altered Dnmt1-mediated DNA Methylation Disrupts Retinal Pigment
Epithelium (RPE) And Photoreceptor Differentiation
Igor O. Nasonkin1A, Shannath L. Merbs2, Krushangi Patel1A, Raymond Enke2,
Verity Oliver2, Robert N. Fariss3, Kapil Bharti4, Enrique J. Rodriguez-Boulan5,
Donald J. Zack6, Anand Swaroop1B. ANNRL, BN-NRL, Bldg 6, 1National Eye
Institute, Bethesda, MD; 2Ophthalmology, Oncology, Wilmer Eye Institute, Johns
Hopkins Univ, Baltimore, MD; 3Biological Imaging Core, National Eye Inst/NIH,
Bethesda, MD; 4NINDS, National Institutes of Health, Bethesda, MD;
5
Ophthalmology, Weil Med Coll-Cornell Univ, New York, NY; 6Ophthalmology,
Wilmer Eye Inst, Johns Hopkins Univ, Baltimore, MD.
Purpose: DNA methylation is an essential epigenetic modification of DNA that
governs chromatin remodeling and patterns of gene expression. DNA methylation
is generally associated with stable silencing of gene expression and chromatin
compaction. The goal of this project is to determine the role of Dnmt1 in
influencing the development of neural retina (nr) and RPE.
Methods: Dnmt12lox, Rx-Cre mice were generated to produce conditional ablation
of Dnmt1 exons 4 and 5 in developing nr and RPE. We performed histological
evaluation of mutant retinas by light and electron microscopy, and
immunolocalization studies on retinal sections using cell-specific antibodies to
photoreceptors (PRs), RPE, Muller cells, and second order neurons. Quantitation of
DNA methylation was performed by bisulfite conversion followed by
pyrosequencing analysis of genomic DNA of L1 elements, amplified from mouse
tail and retina.
Results: Excision of Dnmt1 exons by Cre resulted in additional exon 6 skipping
(Δ4-6) and a predicted mutant DNMT1 protein lacking the PCNA binding domain.
We obtained variable DNA hypomethylation and severe morphological changes in
the retina and RPE of Dnmt1 mutant mice [lack of PR outer segment elongation,
retinal folding and pseudorosette formation, and abnormalities in RPE development
and maturation]. Notably, the general organization of the retina was unaffected and
all retinal cell types (except S-cones) were produced.
Conclusions: Our data suggest that DNA hypomethylation and altered DNMT1
function during early differentiation can lead to dramatic alterations in the
morphogenesis of PRs and RPE.
Commercial Relationships: Igor O. Nasonkin, None; Shannath L. Merbs,
None; Krushangi Patel, None; Raymond Enke, None; Verity Oliver,
None; Robert N. Fariss, None; Kapil Bharti, None; Enrique J. RodriguezBoulan, None; Donald J. Zack, None; Anand Swaroop, None
Support: None
Program Number: 5988 Poster Board Number: A137
Presentation Time: 11:15 AM - 1:00 PM
Epigenetic Dysregulation In The Retinas Of Diabetic Mice
Evgenya Popova, Colin Barnstable. Neural and Behavioral Sciences, College of
Medicine, Penn State Univ, Hershey, PA.
Purpose: The overall goal of this research project is to determine whether there are
specific sets of molecular changes of epigenetic postranslational modification of
core histones H3 and H4 that occur in a mouse model of Type I diabetes.
Methods: Animal use was in accordance with ARVO / IACUC guidelines.
C57BL/6J-Ins2 Akita/+ mice (Jackson Lab) were continuously maintained by
crossing with C57BL/6j stock at each generation. We developed ChIP-Seq
procedures for solid primary tissue and applied them to obtain comprehensive
chromatin profiling to compare epigenetic marks in retina of Akita mice and wild
type littermates. ChIP-Seq analysis of retinal chromatin was performed with
antibodies against H3K4me2, H3K27ac, H3K27me3, H3K9me3, H3K9ac,
H4K20me3 and H4K12ac. ChIP DNA libraries were sequenced on a SOLiDTM 3
system. Data were analyzed with NextGENe.
Results: Electron microscopy studies show no changes in nuclear morphology or
distribution of heterochromatin in rod nuclei in Akita mice compared with healthy
littermates. Immunohistochemical studies show different distributions of several
epigenetic marks between the retinas of Akita mice and healthy littermates. Overall
Chip-Seq analysis suggests that H3K27ac and H3K4me2, activation epigenetic
markers are upregulated, while H3K27me3 and H3K9me3, inhibitory markers are
downregulated in Akita mice retinas compared with wild type littermates. Detailed
analysis of promoter region occupancy with different epigenetic markers was
performed for one hundred genes. Immunohistochemistry shows increased staining
in rod photoreceptor nuclei with anti-H3K27ac antibody in Akita mice, and ChIPSeq with the same antibody demonstrated an increase in occupancy of the set of
promoter mostly of non-rod specific genes, suggesting activation of these genes
inside rod photoreceptor in diabetic mice.
Conclusions: We used Akita mice as model to study epigenetic changes in diabetic
retinopathy. Our study suggests epigenetic dysregulation in the retina of diabetic
mice with increase of activation epigenetic marks and decrease of inhibitory marks.
While these changes affect many genes, it is clear that not all genes are affected
equally. These studies will give fundamental information about the range of retinal
changes seen in diabetes and will offer new insights into the treatment of the
diabetes.
Commercial Relationships: Evgenya Popova, None; Colin Barnstable, None
Support: Pennsylvania Department of Health using Tobacco CURE Funds. TSF
09/10, SAP#4100050904
Program Number: 5989 Poster Board Number: A138
Presentation Time: 11:15 AM - 1:00 PM
Ephrina3 Presents A Negative Signal For Growth And Neural Differentiation
Of Adult Retinal Stem Cells
Yuan Fang1,2, Kissaou Tchedre2, Xinghuai Sun1, Kin-sang Cho2, Dongfeng F.
Chen2,3. 1Department of Ophthalmology, Eye & ENT Hosptial, Shanghai, China;
2
Ophthalmology/Harvard, Schepens Eye Res Inst, Harvard Med Sch, Boston, MA;
3
RR&D Center of Excellence, VA Boston Healthcare System, VA Center for
Innovative Visual Rehabilitation, Boston, MA.
Purpose: In adult mammals, retinal stem cells (RCS) found in the ciliary margin
exhibit a limited proliferation and differentiation ability comparing with that in
lower vertebrates, while this mechanism remains unknown. Recently, ephrinA3 has
been identified as a negative regulator of adult neurogenesis in the mammalian
central nervous system. This project was aimed to explore the roles of ephrinA3 in
the regulation of RCS proliferation and differentiation.
Methods: Expression of ephrinA3 on mouse eye was analyzed by
immunohistochemistry and western blot. To determine the functional significance
of ephrinA3 in RSC regulation, mice deficient for ephrinA3 were examined. RSC
proliferation was compared in vivo in adult wild-type and ephrinA3-/- mice using 5bromo-2-deoxyuridine (BrdU) pulse labeling. In vitro, quantification of RSC
proliferation and differentiation were carried out using neurosphere cultures.
Results: Expression of ephrinA3 is increased in the retina and ciliary epithelium
along maturation in mouse, and its expression reached the peak in the adult.
Deletion of ephrinA3 greatly enhanced the proliferation of RSCs in the adult in
vivo and in vitro. Results of RT-PCR show that the neurospheres derived from
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ephrinA3-/- mouse express higher levels of neural progenitor cell markers, such as
Sox2, neurogenin, and photoreceptor progenitor cell markers, including Crx, Nrl
and Nr2e3, as compared to RSCs of wild-type mice. RSCs isolated from ephrinA3/mice exhibit higher potential to differentiate into retinal neurons and
photoreceptor cells as compared to wild-type RSCs.
Conclusions: EphrinA3 is an endogenous inhibitor regulating the proliferation and
neurogenic potential of RSCs in adult mice.
Commercial Relationships: Yuan Fang, None; Kissaou Tchedre,
None; Xinghuai Sun, None; Kin-sang Cho, None; Dongfeng F. Chen, None
Support: The project was supported by grants from NIH/NEI (R01EY017641),
NIDA (R21DA024803), Department of Veterans Affairs (1I01RX000110),
Department of Defense (W81XWH-09-2-0091) to D. F. C and the National
Program Number: 5990 Poster Board Number: A139
Presentation Time: 11:15 AM - 1:00 PM
Expression of Transcription Factors Involved in Murine Stem Cell
Differentiation Induce Photoreceptor Phenotypes in Mouse Embryonic Stem
Cells
Mark A. Fields, Laura Vickers, Hui Cai, Jie Gong, Stephen Tsang, Lucian V. Del
Priore. Ophthalmology, Columbia Univ Eye Inst, New York, NY.
Purpose: Embryonic stem cell (ESC) transplantation is a promising therapeutic
approach for the replacement of degenerated retinal cells. Previously we have used
microarray analysis to identify key transcription factors in murine retinal
development. Herein we perform ex vivo gene therapy using a lentiviral delivery
system to express the transcription factor NeuroD1 in ESCs to induce their
differentiation into photoreceptors.
Methods: Retinal samples were collected from E11, E18, P5, P8 and older adult
murine eyes and the transcription factors expressed at different developmental time
points were identified by microarray analysis. DNA of key transcription factors (in
particular NeuroD1) were cloned into the pCDH-EF1-MCST2A expression vector
(System Biosciences, Mountain View, CA) containing a red fluorescent protein
(RFP) or green fluorescent protein (GFP) reporter gene. The cloned NeuroD1-RFP
lentivirus was prepared using a lentiviral packaging system (System Biosciences).
Mouse embryonic stem cells ES-D3 (ATCC, Manassas, VA) were plated into a sixwell culture dish containing a feeder layer of Mitomycin C-treated CF-1 mouse
embryonic fibroblasts cultured at 37°C, 5% CO2 in Dulbecco's Modified Eagle's
Medium (DMEM) supplemented with 10% FBS and β-mercaptoethanol. Cultured
ES-D3 cells were infected with the NeuroD1-RFP and isolated by fluorescent
activated cell sorting (FACS) using a BD Aria II sorter (BD Biosciences, San Jose,
CA). Cells expressing NeuroD1-RFP were plated on laminin-coated dishes and
cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS and βmercaptoethanol. Various antibodies and fluorescence microscopy were used to
identify expression of neural/photoreceptor markers and NeuroD1 protein
expression.
Results: RFP-positive cells were confirmed by immunostaining for NeuroD1;
RFP-positive cells were isolated using FACS. Transduction with NeuroD1 induced
phenotypical changes in ES-D3 cells and positive immunofluorescence staining for
neural markers β-tubulin III. Conversely, we did not observe similar staining from
ES-D3 cells without transduction cultured on laminin plated controls.
Conclusions: Microarray analysis reveals high expression levels of transcriptions
factors involved in mouse retinal development, such as NeuroD1. Ex vivo gene
expression by a lentiviral delivery system of a single gene such as NeuroD1 into
mouse ESCs direct their differentiation towards a neural but not photoreceptor
phenotype.
Commercial Relationships: Mark A. Fields, None; Laura Vickers, None; Hui
Cai, None; Jie Gong, None; Stephen Tsang, None; Lucian V. Del Priore, None
Support: Research to Prevent Blindness, Robert L. Burch III Fund, Retina Society,
Hickeys Family Foundation and the Foundation Fighting Blindness
Program Number: 5991 Poster Board Number: A140
Presentation Time: 11:15 AM - 1:00 PM
Tumor Necrosis Factor Alpha Signaling is Required for Photoreceptor
Regeneration
Craig M. Nelson, Patrick O'Hayer, David R. Hyde. Department of Biology and
Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN.
Purpose: Constant light exposure causes photoreceptor apoptosis in dark-adapted
adult zebrafish, which induces Müller glia proliferation to yield transiently
amplifying pluripotent neuronal progenitor cells that migrate to the ONL and
differentiate into photoreceptors. The signals mediating the initiation of
regeneration remain unknown. We hypothesize that dying photoreceptors generate
a trans-acting signal that induces regeneration. This analysis attempts to identify
this photoreceptor-generated signal that initiates regeneration.
Methods: Light-damaged and undamaged adult zebrafish retinas were
homogenized and the lysate was injected into healthy eyes. Undamaged and lightdamaged protein lysates were analyzed by 2D gel electrophoresis and MALDITOF mass spectrometry to identify candidate signal proteins. Intravitreal injection
and elctroporation of anti-TNFα morpholinos was used to knockdown expression
of TNFα protein. Immunohistochemistry was used to analyze cell death (TUNEL),
proliferation (PCNA), and TNFα expression, which was also quantified on
immunoblots.
Results: Injection of light-damaged retinal lysates significantly increased the
number of dividing Müller glia relative to control lysate-injected eyes. Proteomic
techniques revealed increased expression of TNFα pathway proteins at the time of
photoreceptor death. TNFα expression was increased by photoreceptor cell death
and subsequently localized to the proliferating Müller glia. Knockdown of TNFα
prior to initiating constant light treatment resulted in significantly reduced numbers
of proliferating Müller glia.
Conclusions: A trans-acting signal is generated by dying photoreceptors that
stimulates Müller glia proliferation. Proteins from the TNFα signaling pathway
increased in expression when the photoreceptor cells were undergoing cell death
and knockdown of TNFα significantly decreased the number of proliferating
Müller glia. These data suggest that TNFα signaling is required for Müller glia
proliferation in response to photoreceptor death.
Commercial Relationships: Craig M. Nelson, None; Patrick O'Hayer,
None; David R. Hyde, None
Support: University of Notre Dame Center for Zebrafish Research
Program Number: 5992 Poster Board Number: A141
Presentation Time: 11:15 AM - 1:00 PM
Expression of Epithelial Membrane Protein 2 (EMP2) in Stem Cells
Nicholas H. Cu1A, Fernando A. Fierro1B, Krisztina I. Forward2, Jan Nolta3, Lynn
K. Gordon4, David G. Telander5. AOphthalmology / Stem Cell Program, BStem Cell
Program, 1Univ of California, Davis Sch of Med, Sacramento, CA;
2
Ophthalmology, Univ of California, Davis Sch of Med, Davis, CA;
3
Ophthalmology & Vision Science, University of California, Davis Eye Center,
Sacramento, CA; 4Jules Stein Eye Inst, Univ of California-Los Angeles, Los
Angeles, CA; 5Ophthalmology, University of California, Davis, Sacramento, CA.
Purpose: Epithelial membrane protein-2 (EMP2) is a tetraspan protein that has
been shown to regulate surface expression of integrins. EMP2 has been found in
localized regions of the body, including the eye. In this study, we set out to see if
EMP2 is present in stem cells.
Methods: The human embryonic stem cells (hESC) line H9, human bone marrow
derived - mesenchymal stromal cells (MSC), and the ARPE-19 cell line were
cultured and used in this study. Quantification of EMP2 expression was determined
with immunohistochemistry and Western Blot analysis. Immunohistochemistry and
Western Blot analysis for EMP2 detection was performed with an antibody against
EMP2. F-actin was also identified and labeled in immunohistochemistry with
phalloidin.
Results: Immunohistochemistry demonstrated that EMP2 is expressed in
undifferentiated hESCs, undifferentiated MSCs and ARPE-19 cells. EMP2 was
also found to be expressed in hESC that were 20 days into the differentiation
pathway toward retinal pigment epithelium. Western Blot analysis confirmed that
EMP2 is present and expressed in MSC and ARPE-19 cells.
Conclusions: EMP2 is known to exist in the eye, however, these findings show
that EMP2 is also highly expressed in undifferentiated embryonic stem cells and
mesenchymal stromal cells.
Commercial Relationships: Nicholas H. Cu, None; Fernando A. Fierro,
None; Krisztina I. Forward, None; Jan Nolta, None; Lynn K. Gordon,
None; David G. Telander, None
Support: Foundation for Fighting Blindness (DGT), Macular Degeneration
Foundation (DGT), NIH Grant EY019909 (DGT,LKG)
Program Number: 5993 Poster Board Number: A142
Presentation Time: 11:15 AM - 1:00 PM
Accessory Lacrimal Tissue Gene Expression
Ramesh M. Singa1, Vinay K. Aakalu1, Amy Lin2, Pete Setabutr1, Sandeep Jain1.
1
Department of Ophthalmology and Visual Sciences, University of Illinois at
Chicago Ear and Eye Infirmary, Chicago, IL; 2Department of Pathology, University
of Illinois at Chicago, Chicago, IL.
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Purpose: Extensive research has shown that pancreatic exocrine tissue and salivary
glandular tissue contains slow cycling subsets of precursor cells which may
represent a source of tissue for ex vivo expansion and therapeutic application. The
present study is focused on studying the gene expression of accessory lacrimal
tissue with the intent of identifying cells which express precursor cell markers.
Methods: With IRB approval, conjunctival specimens were obtained from patients
undergoing Müller Muscle-Conjunctival Resection (MMCR) at the Illinois Ear and
Eye Infirmary. Accessory lacrimal glands were identified and processed into a
tissue microarray (TMA). PDX-1 and nestin antibodies (markers associated with
multipotent cells in exocrine tissue) were applied to several sections of the TMA
and confocal microscopy was used to evaluate for fluorescence after secondary
antibody application. A section of the TMA was stained with hematoxylin and
eosin and used as a map to characterize the histopathology of tissue.
Results: Slides stained with nestin and PDX-1 demonstrated immunofluoroescence
in accessory lacrimal gland tissue.
Affinity for nestin (first image) and PDX-1 (second image) in accessory lacrimal
gland acini, as seen by rhodamine red stain.
Conclusions: Cells in the accessory lacrimal gland tissue that may represent
precursor cells as indicated by nestin and PDX-1 marker fluorescence were
identified. Future goals include isolation and culturing of these nestin-and PDX-1expressing precursor cells.
Commercial Relationships: Ramesh M. Singa, None; Vinay K. Aakalu,
None; Amy Lin, None; Pete Setabutr, None; Sandeep Jain, None
Support: None
Program Number: 5994 Poster Board Number: A143
Presentation Time: 11:15 AM - 1:00 PM
Photoreceptor- and RPE-like Cells Derived from Human Pluripotent Stem
Cells Display Characteristic Responses to Small Molecule Stimulation
David M. Gamm1A,1B, Jason S. Meyer2,1B, Kyle A. Wallace1B, Amelia D.
Verhoeven1B, De-Ann M. Pillers1C, Bikash R. Pattnaik1D,1C. AOphthalmology and
Visual Sciences, Eye Research Institute, Stem Cell Regen Med Center, BWaisman
Center, CPediatrics, DOphthalmology and Visual Sciences, Eye Research Institute,
1
University of Wisconsin, Madison, WI; 2Biology, IU Regen Med Center, Indiana
University Purdue University Indianapolis, Indianapolis, IN.
Purpose: To examine the physiological responses of human pluripotent stem cell
(hPSC)-derived retinal cell populations following exposure to selected small
molecules.
Methods: Isolated optic vesicle(OV)-stage neurospheres derived from hPSCs were
differentiated to photoreceptor(PR)- and/or RPE-like cells using an established
protocol. Current responses of individual PR-like cells to applied voltage were
recorded in the presence or absence of agonists and antagonists using standard
whole-cell and perforated patch-clamp configurations. After recording, cells were
backfilled with sulphorhodamine to facilitate identification. To monitor [Ca2+]i
changes in response to ATP, hPSC-RPE cells were first loaded with Fura2AM.
Thereafter, fluorescence imaging was performed to obtain time-dependent, agonistinduced [Ca2+]i measurements.
Results: Differentiating OV neurospheres expressed appropriate gene and/or
protein markers, including those involved in phototransduction. PR-like cells
demonstrated an outward current at depolarizing voltages between -50 and +60 mV
(holding potential = -70 mV) that was blocked by TEA. These PR-like cells
possessed a resting membrane potential of -44±4 mV and a current density of
34±7.5 pA/pF at +40 mV. Upon treatment with membrane-permeable Br-cGMP,
PR-like cells underwent depolarization. hPSC-RPE cells also responded to small
molecule stimulation, consistently showing a transient increase in [Ca2+]i after
exposure to ATP.
Conclusions: hPSC-derived, PR- and RPE-like cells displayed important
functional properties in vitro. Of particular interest, PR-like cells depolarized in the
presence of the phototransduction second messenger cGMP, and RPE cells
demonstrated an increase in [Ca2+]i in response to exogenous ATP, a molecule
postulated to govern the RPE light response. The capacity of hPSC-derived retinal
progeny to respond to physiological stimuli in vitro extends their utility as tools for
basic science and clinical research.
Commercial Relationships: David M. Gamm, None; Jason S. Meyer,
None; Kyle A. Wallace, None; Amelia D. Verhoeven, None; De-Ann M. Pillers,
None; Bikash R. Pattnaik, None
Support: FFB Wynn-Gund Translational Research Acceleration Award, Lincy
Foundation, Macula Vision Research Foundation, NIH P30HD03352, Eye
Research Institute, Retina Research Foundation, ICTR Pilot Grant
Program Number: 5995 Poster Board Number: A144
Presentation Time: 11:15 AM - 1:00 PM
ß-catenin Signaling Is Downregulated In The Regenerating Zebrafish Retina
Ryne A. Gorsuch, Michael Petravick, David R. Hyde. Department of Biology and
Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN.
Purpose: The light-damaged zebrafish retina undergoes a robust regenerative
response that initiates with Müller glia dividing asymmetrically to produce a
proliferating neuronal progenitor cell (NPC) population that restores the lost
neurons. In contrast, the damaged mammalian retina exhibits only a limited number
of proliferating Müller glia that rarely differentiate into the proper cell type.
However, treatment of the damaged rodent retinas with various growth factors can
stimulate significantly more Müller glia to divide and increase the regeneration of
the correct neuronal cell fate. Because Wnt ligands, which activate the canonical
Wnt/ß-Catenin signaling pathway, appear to induce mammalian regeneration, we
examined ß-Catenin expression in the regenerating zebrafish retina.
Methods: The spatial and temporal expression of ß-Catenin during regeneration of
the light-damaged retina was assessed by immunohistochemistry. ß-Catenin
expression was also quantified from light-damaged retinal lysates by immunoblots.
Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of both
ß-catenin paralogs (ctnnb1/2) during regeneration.
Results: Prior to retinal damage, ß-Catenin was localized in INL nuclei, which
likely represent subsets of Müller glia, bipolar and horizontal cells. At 31 hours of
light, ß-Catenin expression was reduced in PCNA-positive INL cells, likely the
proliferating Müller glia. ß-Catenin remains absent from PCNA-positive NPCs
throughout the remainder of the light treatment. Furthermore, cytoplasmic
expression of ß-Catenin increased at 51 hours of light and became strongly
localized to the NPC cortical processes and the outer limiting membrane.
Immunoblot analysis of total retinal lysate confirmed the increased ß-Catenin
expression. qRT-PCR will reveal which ctnnb paralog is differentially expressed at
the transcript level.
Conclusions: Light-damage decreases ß-Catenin nuclear signaling in proliferating
Müller glia, where it is redistributed to the cell cortex. In contrast to the
mammalian retina, these data suggest that either ß-Catenin nuclear signaling must
be downregulated for Müller glial proliferation or is needed to allow Müller glia to
respond correctly to regenerative signals.
Commercial Relationships: Ryne A. Gorsuch, None; Michael Petravick,
None; David R. Hyde, None
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Support: Center for Zebrafish Research
Program Number: 5996 Poster Board Number: A145
Presentation Time: 11:15 AM - 1:00 PM
Multipotent Stem Cells In Murine Sclera
Pei-Chang Wu1A,2A, Chia-Ling Tsai1B,3, M. Elizabeth Fini2B, Songtao Shi3.
A
Ophthalmology, BDentistry, 1Chang Gung Memorial Hospital, Kaohsiung county,
Taiwan; AInstitute for Genetic Medicine, BUSC Institute for Genetic Medicine,
2
Keck School of Medicine of USC, Los Angeles, CA; 3Center for Craniofacial
Molecular Biology, School of Dentistry, USC, Los Angeles, CA.
Purpose: Sclera forms the outer coat of the eyeball and provides a strong
framework composed by fibrous extracelluar matrix. Cells of sclera secrete
specialized extracellular matrix. The aim of this study is to identify scleral
stem/progenitor cells (SSPC) derived from murine sclera.
Methods: Scleras of C57BL6/J mice were enzyme digested after retina and choroid
were removed. Proliferating cells in sclera were studied by clonogenicity, selfrenew ablity, proliferation by Brdu and multipotent differentiation capacity. Flowcytometry, RT-PCR, Western blot analysis and immunofluorescence staining were
used for analysis.
Results: SSPC cells from mouse scleral tissues appeared spindle fibroblast-like and
adherent to the culture plate. SSPCs show abilities of clonal growth, proliferation
and self-renewing capacity. These cells showed colony forming ability in 14 days
of plating. SSPCs showed a population doubling over 40. Immunophenotyping of
these cells by FACS and immunofluorescence showed that the SSPC were positive
for mesenchymal markers and negative for hematopoietic markers (Sca-1, CD90.2,
CD44, CD105, CD73, CD45-, CD11b-, Flk1-, CD34-, CD117 -). These cells
expressed undifferentiated stem cell markers including PAX6, ABCG2, Six2 and
NOTCH1 genes, and some lineage-specific markers like alpha-SMA, Vimentin and
Collagen type 1. These cells had negative gene expression for epithelium and
muscle cell marker like CK12, CK19 and Desmin.The culture of SSPCs can be
differentiated toward the adipogenic, chondrogenic, and neurogenic lineages.
Conclusions: The results indicate that sclera-derived cells are a new source of
multipotent mesenchymal stem cells. These appear to be the first cells from sclera
identified with stem/progenitor potential. Further analysis of these cells will aid
elucidation of molecular mechanism of sclera development, scleritis and myopia.
Commercial Relationships: Pei-Chang Wu, None; Chia-Ling Tsai, None; M.
Elizabeth Fini, None; Songtao Shi, None
Support: None
Program Number: 5997 Poster Board Number: A146
Presentation Time: 11:15 AM - 1:00 PM
In Vitro Expansion Of Mesenchymal Stem Cell-like Cells From The Human
Trabecular Meshwork
Tina T. Wong1,2, Padmapriya Sathiyanathan3, Stephanie Chu2, Li Fong Seet2,
Lawrence W. Stanton3. 1Glaucoma, Singapore National Eye Centre, Singapore,
Singapore; 2Ocular Wound Healing and Therapeutics, Singapore Eye Research
Institute, Singapore, Singapore; 3Stem Cell and Developmental Biology Group,
Genome Institute of Singapore, Singapore, Singapore.
Purpose: Development of primary open-angle glaucoma (POAG) is associated
with increased resistance to aqueous humour outflow through the trabecular
meshwork. The decrease in trabecular meshwork (TM) cell population and cellular
dysfunction resulting in excessive deposition of extracellular matrix proteins, both
characteristics of glaucomatous TM tissue, are believed to contribute to increased
outflow resistance and disease development. Putative stem cells are thought to exist
in the TM. The purpose of the study is to culture cells derived from the TM and
characterize them for stem cell characteristics.
Methods: The TM was stripped from cadaver eyes and treated with collagenase
overnight. The samples were trypsinized and seeded in DMEM (low glucose) with
10% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate and 1 mM non-essential
amino acids. The expanded cells were characterized for markers of TM tissue as
well as stem cells by real-time quantitative PCR (qPCR), immunofluorescent
analyses and flow cytometry.
Results: qPCR showed that the expanded cells were positive for the TM markers,
CHI3L1, ANK3, HMFG-1, MMP1, MGP and LDL-R. Both qPCR and
immunofluorescent analyses confirmed the expression of the stem cell markers,
Nanog, Oct4 and Nestin. qPCR also revealed the expression of the mesenchymal
stem cell (MSC) markers CD73, CD90, CD105, and CD146, and the absence of
CD31, CD34, and CD45 expression which are not found in MSCs. Flow cytometry
analyses confirmed the expression of the MSC markers in a significant population
of the cultured cells. Morphologically, the cells resembled MSCs in appearance.
Conclusions: Cells derived from the TM tissue and cultured in vitro express stem
cell as well as MSC markers. These cells may have the potential to help replace lost
or dysfunctional cells in the TM tissue associated with glaucoma.
Commercial Relationships: Tina T. Wong, None; Padmapriya Sathiyanathan,
None; Stephanie Chu, None; Li Fong Seet, None; Lawrence W. Stanton, None
Support: NMRC/TCR/002-SERI/2008
Program Number: 5998 Poster Board Number: A147
Presentation Time: 11:15 AM - 1:00 PM
A Ciliopathy Gene CC2D2a is Required For Eye Development
Trevor Foskett1A, Shobi Veleri1A, Milton A. English1A,2, Raman Sood2, Rivka
Rachel1A, Lijin Dong1B, Paul Liu2, Anand Swaroop1A. ANeurobiologyNeurodegeneration & Repair Lab, BGenetic Engineering Core, 1National Eye
Institute, Bethesda, MD; 2National Human Genome Research Institute, Bethesda,
MD.
Purpose: CC2D2a (Coiled-Coil & C2 Domain containing 2a) has been identified
as one of the causal genes associated with ciliopathies like Joubert syndrome (JS)
and Meckel-Gruber syndrome (MKS) in patients. Photoreceptor morphology and
function require an intact connecting cilium. Hence, the goal of this project is to
determine the role of CC2D2a in photoreceptor development and function.
Methods: In order to test whether mutations of CC2D2a alone could cause JS- and
MKS-like phenotypes, we sought to knock down its function in zebrafish. This was
accomplished using anti-sense morpholinos (both translation-blocking and spliceblocking). A CC2D2a-KO mouse has been generated to further explore its role in
cilia-related functions with a focus on photoreceptor development.
Results: After injection with anti-sense CC2D2a morpholinos, zebrafish embryos
showed dose-dependent defects. Notably, the eye is smaller than in control
zebrafish, and appeared to be malformed. The morphant zebrafish also showed
convergent extension (CE) defects. Additionally, CC2D2a MO-injected zebrafish
are much smaller than those injected with control morpholinos. The
characterization of CC2D2a-KO mouse is in progress.
Conclusions: Our zebrafish data indicate that CC2D2a plays a role in primary cilia
biogenesis and is required for photoreceptor development.
Commercial Relationships: Trevor Foskett, None; Shobi Veleri, None; Milton
A. English, None; Raman Sood, None; Rivka Rachel, None; Lijin Dong,
None; Paul Liu, None; Anand Swaroop, None
Support: NEI Intramural
Program Number: 5999 Poster Board Number: A148
Presentation Time: 11:15 AM - 1:00 PM
Function of DNA Methyltransferase 1 (Dnmt1) is Essential for Mouse Retinal
Cell Proliferation and Photoreceptor Differentiation
Xian-Jie Yang1, Kun-Do Rhee1, Carrie Y. Zhao1, Juehua Yu2, Guoping Fan2.
1
Ophthalmology, Jules Stein Eye Institute-UCLA, Los Angeles, CA; 2Human
Genetics, Department of Human Genetics-UCLA, Los Angeles, CA.
Purpose: DNA methylation is essential for regulation of gene expression, X
chromosomal inactivation, genomic imprinting, and chromatin modification.
Among the three types of DNA methyltransferases, Dnmt1 is involved in
maintenance of methylation patterns. Germ line Dnmt1 knockout is embryonic
lethal. Brain-specific Dnmt1 conditional knockout (cKO) causes neuronal cell
death and promote astroglial differentiation. In this study, we examine the roles of
Dnmt1 in the developing mouse retina.
Methods: Mice carrying a Dnmt1 loxP allele were crossed with a Chx10-cre
transgenic line, which expresses Cre at the onset of retinogenesis. The resulting
Dnmt1 cKO mutant retinas were analyzed at various postnatal stages by
immunohistochemistry and confocal imaging, western blot analysis, and
fluorescence activated cell sorting.
Results: From birth to the adulthood, Dnmt1 cKO retinas show progressive
thinning, especially of the outer nuclear layer. At birth, Dnmt1 cKO mutant retinas
exhibit a decreased neuroblast layer (ONL). By P3, the mutant retinas show
abnormal distribution of progenitor cells. Cell cycle analyses reveal that Dnmt1
deletion results in delayed G1 to S phase entry, and reduced G2/M phase cells. Cell
marker labeling shows that most retinal cell types are generated in the Dnmt1
mutant retina, albeit at lower numbers. During the period of photoreceptor
differentiation (P3 to P7), significant loss of ONL cells incurs. Photoreceptor fate
specification is not affected, as expression of Otx2 among progenitors appears
normal. However, postmitotic Crx-positive photoreceptor precursors fail to mature
and die quickly at the onset of opsin expression.
Conclusions: Elimination of Dnmt1 function during retinogenesis causes defects in
progenitor cell cycle progression and reduced neuronal production. The absence of
Dnmt1 activity blocks differentiation of photoreceptor precursor and causes rapid
cell death. Therefore, maintaining DNA methylation by Dnmt1 is essential for
proper retinal progenitor cell proliferation and photoreceptor differentiation.
Commercial Relationships: Xian-Jie Yang, None; Kun-Do Rhee, None; Carrie
Y. Zhao, None; Juehua Yu, None; Guoping Fan, None
Support: NIH-NEI , Research to Prevent Blindness, and California Institute of
Regenerative Medicine
Program Number: 6000 Poster Board Number: A149
Presentation Time: 11:15 AM - 1:00 PM
Regulation Of The Key Photoreceptor Differentiation Factor Nrl During
Retinal Development
Marie-Audrey Kautzmann1, Douglas Kim1,2, Anand Swaroop1. 1NEI, NeurobiologyNeurodegeneration and Repair Laboratory, Bethesda, MD; 2Howard Hughes
Medical Institute, Janelia Farm Research Campus, Ashburn, VA.
Purpose: NRL (Neural Retina Leucine zipper) is a bZIP transcriptional regulator
that controls rod versus cone cell fate decision during retinal development. It is
essential and sufficient for rod differentiation. NRL is also required for
maintenance of rod photoreceptor function as it regulates the expression of most
rod-specific genes. This study aims at understanding how expression of the mouse
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Nrl gene is regulated during retinal development.
Methods: Putative mouse Nrl promoter/enhancer regions were identified by
sequences homology of genomic region upstream of the Nrl transcription start site.
Relevant genomic regions were cloned in the pEGFP-N1 vector. The plasmid
constructs were electroporated in newborn CD1 mouse retina. The fluorescent
reporter gene expression was assessed 14 days after in vivo electroporation. In
silico analysis was performed to identify transcription factor binding sites on Nrl
promoter segments. The binding of candidate regulators was assessed by EMSA
using nuclear extracts from P0-2 and adult mouse retina. HEK293 cells were
transiently transfected to assess promoter activation with luciferase reporter gene.
Results: From 0.4 kb to 3.9 kb lengths of Nrl promoter region were electroporated
in P0 mouse retina. Except for the 0.4 kb construct, the three larger constructs
faithfully produced GFP expression in rod photoreceptors. The 0.9 kb sequence
upstream of the Nrl transcription initiation site was sufficient to drive high reporter
gene expression. In silico study revealed binding sequences for bHLH proteins and
several candidate transcription factors. EMSA studies confirm the binding of
regulatory proteins on predicted conserved sequences. Amongst the transcription
factors tested by luciferase assay, MASH1 shows high activation of the Nrl
promoter.
Conclusions: Our data show that the regulatory sequences necessary for the rod
photoreceptor specific expression of Nrl are located between 0.4 kb and 2.8 kb of
upstream region. Further analysis of the promoter/enhancer regions points to
multiple putative transcription factors that may control Nrl expression in
developing and mature retina. The identification of specific Nrl regulators will lead
to better understanding of regulatory networks that dictate the genesis of
photoreceptors from multipotent retinal progenitors.
Commercial Relationships: Marie-Audrey Kautzmann, None; Douglas Kim,
None; Anand Swaroop, None
Support: None
Program Number: 6001 Poster Board Number: A150
Presentation Time: 11:15 AM - 1:00 PM
Melanopsin X Undergoes A Radical Temporo-spatial Switch In Cellular
Expression During Chick Retinal Development
Daniela M. Verra1, Maria A. Contin1, David Hicks2, Mario E. Guido1. 1Biological
Chemistry- Chemistry School, CIQUIBIC/ CONICET - National University of
Cordoba, Cordoba, Argentina; 2Neurobiol Rhythms, CNRS UPR 3212/INCI,
INSERM, Strasbourg, France.
Purpose: Two orthologs of melanopsin (Opn4) have been identified, Opn4m and
Opn4x. We examined expression patterns of photodetection molecules and
assessed the histological localization of both Opn4 isoforms in chicken retina
during development.
Methods: Retinas from embryonic (E) day 4 to 19 or post-hatch day 7 (P7) chicks
were used for western blotting (WB), immunohistochemistry (IHC) or RT-PCR.
WB used anti-Opn4m/x antibodies, and RT-PCR was done for the transcription
factors Pax6, Brn3 and Crx, the G transducin proteins Gq and α-transducin, and
opsins Opn4m, Opn4x, rhodopsin and red opsin. IHC was done on E8-19 and P7
retina using antibodies against Opn4m/x and cell-specific markers.
Results: We observed two onsets of transcription, one at E4 with expression of
Pax6, Brn3, Gq, and Opn4m and the second at E7-12 with Crx, α-transducin,
rhodopsin and red opsin. Strikingly, Opn4x did not appear till E7-E8. WB
confirmed mRNA data for Opn4m and x. Immunolocalization of both Opn4
showed that Opn4m was restricted to the GC layer at all ages. In stark contrast,
Opn4x was also limited to the GC layer and optic nerve at E8, but by E15 its
expression was mainly confined to cells in the outer retina, presumably horizontal
cells; Opn4x remained expressed in the optic nerve of the mature retina.
Conclusions: The initial onset of expression of non-visual photocascade molecules
could confer early photosensitivity to RGCs. Opn4x switches expression to
horizontal cells concomitantly with their birth. These cells play roles in receptive
field construction, and the data suggest the existence of a new type of
photosensitive retinal cell in birds.
Commercial Relationships: Daniela M. Verra, None; Maria A. Contin,
None; David Hicks, None; Mario E. Guido, None
Support: ANPCyT-FONCyT, CONICET, MinCyT-Cba, SECyT-UNC.
Program Number: 6002 Poster Board Number: A151
Presentation Time: 11:15 AM - 1:00 PM
Requirement of DNA Double-strand Break Repair in Retinal Neurogenesis
and Axonogenesis
Enrique J. de la Rosa1, Jimena Baleriola1, Noemi Alvarez1, Jose L. San-Martin1,
Teresa Suarez1, Gloria Terrados2, Beatriz Escudero3, Antonio Bernad3, Luis
Blanco2. 1Centro de Investigaciones Biologicas CSIC, Madrid, Spain; 2Centro de
Biología Molecular CSIC-UAM, Madrid, Spain; 3Centro Nacional de
Investigaciones Cardiovasculares, Madrid, Spain.
Purpose: Programmed cell death is a genuine process of retinal development that
affects all cell types including progenitors, young neurons, as well as mature
neurons and glial cells. However, the process underlying the death of recently
differentiated neurons during neurogenesis is not established. Several geneticallymodified mouse model systems defective in DNA double-strand break repair
present a dramatic effect on early nervous system development, suggesting a
possible involvement of DNA repair in neural development. Hereby, we try to
demonstrate in vivo the requirement of DNA repair for proper retinal neurogenesis.
Methods: We have analyzed the embryonic retina in two mouse models deficient
for DNA repair, namely Pol mu and SCID, as well as in their wild type
counterparts. Whole mount and dissociated retinas were processed for
immunohistochemistry and TUNEL to determine the effect of the mutations on
DNA repair, neuronal differentiation, axonogenesis and cell death.
Results: Both Pol mu and SCID mutants showed distorted DNA repair and an
increase in apoptotic cell death that selectively affected young retinal ganglion
cells. Further, the pattern of axonal growth into the retina and the optic nerve was
also altered.
Conclusions: Our results support the requirement of balanced DNA double-strand
break generation and repair during retinal neurogenesis in order to achieve correct
retinal development. Further, the occurrence of double-strand breaks seems to
underlie the process of early neural cell death.
Commercial Relationships: Enrique J. de la Rosa, None; Jimena Baleriola,
None; Noemi Alvarez, None; Jose L. San-Martin, None; Teresa Suarez,
None; Gloria Terrados, None; Beatriz Escudero, None; Antonio Bernad,
None; Luis Blanco, None
Support: Spanish Ministerio de Ciencia e Innovación Grants SAF2007-66175
(EJdlR) and CSD2007-00015 (LB)
Program Number: 6003 Poster Board Number: A152
Presentation Time: 11:15 AM - 1:00 PM
Midkine-a Functions As An Autocrine Regulator Of The Cell Cycle In Retinal
Progenitors
Jing Luo, Laura Kakuk-Atkins, Peter Hitchcock. Ophthalmology & Vis Science,
University of Michigan, Ann Arbor, MI.
Purpose: Midkine is a secreted heparin binding growth factor that has numerous
biological functions, both during development and following tissue injury. In the
vertebrate retina, midkine is expressed in retinal stem and progenitor cells
(Calinescu et al., 2010; Livesey et al., 2004). There are two midkine orthologs in
zebrafish, Midkine-a (Mdka) and Midkine-b. The purpose of this study was to use
both loss- and gain-of function approaches in zebrafish to investigate the function
of Mdka during early retinal development.
Methods: Morpholino oligonucletides were injected into embryos to knock down
Mdka synthesis. The Tol2 transposon system was used to generate transgenic lines
for conditional gain-of-function. Dividing cells were labeled with either BrdU or
EdU. Analysis of Percent Labeled Mitoses, using EdU to label cells in S-phase and
antibodies to label cells in M-phase, was used to measure the time for cells to
transit from S- to M-phase (G2) of the cell cycle. Standard methods of
immunocytochemistry and in situ hybridization were used to identify labeled cells.
Western blot analysis from whole embryo lysates was used to evaluate the loss of
Mdka and the induction of a Mdka:EGFP fusion protein. Retinas from
experimental and control animals were evaluated between 26 and 72 hours post
fertilization (hpf).
Results: The loss of Mdka does not alter gross embryonic or larval development,
but does result in a transient delay in neuronal differentiation. Labeling with BrdU
shows that at 48hpf there is a paucity of differentiated neurons and all cells are
proliferative, though there are significantly fewer cells in M-phase. This delay in
neuronal differentiation does not result from a delay in initiating molecular
developmental events, but results from a lengthening of the cell cycle. The cell
cycle is significantly longer in morphants than in wild-type retinas. In contrast to
Mdka loss-of-function, the cell cycle is significantly shorter following Mdka gainof-function.
Conclusions: In retinal stem and progenitor cells, Mdka functions as an autocrine
regulator of the cell cycle. This study provides evidence for a novel signaling
pathway during early retinal development.
Commercial Relationships: Jing Luo, None; Laura Kakuk-Atkins,
None; Peter Hitchcock, None
Support: NIH RO1 EY07060; NIH P30 EY07003; Research to Prevent Blindness
Program Number: 6004 Poster Board Number: A153
Presentation Time: 11:15 AM - 1:00 PM
E2f And Mycn Regulate Retinal Differentiation Through Notch Pathway
Danian Chen, Rod Bremner. Vision Sci Res Program, Toronto Western Hospital,
Toronto, ON, Canada.
Purpose: Coordination of the cell cycle and differentiation is essential to produce
the proper numbers and types of cells in the developing retina. The activating E2fs
and Mycn are indispensable for proliferation of mouse retinal progenitors. However
their roles in retinal differentiation are not clear yet. Our goal was to determine the
effect and mechanism of inactivating E2fs and Mycn on retinal differentiation.
Methods: E2f1-/-, E2f2-/-, E2f3 loxP/loxP, MycnloxP/loxP mice were interbred with
alpha-Cre mice. The transgenic alpha-Cre recombinase specifically expresses in the
peripheral retina from E9.5. Retrovirus injection or plasmids electroporation were
applied to deliver expression vectors of GFP-Cre, GFP control, Hes1 and Notch1
intracellular domain (NIC) into the subretinal space of newborn pups. Retinas were
assessed for cell division, death and differentiation by Brdu/PH3/Ki67 labeling,
TUNEL labeling and cell type marker immunofluorescence at various embryonic
and post-natal ages.
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Results: Decreasing progenitor proliferation in E2f1KO, E2f1-3TKO and E2f13/Mycn quadruple null (QKO) causes a switch to early born neurons only in an
extreme case (E2f1-3/Mycn QKO). Inactivating E2f1-3/N-Myc in early retinal
progenitor cells (RPC) resulted in proliferation arrest and cell cycle exit, and those
QKO retinas only generated early born neurons (ganglions, cones, horizontal and
amacrine cells) in the expenses of late-born cells (rod, bipolar and Müller glia).
Notch pathway (Notch1, Hes1, Hes5, and Hey1) was down-regulated, and Math5,
Prox1 and Trb2 were up-regulated in QKO retinas. Inactivating E2f1-3/Mycn in
late RPC by Cre retrovirus or plasmid resulted in more rod cells in the expense of
bipolar and Müller glia. Over-expression Hes1 or NIC can override this defect.
Conclusions: 1. Above a low threshold, proliferation rate per se does not influence
neurogenesis. 2. E2f1-3 and Mycn redundantly regulate the cell cycle machinery
and Notch pathway in RPCs, thus control and coordinate retinal proliferation and
differentiation.
Commercial Relationships: Danian Chen, None; Rod Bremner, None
Support: CIHR
Program Number: 6005 Poster Board Number: A154
Presentation Time: 11:15 AM - 1:00 PM
Molecular Mechanisms Underlying the Delay of Rhodopsin Expression in
Immature Rods
Hyun-Jin Yang, Anand Swaroop. Neurobiol-Neurodegen & Repair Lab, NEI,
Bethesda, MD.
Purpose: The proper formation and function of rod photoreceptors largely depend
on rhodopsin, a structural and phototransduction protein. Although differentiating
rods begin to express transcription activators of rhodopsin shortly after their final
mitosis, the onset of rhodopsin expression exhibits five to ten days of delay in
mouse. The purpose of this study is to identify regulatory sequences and
transcription repressor(s), which control rhodopsin expression in the developing
retina.
Methods: Multi-species alignment was employed to identify conserved elements of
the rhodopsin regulatory region. Luciferase reporter constructs were generated with
a full length or a series of 5’ truncated rhodopsin regulatory region. P0 mouse
retinas were then electroporated in vivo with each rhodopsin reporter gene, and the
reporter activity was assessed either at P2 or P6, before or after the normal onset of
rhodopsin expression.
Results: The regulatory sequences that negatively regulate rhodopsin expression in
immature rod precursors were identified using in vivo reporter gene assays. Five
conserved elements including the previously characterized rhodopsin proximal
promoter region and rhodopsin enhancer region were identified by in silico
analysis. In vivo reporter gene assays show that a 100 bp sequence located about
1.5 kb upstream of the rhodopsin transcription start site negatively regulates
reporter gene expression at P2 but not at P6. In silico analysis and gene expression
profile analysis were employed to identify candidate transcriptional repressors,
which have a binding site within the 100 bp region and are down-regulated after the
onset of rhodopsin expression. The transcription factors that meet both criteria are
now being validated by biochemical analysis.
Conclusions: Our data show that a 100 bp region about 1.5 kb upstream of
transcription start site imposes a negative regulatory effect on rhodopsin expression
selectively at P2. Our findings should provide insights into mechanisms that trigger
maturation of rod cells.
Commercial Relationships: Hyun-Jin Yang, None; Anand Swaroop, None
Support: NEI intramural program
Program Number: 6006 Poster Board Number: A155
Presentation Time: 11:15 AM - 1:00 PM
Regulation Of Retinal Neurogenesis By Frizzled Receptors
Chunqiao Liu1A, Hirva Bakeri2, Anand Swaroop1B. AN-NRL, BN-NRL, Bldg 6,
1
National Eye Institute, Bethesda, MD; 2College of Human Ecology, Cornell
University, New York, NY.
Purpose: To examine the role of Wnt-Frizzled signaling in retinal development.
Methods: Fz5 and Fz8 compound mutant mice were generated. Retinal
neurogenesis was analyzed by immunohistochemistry for early born neurons. BrdU
pulse was used to study cell cycle exit rate. Frizzleds’ function was investigated
with acute retinal explants, in vitro. Phalloidin was used to stain neuroblast F-actin,
laminin was used to study ECM deposition. Hes1 expression was examined by in
situ hybridization to monitor changes in Shh and or Notch signaling pathway.
Results: Embryos lacking both Fz5 and Fz8 die in early development. A majority
of triallelic Fz5-/-;Fz8+/- mutants that survive until birth develop severe retinal
coloboma and microphthalmia with full penetrance. Ganglion and amacrine cell
populations are disproportionally large at E17.5. Increased cell cycle exit of retinal
progenitors was detected at E13. Neuroblast organization and apical basal polarity
are perturbed. Hes1, a common target of Shh and Notch signaling, is
downregulated. In vitro blocking of Frizzled receptors leads to randomized
divisions of progenitors along the retinal neuroblast layer.
Conclusions: Fz8 and Fz5 signaling in a dose-dependent manner controls retinal
progenitor expansion.
Commercial Relationships: Chunqiao Liu, None; Hirva Bakeri, None; Anand
Swaroop, None
Support: NEI intramural program
Program Number: 6007 Poster Board Number: A156
Presentation Time: 11:15 AM - 1:00 PM
App Involvement In Retinogenesis
Frederic B. Mascarelli1, Na An1, Giuseppe Ciccotosto2A, Shayne Bellingham2B,
Andrew Hill2B, Olav Andersen3, Anders Nykjaer3, Roberto Cappai2A, Virginie
Dinet1. 1Centre de Recherches des Cordeliers, INSERM, Paris, France;
A
Department of Pathology, BDepartment of Biochemistry & Molecular Biology,
2
Bio21 Molecular Science and Biotechnology Institute, Melbourne, Australia;
3
Department of Medical Biochemistry, MIND-center, Aarhus, Denmark.
Purpose: The amyloid precursor protein (APP), expressed most abundantly in the
brain, is known to play, together with its proteolytic fragments, numerous and
varied roles in cell physiology and pathology, including in Alzheimer disease (AD).
Amyloid beta, the main proteolytic fragment of APP has been recently detected in
retinas of AD patients.Our understanding of APP's normal functions remains
however very limited. We therefore investigated the spatiotemporal patterns of
expression and the roles of APP in the mice developing neuroretina and in adults.
Methods: We identified the cell types that expressed APP by PCR, in situ
hybridization and immunochemistry. APP knockout mice were used to define the
functional role of APP in retinal cell differentiation. To determine the mechanism
of action of APP, retinal differentiation and APP expression were investigated in
mice knocked out for sorLA, a recently identified regulator of APP.
Results: APP695, APP751 and APP770 are expressed in the mouse retina from
embryonic stage through adulthood. APP mRNA and protein are expressed
according to the different waves of retinal differentiation. Depletion of App led to
alteration of the inner synaptic layer, only half as many glycinergic amacrine cells
and a 50% increase in the number of horizontal cells. We identified Ptf1a as a
downstream effector of APP. A similar phenotype in mice knocked out for sorLA
was observed.
Conclusions: APP functions through sorLA to control the determination of AII
amacrine and horizontal cell fate. These findings provide novel insights that
indicate that APP plays an important role in retinal differentiation.
Commercial Relationships: Frederic B. Mascarelli, None; Na An,
None; Giuseppe Ciccotosto, None; Shayne Bellingham, None; Andrew Hill,
None; Olav Andersen, None; Anders Nykjaer, None; Roberto Cappai,
None; Virginie Dinet, None
Support: Supported by a European grant LSHG-CT-2005-512036 from EVIGenoRET, the Ministère de la Recherche and the ANR and the NHMRC.
Program Number: 6008 Poster Board Number: A157
Presentation Time: 11:15 AM - 1:00 PM
Onecut Transcription Factors are Potential Regulators of Retinal
Development
Xiuqian Mu1,2, Fuguo Wu1, Renzhong Li1, Darshan Sapkota1. 1Department of
Ophthalmology/Ross Eye Institute, Department of Biochemistry, University at
Buffalo, Buffalo, NY; 2SUNY Eye Institute, Buffalo, NY.
Purpose: Our current study investigates whether two members of the onecut
transcription family, Oc1 (also known as Hnf-6) and Oc2, play a role in mouse
retinal development.
Methods: We performed immunofluorescence staining with anti-Oc1 and anti-Oc2
on mouse retinal sections of different developmental stages and genotypes and
examined their relationship with other retinal and cell cycle markers.
Results: The onecut transcription factors are defined by a ‘cut’ domain and an
atypical homeo domain. There are three onecut members, Oc1, Oc2 and Oc3, in the
mouse. They function in the development of the liver, pancreas, intestine and
lymphocytes. They have also been reported to be expressed in the central nervous
system such as the spinal cord. To investigate whether they regulate retinal
development, we performed immunofluorescence staining for Oc1 and Oc2 on
mouse retinas of different developmental stages. We found that these two factors
had almost identical expression patterns throughout development. Both factors
started to be expressed in the retina at around E11.5. At early stages (E11.5 and
E12.5), they were expressed in both the progenitor cell layer and ganglion cell
layer. As development progressed, their expression diminished in the progenitor
cells and became more restricted to the ganglion cell layer. By P5, Oc1 and Oc2
were expressed at very low levels in ganglion cells, but very strongly in horizontal
cells. Co-labeling of Oc1 with cell cycle markers showed that Oc1 was expressed
in both proliferating retinal progenitors and postmitotic retinal cells. Co-labeling of
Oc1 with Math5, Pou4f2 and Isl1, three transcription factors required for ganglion
cell development, revealed that Oc1 coincided with Math5 at E11.5 and E12.5, but
not at E14.5, in the progenitor cell layer, and overlapped with Pou4f2 and Isl1 in
the ganglion cell layer. Furthermore, we found that expression of Oc1 and Oc2 did
not change in retinas of Math5-, Pou4f2- and Isl1-null mice as compared to that in
wild-type retinas.
Conclusions: Oc1 and Oc2 are expressed in developing ganglion cells and their
progenitors; they may regulate the formation of ganglion cells in a pathway
independent of Math5, Pou4f2 and Isl1. The two factors are also expressed in
developing horizontal cells, suggesting that they function in the genesis of
horizontal cells as well.
Commercial Relationships: Xiuqian Mu, None; Fuguo Wu, None; Renzhong
Li, None; Darshan Sapkota, None
Support: The Glaucoma Foundation, The Whitehall Foundation, Unrestricted
Deparmental Grant from RPB
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Program Number: 6009 Poster Board Number: A158
Presentation Time: 11:15 AM - 1:00 PM
Ascl1 Expression Defines A Subpopulation Of Lineage-restricted Progenitors
In The Mammalian Retina
Joseph A. Brzezinski, IV1, Euiseok J. Kim2, Jane E. Johnson3, Thomas A. Reh1.
1
Department of Biological Structure, University of Washington, Seattle, WA;
2
Division of Biological Sciences, University of California at San Diego, San Diego,
CA; 3Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX.
Purpose: The seven principal cell types of the neural retina derive from a
population of multipotent progenitors during development. It is thought that
differences in progenitor gene expression are responsible for differences in
progenitor competence (potential) and subsequently, fate diversification. We
investigated whether basic helix-loop-helix (bHLH) transcription factor expression
heterogeneity in retinal progenitors correlated with different fate outcomes during
murine retinal development.
Methods: We assayed the expression of the bHLH transcription factors Ascl1
(Mash1), Ngn2 (Neurog2), and Olig2 in retinal progenitors using an Ascl1GFP/+
knock-in mouse line and immunohistochemistry. To determine whether progenitor
transcription factor expression heterogeneity correlated with different cell fate
outcomes, we conducted Ascl1 and Ngn2 inducible expression fate mapping using
the CreERTM/LoxP system. We examined the number of retinal ganglion cells
(RGCs) in Ascl1GFP/GFP null mice to test whether Ascl1 repressed RGC
development.
Results: We observed that progenitors were heterogeneous, expressing every
possible combination of Ascl1-GFP, Ngn2, and Olig2. Lineage tracing experiments
showed that Ngn2+ and Ascl1+ cells gave rise to markedly different distributions
of cells. The Ngn2 lineage comprised all seven retinal cell types, whereas the Ascl1
lineage lacked a single cell type, RGCs. We also observed that Ascl1 null mice had
normal numbers of RGCs during development.
Conclusions: We show that Ascl1 expression defines a competence-restricted
progenitor lineage in the retina. Nonetheless, Ascl1 itself is not required to restrict
RGC competence. Our results directly correlate progenitor transcription factor
expression heterogeneity with cell fate diversification during retinal development.
Commercial Relationships: Joseph A. Brzezinski, IV, None; Euiseok J. Kim,
None; Jane E. Johnson, None; Thomas A. Reh, None
Support: NIH Grant F32 EY19227, NIH Grant R01 EY013475, and NIH Grant
R01 NS32817
Program Number: 6010 Poster Board Number: A159
Presentation Time: 11:15 AM - 1:00 PM
Function of Transcription Factor Sox11 During Mouse Retinal Development
Ayumi Usui1,2, Shinya Satoh2, Minoru Tanaka1, Akira Murakami3, Sumiko
Watanabe2. 1Ophthalmology, Juntendo University Urayasu Hospital, Urayasu,
Japan; 2Department of Molecular and Developmental Biology, The Institute of
Medical Science, The University of Tokyo, Tokyo, Japan; 3Dept of
Ophthalmology, Juntendo Univ School of Med, Bunkyo-Ku, Japan.
Purpose: Sry-related HMG box (Sox) proteins play diverse and critical roles in a
variety of morphogenetic processes during embryonic development. We focused on
Sox11, which is a member of the SoxC subtype, due to its unique expression
pattern revealed by our microarray analysis across different subsets of retinal
progenitor cells. To elucidate the function of Sox11 in mouse retinal development,
we used gain- and loss-of-function analyses.
Methods: The detailed spatial and temporal expression pattern of Sox11 in the
developing mouse retina was examined by immunohistochemistry, in situ
hybridization and RT-PCR. We then over-expressed Sox11 by retrovirus-mediated
gene transfer in retinal explant culture prepared from E17 mouse embryos to
examine its role in retinal development. In addition, we analyzed retinal cell
differentiation, proliferation and survival in Sox11 knock out (Sox11-/-) mouse
retina by immunohistochemistory, RT-PCR, and microarray.
Results: Sox11 is expressed in early progenitor cells and early arising retinal
neurons. The expression level of Sox11 mRNA in the retina is very high in the
early stage, then decreases as development proceeds. Over-expression of Sox11 in
retinal progenitor cells resulted in aberrant sub-retinal distribution of cells in
explant culture. Moreover Muller glia cells failed to differentiate. The eyes of
Sox11-/- mice were far smaller than those of control (WT). Proliferation and
survival of Sox11-/- retinal cells were impaired in stasge-specific manner.
Neurogenesis of early arising subtypes was severely suppressed at early stages.
However, their differentiation overtakes WT at later developmental stages. We
found that the SoxC member, Sox4 is also strongly expressed in developing mouse
retina, and its expression level in retina increase as that of Sox11 subsides. Overexpression of Sox4 in retinal progenitor cells resulted in a phenotype similar to that
of Sox11.
Conclusions: Sox11 plays roles in neurogenesis, proliferation and cell survival in
retinal development and promotes differentiation of retinal neurons. The data
suggest redundancy in the activities of Sox4 and Sox11, leading us to propose
coordinated regulation of the retina by members of the SoxC gene family.
Commercial Relationships: Ayumi Usui, None; Shinya Satoh, None; Minoru
Tanaka, None; Akira Murakami, None; Sumiko Watanabe, None
Support: None
Program Number: 6011 Poster Board Number: A160
Presentation Time: 11:15 AM - 1:00 PM
Comparative Analysis of Histone Acetyl Transferases And Deacetylases In
Developing Murine And Chick Optic Cup
Deborah C. Otteson1, Teri L. Belecky-Adams2, Mahesh Shivanna2. 1Optometry,
University of Houston, Houston, TX; 2Dept Biology, Ctr Regenerative Bio & Med,
Indiana Univ-Purdue Univ Indianapolis, Indianapolis, IN.
Purpose: Histone acetyltransferases (HATs) and histone deacetylases (HDACs)
are known to regulate transcription by modifying the histones, thereby enabling or
restricting the access of transcription factors to genomic DNA. We examined HAT
and HDAC expression in developing murine retina and chick optic cup as a first
step in understanding the epigenetic mechanisms regulating retinal development
and disease.
Methods: Expression of HATs (p300, P/CAF) and class I (HDAC1, 2, 3 and 8) and
class IV HDAC (HDAC11) in the optic cup was determined at E15 (embryonic day
15) and P30 (postnatal day 30) in mice and E5, E8, and E18 in chick. For
immunostaining, eyes were fixed in paraformaldehyde and embedded in 20%
sucrose:OCT mixture (3:1 ratio) prior to cryosectioning at 10 μm. Immunostained
sections were imaged by confocal fluorescence microscopy. For western blots, total
proteins were isolated from retinas at the same developmental stages, separated on
denaturing SDS polyacrylamide gels and detected using chemiluminescence.
Results: In both mouse and chick, HDACs showed nuclear localization, with the
highest staining intensity throughout the inner retinal layers and decreasing
expression in proliferating cells in the outer half of the optic cup. Immunostaining
for p300 and P/CAF, was weaker at all stages examined, compared to the HDACs.
Western blot analysis of developing chick retina confirmed that HDAC expression
decreased with age, while HAT levels were low in early stages and increased with
age.
Conclusions: The expression patterns of class I and IV HDACs and the HATs,
P/CAF and p300, in the developing mouse and chick optic cup suggest a role for
dynamic changes in histone acetylation and deacetylation during retinal
differentiation.
Commercial Relationships: Deborah C. Otteson, None; Teri L. BeleckyAdams, None; Mahesh Shivanna, None
Support: American Health Assistance Foundation G2008-113, NIH grant
R01EY019525-01
Program Number: 6012 Poster Board Number: A161
Presentation Time: 11:15 AM - 1:00 PM
The Proneural Target Gene Sbt1 Regulates Neurogenesis By Governing
Proliferation In The Xenopus Retina
Kathryn B. Moore1, Mary A. Logan2, Issam Al Diri1, Monica L. Vetter1.
1
Neurobiology and Anatomy, University of Utah, Salt Lake City, UT; 22Jungers
Center for Neurosciences Research Department of Neurology, Oregon Health and
Science University, Portland, OR.
Purpose: Proneural basic helix-loop helix (bHLH) transcription factors are key
regulators of retinal neurogenesis, activating the expression of target genes that
execute a program of neuronal differentiation within progenitors. Our study focuses
on understanding this differentiation program in Xenopus retinal progenitors by
examining the function of bHLH target genes.
Methods: We performed a screen for proneural target genes, identifying a novel
gene called sbt1 (shared bHLH target 1). sbt1 encodes a novel protein with no
conserved functional motifs that is conserved across vertebrate species. We used in
situ hybridization and gain and loss of function approaches to elucidate the spatial
and temporal expression of sbt1 and to gain insight into its function during retinal
development.
Results: In situ hybridization analysis showed that sbt1 is transiently expressed in
late proliferating and early differentiating cells in the Xenopus retina and is
localized both at the membrane and in the nucleus. Overexpression of either mouse
or Xenopus sbt1 in progenitors promoted differentiation of early born retinal
neurons, and also enhanced the ability of the bHLH factor Ath5 to promote
neurogenesis. Conversely, inhibition of SBT1 translation in Xenopus retinal
progenitors by injection of antisense morpholino into cleavage-stage blastomeres
prevented/delayed retinal neuron differentiation, resulting in an increase in Müller
glia and progenitors. Loss of function of sbt1 in retinal progenitors blocked the
expression of markers for differentiated retinal neurons suggesting that it is
required for full proneural function. In addition, overexpression of sbt1 caused a
reduction in mitotic cells in the optic vesicle as measured by phospho-histone H3
staining. We performed a yeast 2-hybrid screen for SBT1 interactors and have
isolated several potential protein partners, including proteins involved in cell cycle
regulation.
Conclusions: Based on these results, we propose that sbt1 is expressed in retinal
progenitors as they initiate neuronal differentiation, and may function in regulating
cell cycle exit downstream of proneural bHLH factors during retinal development.
Commercial Relationships: Kathryn B. Moore, None; Mary A. Logan,
None; Issam Al Diri, None; Monica L. Vetter, None
Support: Supported by NEI WY012274 to MLV
Program Number: 6013 Poster Board Number: A162
Presentation Time: 11:15 AM - 1:00 PM
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Neil3 is Genetically Downstream of Rx and Essential for Normal Retinal
Development
Heithem M. El-Hodiri, Yi Pan. Molecular & Human Genetics, Nationwide
Children's Research Institute, Columbus, OH.
Purpose: NEIL3 is a member of the nei family of DNA glycosylases. Unlike other
family members, the NEIL3 gene encodes two putative DNA-binding zinc fingers
in addition to the canonical DNA glycosylase domain found in all nei family
members. We identified NEIL3 in a microrarray screen for genes dependent on
expression of the Retinal Homeobox (Rx) gene. The purpose of this study is to
define the genetic relationship between Rx and NEIL3 and to investigate the
involvement of NEIL3 in retinal development.
Methods: Gene expression was visualized by non-radioactive in situ hybridization
using wholemount or sectioned embryos and antisense riboprobes or quantified by
quantitative RT-PCR. NEIL3 antisense morpholino oligonucleotides (MOs) were
designed by and procured from Gene Tools LLC. For phenotypic analysis and
phenotype rescue experiments, NEIL3 and/or Rx antisense MOs or RNA were
microinjected into 2 - 4 celled Xenopus laevis embryos. For histology and
immunohistochemistry, embryos were fixed, paraffinized, and sectioned at 8
micron thickness.
Results: We verified that NEIL3 expression is dependent on Rx: (1) NEIL3 is
expressed in a subset of the Rx expression pattern during retinal development; (2)
NEIL3 expression is diminished upon Rx knockdown; and (3) NEIL3 expression is
induced by Rx expression in naïve ectoderm. Notably, exogenous expression of
NEIL3 largely rescues the Rx knockdown phenotype. Rescue is diminished by
perturbation of the canonical NEIL3 domains, the DNA glycosylase domain and
DNA-binding (GRF-type) zinc fingers. NEIL3 knockdown results in small or no
eye (microphthalmia or anophthalmia). Partial knockdown of both Rx and NEIL3
results in a more severe phenotype than knockdown of either gene product alone.
Small eyes generated by NEIL3 knockdown exhibited highly disorganized retinas
that contained few and sporadically distributed differentiated cells and persistent
proliferating cells.
Conclusions: NEIL3 is genetically downstream of Rx and is necessary for normal
retinal development. All putative functional domains are necessary for NEIL3
function. Our data is consistent with a role for NEIL3 in gene regulation,
downstream of Rx, in the maturation of retinal progenitor cells during normal
retinal development.
Commercial Relationships: Heithem M. El-Hodiri, None; Yi Pan, None
Support: NIH Grant EY015480
Program Number: 6014 Poster Board Number: A163
Presentation Time: 11:15 AM - 1:00 PM
Gsk3β Regulates Ganglion Cell Differentiation During Mouse Retinal
Development
Eleanor R. Trousdale, Jerome E. Roger, Keerthi Ranganath, Anand Swaroop.
Neurobiol-Neurodegen & Repair Lab, NEI, Bethesda, MD.
Purpose: Signaling pathways play an important role in cell fate determination,
often by modulating the phosphorylation state of the regulators. Glycogen Synthase
Kinase 3 beta (GSK3β) is a key protein kinase involved in the regulation of many
biological functions including cell division, proliferation, motility, and apoptosis.
This study aims to determine the role of GSK3β in mammalian retinal
development.
Methods: Gsk3b-floxed mice were mated with Rx-Cre mice to delete Gsk3b in
early retinal progenitors. Histological changes were evaluated by
immunohistochemistry (IHC) and quantified by cell counting. Changes in gene
expression were assessed by qPCR.
Results: The lamination of the retina in Gsk3b-/- is identical to wild type mice. The
number of rhodopsin-positive cells is not affected by the lack of Gsk3b. In contrast,
Muller cells are activated (GFAP positive), with displaced cell bodies in the inner
nuclear layer (INL) as shown with Sox-9 staining. Interestingly, an increase
(around 12%) in the number of Brn3a- and NF L-positive cells is observed in the
INL, suggesting that they correspond to displaced ganglion cells (GC). However
there is no difference in the number of Brn3a positive cells found in the ganglion
cell layer (GCL). At E12.5, more GC are already differentiated in Gsk3b-/- mice.
By qPCR, GFAP activation is confirmed and suggests that the retina is under stress.
Although expression of Nrl, Nr2e3, Rorb and Crx is not altered, several
phototransduction genes (e.g. Cngb3, Gnat1, Gnat2) are down regulated,
suggesting a potential alteration of photoreceptor function in Gsk3b-/- mice.
Conclusions: We demonstrate that the lack of Gsk3b expression leads to Muller
cell activation, and altered ganglion cell differentiation. However, it appears that
Gsk3a can compensate for Gsk3b. We are currently generating Gsk3a and b
double-knockout mice to fully decipher the function of Gsk3 in retinal
development.
Commercial Relationships: Eleanor R. Trousdale, None; Jerome E. Roger,
None; Keerthi Ranganath, None; Anand Swaroop, None
Support: NIH/NEI intramural funding
Program Number: 6015 Poster Board Number: A164
Presentation Time: 11:15 AM - 1:00 PM
Analysis of the Role of Pax6 Targeting miRNA During Mouse Retinal
Development
Yasuo Ouchi1, Hiroyuki Mano2, Sumiko Watanabe3. 1The Department of
Biomedical Sciences, Chubu University, Aichi, Japan; 2Division of Functional
Genomics, Jichi Medical University, Tochigi, Japan; 3Dept Molecular &
Developmental Biology, Univ of Tokyo, Inst Med Science, Tokyo, Japan.
Purpose: MicroRNAs represent a class of small (20-25 nucleotides), non-coding
RNAs that are key regulators of many cellular events during tumorigenesis and
organ development. Although recent studies provide an important role of miRNA
in regulation of gene expression, the contributions of miRNAs to retinal
development and function are largely unknown. To clarify the role of miRNAs in
mouse retinal development, we used gain- and loss-of-function analyses.
Methods: Based on the solexa sequencing expression profile of miRNAs, we
focused on five kinds of miRNAs which characteristically expressed in mouse
retinal development. In order to elucidate the function of the miRNAs, we used
retroviral vector based gain- and loss-of-function approach in E17 mouse retinal
explant culture. To identify target gene(s) of miRNAs, we used bioinfomatic tools,
such as Targetscan, miRTar and miRanda. The suppression of the target gene was
confirmed by immunohistochemical analysis and luciferase reporter gene assay.
Results: We found that forced expression of miR-7a significantly down-regulates
proliferation of the retinal progenitor cells. In accordance with this result, inhibition
of miR-7a using overexpression of decoy sequence slightly decreased proliferationactivity of the retinal progenitor cells. We next examined the expression pattern of
miR-7a, and strong expression was detected in neuroblast and ganglion cells at E14
mouse retina. As the development proceeds, expression in neuroblast became weak,
suggesting the role of miR-7a in retinal progenitor cells. To identify target gene(s)
of miR-7a using bioinfomatic tools, we found evolutionary conserved 3’UTR
region of Pax6 gene in human, mouse and zebrafish is a possible target of miR-7a.
To determine whether Pax6 is a direct target of miR-7a, we cloned the 3' UTR of
Pax6 into the 3' UTR of a SV40-driven luciferase reporter. In the presence of the
Pax6 3' UTR, miR-7a repressed luciferase activity, and this repression was
diminished by mutation of miR-7a binding sites. Furthermore, number of Pax6
positive cells was significantly decreased by miR-7a overexpression in retinal
explant. As expected, inhibition of miR-7a in retinal progenitor cells increased
Pax6 positive cells.
Conclusions: These results suggest that miR-7a regulates the Pax6 expression in
retinal progenitor cells and regulates timing of differentiation of retinal progenitor
cells during the retinal development.
Commercial Relationships: Yasuo Ouchi, None; Hiroyuki Mano,
None; Sumiko Watanabe, None
Support: None
Program Number: 6016 Poster Board Number: A165
Presentation Time: 11:15 AM - 1:00 PM
Redundant Roles for Transcription Factors AP-2α and AP-2β in Amacrine
and Horizontal Cell Development
Judith A. West-Mays1, Trevor Williams2, Erin Bassett1. 1Dept. of Pathology and
Molecular Medicine, McMaster University, Hamilton, ON, Canada; 2Depts. of
Craniofacial Biology and Cell and Developmental Biology, University of Colorado,
Denver, CO.
Purpose: Our previous studies in mouse models have demonstrated a requirement
for transcription factor AP-2α (Tcfap2a) in the development of multiple ocular
tissues, including the lens, cornea and more recently, the optic cup. Although we
have demonstrated that AP-2α is expressed in post-mitotic developing and mature
amacrine cells, we failed to detect retinal defects in conditional knockouts with
neural retina (NR)-specific deletion of Tcfap2a. However, the closely related
family member AP-2β showed a spatiotemporal expression pattern in the NR that
resembled, and significantly overlapped with, that of AP-2α. We therefore created
double mutants lacking both Tcfap2a and Tcfap2b in the developing NR, in order
to examine their potentially redundant roles in retinogenesis.
Methods: Double Tcfap2a/b retinal mutants with both Tcfap2a and Tcfap2b
deleted from the developing retina were generated by crossing retina-specific
Tcfap2a conditional knockouts onto a Tcfap2b germ-line knockout background.
The developing NR was examined using histological and immunofluorescent
techniques, until the death of double mutants at birth.
Results: In addition to amacrine cells, AP-2α and AP-2β were also co-expressed in
horizontal cells, although AP-2α expression was only transiently detected in
immature horizontal cells. Tcfap2a/b-deficient retinas had multiple defects that
were not detected upon deletion of either family member alone. These included a
near complete lack of horizontal cells, assessed by loss of the horizontal cell
markers PROX1, LIM1, PGP9.5 and neurofilament medium chain. Additionally,
amacrine cells were misplaced and did not begin to form orderly mosaics. By late
embryogenesis, BHLHB5-positive GABAergic amacrines failed to position
themselves within the inner nuclear layer, and the SOX2/ISL1-positive cholinergic
amacrine cells did not achieve the regular mosaic spacing observed in control
littermates.
Conclusions: This work demonstrates critical overlapping roles for AP-2α and AP2β in neural retinogenesis. Our studies of double Tcfap2a/b retinal mutants suggest
that AP-2α/β influences the intrinsic programs required for amacrine and horizontal
cell development.
Commercial Relationships: Judith A. West-Mays, None; Trevor Williams,
None; Erin Bassett, None
Support: NIH EY11910 (JWM); FFB Canada (JWM)NIH DE-12728 (TW)
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Program Number: 6017 Poster Board Number: A166
Presentation Time: 11:15 AM - 1:00 PM
A SNP in the Isl1 Promoter May be the Source of cis-Modulated Differences in
Expression and Impact Horizontal Cell Number
Irene E. Whitney1A,1B, Daniel C. Ciobanu2, Robert W. Williams3, Benjamin E.
Reese1A,1C. ANeuroscience Research Institute, BMolecular, Cellular, and
Developmental Biology, CPsychology, 1University of California Santa Barbara,
Santa Barbara, CA; 2Animal Science, University of Nebraska-Lincoln, Lincoln,
NE; 3Anatomy and Neurobiology, University of Tennessee, Memphis, TN.
Purpose: We previously reported a two-fold increase in horizontal cell number
from the A/J to the C56BL/6J (B6/J) strain (Reese et al., ARVO Abs. 2008,
49:3738). Using recombinant inbred strains we had mapped this variation to the
distal end of Chr 13. Analysis of the interval underlying this QTL identified the
transcription factor Isl1 as a promising candidate. Conditional knockout mice of
Isl1 were shown to have a significant increase in horizontal cell number. The
current study focuses on multiple lines of evidence supporting Isl1 as the causative
gene.
Methods: Sequencing of the Isl1 cDNA was conducted following 5’ and 3’ UTR
RACE. qPCR was used to determine the abundance of Isl1. Allelic-specific
expression (ASE) of Isl1 was measured in retinas of reciprocal F1 crosses of B6/J
and A/J. Two predictive programs of transcription factor binding, TESS and
MATCH, were used to identify the functional impact of sequence differences
between B6/J and A/J.
Results: We identified two SNPs in the promoter of Isl1 which may affect
differences in the level of transcription between the two strains. Indeed, Isl1
expression is significantly higher in A/J relative to B6/J. Analysis of ASE showed
significantly greater expression of Isl1 A alleles relative to B alleles, in both B6AF1
and AB6F1 samples, confirming the cis-regulation of this gene. Both predictive
programs showed the binding of bHLH factors being affected by one of the
promoter SNPs (rs545658554). This family of transcriptional regulators targets the
sequence known as an E-box (CANNTG), present in the B allele and not the A
allele. Interestingly, A is the conserved allele, seen in multiple other mammalian
species and in several wild-derived mouse strains.
Conclusions: The creation of a novel E-box sequence within the promoter of B6/J
provides a putative target for bHLH transcriptional regulators such as Neurod1 and
E2a, each with documented roles in retinal neurogenesis. This differential control
of Isl1 between B6/J and A/J may underlie the variation in horizontal cell number
seen between the strains.
Commercial Relationships: Irene E. Whitney, None; Daniel C. Ciobanu,
None; Robert W. Williams, None; Benjamin E. Reese, None
Support: NIH Grants EY011087 & EY019968
Program Number: 6018 Poster Board Number: A167
Presentation Time: 11:15 AM - 1:00 PM
The Roles Of Crb Proteins During Neurulation And Early Retinal
Morphogenesis In Zebrafish
Jian Zou, Xiangyun Wei. Department of Ophthalmology, University of Pittsburgh,
Pittsburgh, PA.
Purpose: Many epithelium-derived tissues express the Crumbs (Crb) polarity
proteins, which form complexes with other polarity proteins such as Nok and Lin7
to maintain epithelial polarity and regulate tissue morphogenesis. Several crb genes
have been identified in zebrafish. However their functional difference during
neuroepithelial morphogenesis remains unclear. This study was undertaken to
investigate the roles of Crb1, Crb2a, and Crb2b in neurulation and early retinal
morphogenesis in zebrafish.
Methods: Specific antibodies against Crb1, Crb2a, and Crb2b were generated and
used to analyze the expression patterns of Crb proteins in the neuroepithelium by
immunohistochemistry. Morpholinos were used to analyze the loss-of-function
phenotypes of the crb genes. Pulldown assays and mammalian cell culture assays
were performed to determine the physical adhesion porperties of the extracellular
domains of Crb proteins. The crb mRNAs were injected into 1-4-cell stage
embryos for rescue or overexpression assays.
Results: Crb1, Crb2a and Crb2b are expressed in distinct and yet overlapping
temporal and spatial patterns earlier in the neural tube, as well as later in the
developing brain, retina, and the spinal cord. For example, Crb2a is the only Crb
protein expressed in undifferentiated retinal neuroepithelium. The results of
biochemical pull-down and HEK293 cell culture expression assays suggested that
Crb proteins play redundant roles in the neuroepithelium by mediating cell-cell
adhesion at the apical surface. The severity of the disruption of epithelial polarity is
correlated with the total Crb protein level in the neuroepithelium.
Conclusions: The authors demonstrated that the novel adhesion function mediated
by the Crb proteins and their intracellular partners play an important role during
neurulation and early retinal morphogenesis. The temporal and spatial regulation of
Crb-mediated cell-cell adhesion may regulate the balance between tissue plasticity
and rigidity. This regulation might underlie dynamic tissue morphogenesis, which
requires the tissue to bear a certain amount of plasticity to accommodate shape and
position changes of individual cells as well as intercellular cohesiveness to
maintain tissue integrity and architecture.
Commercial Relationships: Jian Zou, None; Xiangyun Wei, None
Support: NIH Core Grant 5P30EY008098-17 and grant R01EY016099
Program Number: 6019 Poster Board Number: A168
Presentation Time: 11:15 AM - 1:00 PM
mGluR6 In The Zebrafish Retina
Stephan C. Neuhauss, Marion Haug, Matthias Gesemann. Institute of Molecular
Life Sciences, University of Zurich, Zurich, Switzerland.
Purpose: Metabotropic glutamate receptors (mGluRs) have been identified at all
synapses of the vertebrate retina, where they likely regulate neurotransmitter
release. The only example of an mGluR functioning in direct synaptic transmission
is mGluR6. It is expressed on ON-bipolar cell dendrites and mediates the ONresponse in the mammalian retina which provides information about brighter than
background stimuli to the ganglion cells. Data from lower vertebrates propose a
division of labor of the ON-bipolar cell response: mGluR6 is suggested to be fully
responsible for the scotopic ON-response, whereas the photopic ON-response is
involves mGluR6 together with a glutamate transporter. We use the cone dominant
retina of zebrafish to assess this hypothesis and to define the functional role of
mGluR6 in teleost vision.
Methods: Expression of mGluR6 in larval zebrafish retina was assessed by RNA
in situ hybridization and immunohistochemistry. A functional analysis of mGluR6b
was accomplished by measuring ERG and OKR on morpholino antisense injected
larvae.
Results: Phylogenetic analysis of mGluR6 reveals the occurrence of two zebrafish
mGluR6 paralogs, mGluR6a and -6b. Interestingly, we located the RNA of both
mGluR6 paralogs predominantly in retinal ganglion cells of larval zebrafish and in
the inner part of the inner nuclear layer (INL). Only one paralog, mGluR6a, is
expressed in the region where bipolar cell bodies are located. An
immunohistochemical analysis of mGluR6a is consistent with the RNA pattern and
shows a postsynaptic location in the outer plexiform layer, a saining in two layers
of the IPL and in single cells around it. Behavioral analysis after depletion of
mGluR6b with morpholino antisense injected larvae revealed a decreased ERG bwave indicating a decrease in ON-bipolar cell response but had no effect on general
visual performance detected with OKR measurements.
Conclusions: Those results provide evidence that mGluR6b plays a crucial role in
the zebrafish cone ON-bipolar cell pathway. The diminished but not erased b-wave
supports the hypothesis of two receptors involved in the photopic ON-pathway.
Commercial Relationships: Stephan C. Neuhauss, None; Marion Haug,
None; Matthias Gesemann, None
Support: SNF 3100A0-117782
Program Number: 6020 Poster Board Number: A169
Presentation Time: 11:15 AM - 1:00 PM
Genetic Interactions Between Brn3 Transcription Factors In Retinal Ganglion
Cell Type Specification
Melody Shi, Oluwaseyi Motajo, Sumit Kumar, Tudor C. Badea. NeurobiolNeurodegen & Repair Lab, NEI, Bethesda, MD.
Purpose: Retinal Ganglion Cells (RGCs) are a diverse group of neurons consisting
of about 20 distinct cell types, which convey visual information via distinct
channels to the brain. The developmental mechanisms by which distinct cell types
acquire their very different dendritic arbor shapes and sizes and their specific
axonal targeting to the different retinorecipient nuclei of the brain are largely
unexplored.
During early RGC development, the Brn3 family of transcription factors helps
specify RGCs, by instructing various aspects of dendrite and axon formation.
Previous published results suggest that Brn3b functions upstream of Brn3a and
Brn3c and works to ensure both RGC survival and Brn3a and Brn3c expression.
Brn3a is believed to be able to substitute for Brn3b when it is expressed from the
Brn3b locus, and double mutants of Brn3c and Brn3b show more severe RGC loss
than Brn3b single mutants. More recently, we reported that Brn3a may play a role
in the specification of RGCs with a particular set of arbor morphologies. In
addition, we also determined that Brn3a, Brn3b and Brn3c are expressed in
overlapping, but not identical sets of adult RGC cell types. Taken together, these
results raise the possibility that Brn3 transcription factors are part of a
combinatorial code that regulates the formation of distinct RGC cell types. To
explore the genetic interactions between the three transcription factors, we
generated pair wise double-knockout combinations.
Methods: Here we describe genetic interactions between Brn3a and Brn3b with
respect to their role in RGC formation.
Retinas and brains from adult mice of a) Pax6αCre; Brn3b -/-; Brn3a CKOAP/+ b)
Pax6αCre; Brn3b +/-; Brn3a CKOAP/+ c) Pax6αCre; Brn3b -/-; Brn3a CKOAP/and d) Pax6αCre; Brn3b +/-; Brn3a CKOAP/- genotypes were analyzed by
Alkaline Phosphatase histochemistry and indirect immunofluorescence.
Results: We find that deleting Brn3b dramatically reduces but does not completely
remove Brn3a positive RGCs from the retina and that surviving Brn3aAP/- RGCs
exhibit abnormal dendritic arbor morphologies. In Brn3b-/-; Brn3aAP/- retinas,
essentially no Brn3aAP RGCs can be seen, however some AP negative,
Neurofilament positive RGCs survive.
Conclusions: We conclude that Brn3b is not required for Brn3a expression nor for
the survival of Brn3a positive RGCs in a significant fraction of Brn3aAP RGCs,
and that Brn3a has additional, Brn3b independent roles in RGC development and
survival. These results are consistent with a combinatorial code hypothesis, in
which various combinations of Brn3 transcription factors help to specify distinct
RGC cell types.
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
Commercial Relationships: Melody Shi, None; Oluwaseyi Motajo,
None; Sumit Kumar, None; Tudor C. Badea, None
Support: NIH/NEI Intramural Program for T. Badea
Program Number: 6021 Poster Board Number: A170
Presentation Time: 11:15 AM - 1:00 PM
Expression of Receptors for Luteinizing Hormone-Releasing Hormone (LHRH) in Human Uveal Melanomas
Zita Steiber1A,1B, Andrea Treszl1B, Gabor Olah1B, Armin Buglyo1B, Bernadett
Rozsa1B, Andras Berta1A, Gabor Halmos1B. AOphthalmology, BBiopharmacy,
1
University of Debrecen, Debrecen, Hungary.
Purpose: Treatment options available for patients with advanced uveal melanoma
are limited. The presence of luteinizing hormone-releasing hormone (LH-RH)
receptors has been demonstrated in various human cancers. Consequently, we
investigated the expression of LH-RH ligand and the presence of receptors for LHRH in tissue samples and cell lines of human uveal melanoma as potential
molecular targets for cancer therapy.
Methods: The expression of LH-RH ligand and LH-RH receptor transcript forms
were investigated by RT-PCR using gene specific primers in a panel of human
uveal melanoma specimens and 3 cell lines. The presence of LH-RH receptor
protein was evaluated by Western blotting.
Results: The expression of mRNA for LH-RH receptor transcript forms was
detected in 17 out of the 38 tissue specimens (47%) and in 2 of 3 cell lines tested.
mRNA for LH-RH ligand could be detected in 76% of tissue samples (29 out of 38)
and in all 3 cancer cell lines. No PCR products for pituitary type of LH-RH
receptors were found. The presence of specific, full lenght LH-RH receptor protein
was demonstrated in tumor specimens by Western blotting.
Conclusions: We were able to detect the expression of LH-RH and transcript
forms of LH-RH receptors in human uveal melanomas. Our results support the
merit of further investigation of the expression of LH-RH receptors and its
transcript forms in human ophthalmological tumors as well as the application of
cytotoxic LH-RH analogs for receptor-based targeted therapy of such malignancies.
These observations signify the importance of the development of new LH-RH
analogs with potential therapeutical applications.
Commercial Relationships: Zita Steiber, None; Andrea Treszl, None; Gabor
Olah, None; Armin Buglyo, None; Bernadett Rozsa, None; Andras Berta,
None; Gabor Halmos, None
Support: Hungarian Scientific Research Fund (OTKA) K 81596 (G.H.) and
TAMOP 4.2.1./B-09/1/KONV-2010-0007 project (G.H.), Zoltan Magyary
Postdoctoral Fellowship supported by EEA Grants and Norway Grants (A.T.)
Program Number: 6022 Poster Board Number: A171
Presentation Time: 11:15 AM - 1:00 PM
Molecular Regulation of the Map3k1 Gene Promoter
Qinghang Meng, Maureen Mongan, Ying Xia. Environmental Health, University of
Cincinnati, Cincinnati, OH.
Purpose: The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is
crucial for eyelid morphogenesis. Loss of MAP3K1 in mice results in defective
embryonic eyelid closure and an eye-open at birth (EOB) phenotype. We have
previously shown that MAP3K1 regulates eyelid development at two levels. One
level is the expression of MAP3K1, which is induced in the developing eyelid
epithelial cells. Another level is the activity of MAP3K1, which is required to
activate a JNK-c-JUN signaling cascade for eyelid closure. Previous in vitro studies
show that RhoA, a member of the Rho GTPase family, may act on the second level,
activating MAP3K1-mediated JNK pathways. This idea has never been tested in
vivo. The current study uses an in vivo compound and cell type-specific gene
knockout system and in vitro molecular biology to investigate the interaction
between RhoA and MAP3K1 in eyelid development.
Methods: The double transgenic mice containing RhoAflox conditional allele and
Le-cre driver were mated with the Map3k1+/ΔKD mice, in which the Map3k1ΔKD
allele contains a bacterial β-galactosidase gene knocked-in into the Map3k1 locus,
replacing the exons coding for MAP3K1 kinase domain. As a result, the
Map3k1ΔKD allele produces a protein that lacks kinase activity and has its Nterminal domain fused to β-gal. The eyelid development in fetuses was examined at
E17.5. MAP3K1 expression in the developing eyelids was evaluated by whole
mount X-gal staining. Transient transfection was done using 293 cells with
Map3k1-promoter-luc and various mammalian expression vectors for active and
dominant negative RhoA and ROCKI and II, a protein kinase downstream of RhoA
The cells were treated with colchicine, stimulus known to induce MAP3K1
expression, and luciferase activity was determined.
Results: We found that MAP3K1 functionally interacted with RhoA in eyelid
development. When RhoA is deleted in the developing eyelid epithelial cells,
MAP3K1 became haploinsufficient for eyelid closure. Correspondingly, the RhoA
ablation reduced MAP3K1 expression in the developing eyelids. In 293 cells,
active RhoA stimulated the exogenous Map3k1-promoter-luc and endogenous
MAP3K1 expression, whereas, dominate negative RhoA reduced Map3k1promoter-luc activity induced by colchicine. Furthermore, ROCK also induced
Map3k1-promoter-luc activity and the ROCK inhibitor blocked colchicines
induced promoter activity and MAP3K1 expression.
Conclusions: In addition to activating the MAP3K1-JNK pathways, RhoA can also
crosstalk with MAP3K1 by activating its gene promoter and induces its expression.
Commercial Relationships: Qinghang Meng, None; Maureen Mongan,
None; Ying Xia, None
Support: NIH Grant EY15227
Program Number: 6023 Poster Board Number: A172
Presentation Time: 11:15 AM - 1:00 PM
Expression And Function Of Planar Cell Polarity Gene Prickle1 During
Retinal Development
Dustin T. Whitaker, Hirva Bakeri, Chunqiao Liu, Anand Swaroop. NeurobiolNeurodegem & Repair Lab, NEI, Bethesda, MD.
Purpose: We have previously shown that the expression of Prickle1 (Pk1), a core
component of the planar cell polarity (PCP) signaling pathway, is down-regulated
in cone-like photoreceptors of the Nrl-knockout retina (Akimoto et al., 2006). To
investigate it’s potential role in rod photoreceptor polarity and morphogenesis, we
have studied Pk1 expression during retinal development in the mouse. We have
also investigated its potential function using shRNA knockdown technology in the
postnatal retina.
Methods: In situ hybridization (ISH) was used to study the spatial and temporal
expression of Pk1 in the retina and the brain. In vivo electroporation was used to
introduce shRNA into postnatal retina to knockdown Pk1 expression.
Immunohistochemistry (IHC) was performed to study retinal differentiation.
Results: Pk1 is expressed in postmitotic neurons of the central nervous system
prominently in the hippocampus, striatum, and olfactory cortex. In the retina, Pk1
is expressed in the apical, likely postmitotic, cells in the retina at an early postnatal
age. In the adult retina, it is weakly expressed in the inner nuclear and retinal
ganglion cell layer (GCL). Knockdown of Pk1 by shRNA led to sustained Ki67
expression and down-regulation of rhodopsin expression.
Conclusions: Pk1 has a potential role in coordination of photoreceptor
differentiation in early postnatal retina. It might be involved in maintaining a state
of differentiation in immature photoreceptor cells.
Commercial Relationships: Dustin T. Whitaker, None; Hirva Bakeri,
None; Chunqiao Liu, None; Anand Swaroop, None
Support: NIH/NEI Intramural
Program Number: 6024 Poster Board Number: A173
Presentation Time: 11:15 AM - 1:00 PM
Candidate Genes Critical for Normal Axonal Navigation at the Optic Chiasm
of Oculocutaneus and Ocular albino Mice
Alejandra Young1, Benjamin E. Reese2, Novrouz Akhmedov1, Debora B. Farber3.
1
Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA;
2
Neuroscience Research Institute and Department of Psychology, UCSB, Santa
Barbara, CA; 3Jules Stein Eye Inst, CHS/UCLA, Los Angeles, CA.
Purpose: To define the differential gene expression profiles associated with
embryonic retinal cells from c2J and Oa1-/- mice (models for Oculocutaneous and
Ocular albinism, respectively) and their corresponding control mice (C57Bl/6 and
NCrl), using microarray analysis. Since melanin biosynthesis plays an important
role in the ipsilateral/contralateral decision of ganglion cell axons at the optic
chiasm, clustering of the axonal guidance genes from control and diseased retinas
hybridized differently in the microarrays will allow us to identify genes influencing
normal/abnormal decussation at the chiasm and, directly or indirectly, RPE
pigmentation.
Methods: 12.5 dpc mouse embryos (7-13/litter) were collected from c2J, Oa1-/and the corresponding (C57Bl/6 and NCrl) females. Total RNA was isolated from
eyes of each litter and stored as one biological sample at -80ºC. Samples were
hybridized to mouse Affymetrix gene arrays at the UCLA Microarray core facility.
RT-PCR was used to confirm the differential expression of those genes involved in
the Axon Guidance Pathway common to both c2J and Oa1-/- mice or specific to
each of these animal models of albinism.
Results: Analysis of the microarrays from Oa1-/- vs. control NCrl mice gave 37
significant, differentially expressed genes from which 7 were involved in axon
guidance: 4 up-regulated and 3 down-regulated. Similarly, analysis of c2J vs.
C57B6 samples gave 209 significant differentially expressed genes from which
only 2 down-regulated genes were related to axon guidance. QPCR confirmed the
differential expression of the selected axon guidance genes.
Conclusions: Microarray analysis of the RNA from 12.5dcp embryonic eyes of
albino Oa1-/- and c2J mice, which have common visual abnormalities, and their
corresponding control pigmented mice, has provided the first comprehensive list of
genes potentially involved in normal/abnormal optic axon guidance. These genes
may also influence normal melanogenisis and the abnormal pigmentation
associated with oculocutaneous and/or ocular albinism.
Commercial Relationships: Alejandra Young, None; Benjamin E. Reese,
None; Novrouz Akhmedov, None; Debora B. Farber, None
Support: Vision of Children Foundation
Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.