Download ARVO 2014 Annual Meeting Abstracts 124 Retinal Development

ARVO 2014 Annual Meeting Abstracts
124 Retinal Development
Sunday, May 04, 2014 1:30 PM–3:15 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 691–723/C0234–C0266
Organizing Section: Retinal Cell Biology
Program Number: 691 Poster Board Number: C0234
Presentation Time: 1:30 PM–3:15 PM
Characteristics of Normal Foveal Development in Infants and
Young Children as Imaged Using Hand-Held Optical Coherence
Tomography
Helena Lee, Ravi Purohit, Aarti Patel, Eleni Papageorgiou, Mashal
Bibi, Viral Sheth, Gail Maconachie, Rebecca McLean, Frank A.
Proudlock, Irene Gottlob. Ophthalmology, University of Leicester,
Leicester, United Kingdom.
Purpose: To characterise the time course of normal foveal
development in vivo in full term infants and children using hand-held
high resolution spectral domain optical coherence tomography (HHSDOCT).
Methods: 256 children with a mean age of 2.2 years (range 0-6.9
years) and 39 older children and adults with a mean age of 15 years
(range 7.1-27 years) were recruited. Each participant had a full
ophthalmological examination and HH-SDOCT scans. The OCT
scans were segmented using a customised macro in ImageJ. The
thickness and angle of each retinal layer at the fovea and parafovea
were quantified and correlated with log gestational age (logGA) and
visual acuity (VA).
Results: The central macular thickness (CMT) increases linearly with
logGA by 85% between birth and three years of age, after which it
plateaus. This relationship is described by: CMT (mm) = 61log(GA-1)
+ 111. In the parafovea, there is a more gradual 20% increase in
retinal thickness over the same time period. The foveal outer segment
(OS) and inner segment (IS) of the photoreceptors and the outer
nuclear layer (ONL) also follow a linear pattern, with a 330%,
24% and 55% increase in thickness respectively, between birth and
eighteen months of age, after which they plateau. In the parafovea,
this increase is more gradual with the OS (76%), IS (12%) and ONL
(15%). The foveal outer plexiform (OPL), inner nuclear (INL), inner
plexiform (IPL) and ganglion cell layers (GCL) decrease in thickness
with GA. Interestingly, the parafoveal thickness of the retinal nerve
fibre layer (RNFL), GC complex (GCL and IPL) initially decrease in
thickness until two years of age, followed by a gradual increase. The
age adjusted CMT, ONL and IS are significant predictors of VA, with
r 2 = 0.739 and p = 0.000 for CMT and r2 = 0.748 and p= 0.000 for
the ONL and IS. The age adjusted angle between the fovea and the
upper border of the ONL at 1000 mm is also a significant predictor
of VA, with r 2 = 0.777 (p = 0.001) and 0.765 (p = 0.045) for the
temporal and nasal angles respectively.
Conclusions: We have characterised the time course of normal foveal
development in infants and young children using the HH-SDOCT
and several predictors of visual acuity have been identified. This is
important as the HH-SDOCT will play an increasingly prominent
diagnostic and prognostic role in children with retinal pathology.
Commercial Relationships: Helena Lee, None; Ravi Purohit,
None; Aarti Patel, None; Eleni Papageorgiou, None; Mashal
Bibi, None; Viral Sheth, None; Gail Maconachie, None; Rebecca
McLean, None; Frank A. Proudlock, None; Irene Gottlob, None
Support: Medical Research Council, London, UK (grant number:
MR/J004189/1), Ulverscroft Foundation, Leicester, UK and
Nystagmus Network UK
Program Number: 692 Poster Board Number: C0235
Presentation Time: 1:30 PM–3:15 PM
Identifying surface markers to distinguish cells types in the
human fetal retina
Jennifer Aparicio, Anthony Choi, Victor Liao, David Cobrinik.
Ophthalmology, Children’s Hospital Los Angeles, Los Angeles, CA.
Purpose: In light of recent advances in stem cell biology and
prospects of cell transplantation in the field of ophthalmology, it is
increasingly important to characterize retinal cells, especially in the
early stages of eye development. Our primary objective is to define
surface markers to enable isolation of pure cell populations in the
human fetal retina and human embryonic stem cell (ES)-derived
retinal tissue for further study.
Methods: To demarcate unique cell types in the developing
human retina, we screened a fetal retina at 20 weeks gestation,
with a commercially available cell surface marker antibody panel.
Dissociated retinal cells were co-labeled with CD133 and each of
251 monoclonal antibodies, and analyzed by flow cytometry. CD133,
a marker of progenitors and photoreceptors, was employed to aid
in defining populations marked by panel antibodies. Dissociated
ES-derived retinal cells were analyzed for expression of a subset
of markers present in fetal retina. In addition, immunofluorescence
microscopy was used to examine the pattern of expression of many of
the antibodies in fetal retina 15 to 21 fetal weeks in age.
Results: Of the surface markers analyzed by flow cytometry, 112
were expressed in some cells in the human fetal retina. The 55
markers most frequently expressed on retinal cells exhibited one of
eight different patterns of expression when analyzed along with cell
size and CD133 expression. Patterns detected by antibodies to CD73,
CD15, and CD44 are consistent with patterns previously described
in the murine retina, a photoreceptor precursor marker, and early and
late progenitor markers, respectively. Furthermore, 49-day-old ESderived retina exhibited expression of 45 of 50 antigens expressed in
fetal retina, suggesting that those not expressed are displayed on more
mature cell types. Immunofluorescence analysis demonstrated that
some surface markers differed in their expression levels from central
to peripheral retina indicative of cells of differing maturational states.
Conclusions: Commonly available cell surface antigens can be
used to separate disassociated single cell suspensions of retinal cells
into distinct subpopulations. These markers may enable purification
of retinal cell types from heterogeneous populations, including
photoreceptor precursors, ganglion cells, and interneurons from both
human fetal retina and ES-derived retina.
Commercial Relationships: Jennifer Aparicio, None; Anthony
Choi, None; Victor Liao, None; David Cobrinik, None
Program Number: 693 Poster Board Number: C0236
Presentation Time: 1:30 PM–3:15 PM
Cup-to-disc and arteriole-to-venule ratios in preterm birth
Da Ye Choi, Jaeryung Kim, Kyung-Ah Park, In-Jeong Lyu, Sei Yeul
Oh. Ophthalmology, Samsung medical center, Seoul, Republic of
Korea.
Purpose: To investigate the influence of preterm birth on the optic
disc and retinal vessels by measurements of cup-to-disc (C/D) ratio
and arteriole-to-venule (A/V) ratio.
Methods: Eighty-three eyes of 42 preterm birth were included in this
study. In the age and sex-matched control group, 83 eyes of 42 fullterm birth were used. Fundus color photographs were taken. ImageJ
software was used to calculate C/D and A/V ratios from the fundus
images.
Results: Fundus photographs were taken at an age of 8.01 ± 2.22
years for the preterm group and 8.01 ± 2.13 years for the control
group. The mean gestational age of the preterm group was 27.57
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
weeks (range, 24 to 34 weeks). The preterm group had significantly
larger C/D ratios and smaller A/V ratios (mean ± standard deviation;
0.46 ± 0.12 and 0.71 ± 0.09, respectively) than the control
group (mean ± standard deviation; 0.36 ± 0.07 and 0.82 ± 0.08,
respectively) (p < 0.001 and p < 0.001, respectively) after age, sex
and spherical equivalent refractive error were adjusted.
Conclusions: Preterm birth is significantly associated with larger
C/D ratio and smaller A/V ratio. These findings show the effect of
preterm birth on the development of optic disc and retinal vessel
development.
Commercial Relationships: Da Ye Choi, None; Jaeryung Kim,
None; Kyung-Ah Park, None; In-Jeong Lyu, None; Sei Yeul Oh,
None
Program Number: 694 Poster Board Number: C0237
Presentation Time: 1:30 PM–3:15 PM
Contrasting Foveal Specialization in Disorders Associated with
Foveal Hypoplasia
Melissa A. Wilk1, Brian Higgins2, Robert F. Cooper3, Drew H. Scoles4,
Kimberly E. Stepien2, C Gail Summers5, 6, Alfredo Dubra2, 7, Deborah
M. Costakos2, Joseph Carroll1, 2. 1Cell Biology, Neurobiology,
& Anatomy, Medical College of Wisconsin, Milwaukee, WI;
2
Ophthalmology, Medical College of Wisconsin, Milwaukee, WI;
3
Biomedical Engineering, Marquette University, Milwaukee, WI;
4
Biomedical Engineering, University of Rochester, Rochester, NY;
5
Ophthalmology & Visual Neurosciences, University of Minnesota,
Minneapolis, MN; 6Pediatrics, University of Minnesota, Minneapolis,
MN; 7Biophysics, Medical College of Wisconsin, Milwaukee, WI.
Purpose: While foveal specialization has been well characterized
in albinism, less is known regarding foveal morphology in other
disorders associated with foveal hypoplasia. Here we sought to
quantify foveal specialization in patients with aniridia or a history of
premature birth using spectral domain optical coherence tomography
(SD-OCT) and adaptive optics scanning light ophthalmoscopy
(AOSLO), and compare these findings to those of patients with
albinism.
Methods: Subjects with a diagnosis of albinism (n=5), aniridia
(n=3), or a history of premature birth (n=3, birth was between 25
and 30 weeks’ gestation) were recruited for this study. Volumetric
SD-OCT scans of the macula were acquired, and custom MATLAB
software was used to derive estimates of foveal pit depth, diameter,
and volume. Additionally, high-resolution linear SD-OCT scans were
acquired and manually segmented to obtain measurements of relative
foveal cone inner and outer segment (IS and OS, respectively) length.
Images of the photoreceptor mosaic and foveal avascular zone
(FAZ) were acquired using AOSLO. When possible, cone density
was measured using a semi-automated cone counting program,
and FAZ area and diameter were measured using semi-automated
segmentation.
Results: Despite having reduced FAZ areas, all 3 subjects with
a history of premature birth displayed normal foveal pit metrics,
normal foveal cone OS elongation, and normal cone packing.
Consistent with previously reported results from 32 subjects with
albinism and the additional 5 subjects reported here, the subjects with
aniridia had variable OS lengthening. Clear evidence of cone packing
was seen in one subject with aniridia, though due to the presence of
severe nystagmus, it was not possible to quantify cone density in the
remaining two subjects.
Conclusions: Contrary to previous observations in patients with
a history of premature birth, our subjects displayed normal foveal
specialization. Further work contrasting foveal specialization across
these disorders may be useful to better understanding normal foveal
development. While previous work from our group has shown that it
is possible to obtain high quality images in patients with nystagmus,
AOSLO hardware improvements and some form of eye tracking are
needed to enable imaging of patients with severe nystagmus, such as
that seen in aniridia.
Commercial Relationships: Melissa A. Wilk, None; Brian Higgins,
None; Robert F. Cooper, None; Drew H. Scoles, None; Kimberly
E. Stepien, None; C Gail Summers, None; Alfredo Dubra, Canon
USA, Inc. (C), US Patent 8,226,236 (P); Deborah M. Costakos,
None; Joseph Carroll, None
Support: Supported by Vision for Tomorrow, Research to
Prevent Blindness, Burroughs Wellcome Fund, and NIH Grants
P30EY001931, UL1RR031973, T32EY014537, & T32GM007356.
Supported, in part, by an unrestricted grant to the Department
of Ophthalmology & Visual Neurosciences at the University of
Minnesota from Research to Prevent Blindness, Inc., New York, NY.
Program Number: 695 Poster Board Number: C0238
Presentation Time: 1:30 PM–3:15 PM
Viability of human fetal retina/RPE sheets after 4 days cold
storage
Magdalene J. Seiler1, Gabriel Nistor2, 1, Norman D. Radtke3, Robert
B. Aramant1. 1Anatomy & Neurobiology, Univ of California, Irvine,
Irvine, CA; 2California Stem Cell Inc., Irvine, CA; 3Retina Vitreous
Resource Center, Louisville, KY.
Purpose: For development of clinical retinal transplantation trials,
it would be important to maintain viability of the donor tissue for
several days after dissection, and to ship the tissue in temperature
controlled containers.
Methods: Permission to use fetal tissue for research was obtained
from the Western Institution Review Board, Norton Healthcare
Research office, and the hSCRO committee of UC Irvine. Retina
together with its RPE was dissected from 6 fetal eyes (age 10.5 – 12.5
weeks post-conception, 4 donors) that were received at 1 day (d) after
abortion. All tissues had been placed into cold custom-made CO2independent hibernation medium with B27 supplements immediately
after harvest. Eyecups were treated with dispase for 15-20 minutes to
separate choroidal vessels from RPE. After dissection, retinas with
RPE were cut into 3-5 tissue pieces (size 2-6 mm2). Of each eye,
pieces were either fixed immediately, or placed inside plastic nozzles
in hibernation medium in shipping tubes (18 pieces). The tubes were
placed into shipping containers, and their temperature monitored
(between 8 and 2.5 sC). After shipping for 1, 2, or 3 d, tissues were
fixed (= 4 d after harvest). Cell viability in the retinas was tested by
TUNEL staining (counting TUNEL-stained(+) cells per mm2 and %
TUNEL-stained cells of total cells in 10 μm cryostat cross-sections).
This analysis was performed for all cells, and separately for the
inner retinal layers (containing differentiating neurons) and outer
neuroblastic layers (that would develop into photoreceptors and other
retinal cells).
Results: Of the 18 pieces placed into shipping containers, 14 pieces
retained retina-RPE contact. - A low percentage of cells (0.3%) were
TUNEL-positive after dissection. Although the number of TUNEL+
cells increased with time, the percentage of TUNEL+ cells remained
very low (up to 0.9% on d 2 of shipping). In the inner retinal layers,
there was a higher percentage of TUNEL+ cells (0.5-1.9%) than
the 0.3-0.6% TUNEL+ cells in the outer neuroblastic layers. The
number of TUNEL + cells increased significantly in the inner layers
after 1 d shipping, whereas the number of TUNEL+ cells in the outer
neuroblastic layers did not increase significantly (from 0.3 to 0.6%)
until 3 d of shipping (= 4 d after harvest).
Conclusions: These results demonstrate that intact fetal-derived
sheets of retina with RPE remain viable for several days with careful
dissection and handling.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
Commercial Relationships: Magdalene J. Seiler, Ocular
Transplantation LLC (C), Ocular Transplantation LLC, patent #
5,941,250; # 8,057,483 (P); Gabriel Nistor, None; Norman D.
Radtke, None; Robert B. Aramant, Ocular Transplantation LLC
(E), Ocular Transplantation LLC patent # 5,941,250; # 8,057,483 (P)
Support: Lincy Foundation
Program Number: 696 Poster Board Number: C0239
Presentation Time: 1:30 PM–3:15 PM
In vivo retinal development using hand held ultra-high resolution
spectral domain optical coherence tomography in premature and
full term infants
Samira Anwar1, 2, Aarti Patel1, Helena Lee1, Frank A. Proudlock1,
Jonathan Cusack3, Irene Gottlob1. 1Ophthalmology Group,
University of Leicester, Leicester, United Kingdom; 2Department of
Ophthalmology, University Hospitals of Leicester, Leicester Royal
Infirmary, Leicester, United Kingdom; 3Department of Neonatology,
University Hospitals of Leicester, Leicester Royal Infirmary,
Leicester, United Kingdom.
Purpose: To describe in vivo retinal changes during development in
premature infants and normal aged matched controls using hand-held
spectral domain optical coherence tomography (HH-SDOCT).
Methods: Cross sectional and longitudinal images HH-SDOCT were
collected from 33 premature infants ranging in age from 31 – 42
weeks gestational age. Participants were recruited from the neonatal
unit while also undergoing ophthalmic screening for retinopathy of
prematurity (ROP). Control infants ranged in age from 37 weeks
to 41 weeks gestation and were recruited from the maternity unit.
Premature infants were examined at 1-2 weekly intervals until up to
42 weeks. Normal controls were examined within 1 week of birth.
No premature infants had received treatment for ROP at time of
scanning. HH-SDOCT (Bioptigen, 2.6μm axial resolution) scans
were performed without sedation and after dilatation of pupils.
Customised Image J manual segmentation of retinal images including
thickness and angle of each layer centrally and at 1000μm nasally
and temporally was performed and quantified. Statistical analysis was
completed using SPSS v20.
Results: The central retina in the premature group progressively
thinned by 1.74μm/week in contrast to 5μm/week thickening of the
nasal and temporal retina located at 1000μm from the foveal centre.
Changes in central retina were due to a combination of inner layer
migration (reduction of approximately 4.4μm/week) in conjunction
with increased thickening of the outer retinal layers. Changes in
temporal and nasal retina consisted of thickening of both inner layers
and photoreceptor layers. Increase in thickness was primarily seen in
nuclear layers (GCL and INL) whereas plexiform layers did not show
any significant changes. The premature group demonstrated thicker
central retina in comparison to age-matched controls because of
disrupted inner layer migration. Outer retinal photoreceptive elements
were easier to define in the normal group compared to the premature
group.
Conclusions: Premature infants maintain thicker inner retina
centrally with decreased inner retinal migration compared to agematched normal infants. Outer retinal layers were thinner in the
premature group suggesting slower development of these layers.
Commercial Relationships: Samira Anwar, None; Aarti Patel,
None; Helena Lee, None; Frank A. Proudlock, None; Jonathan
Cusack, None; Irene Gottlob, None
Support: Medical Research Council, London, UK (grant number:
MR/J004189/1) Ulverscroft Foundation, Leicester, UK Nystagmus
Network UK
Program Number: 697 Poster Board Number: C0240
Presentation Time: 1:30 PM–3:15 PM
Microphthalmia-associated Transcription Factor (MITF) affects
optic vesicle cell proliferation and retinal pigment epithelium
maturation in human ES cell (hESC) cultures
Anna Petelinsek1, Elizabeth E. Capowski1, Sara Howden2, Lynda S.
Wright1, Isabel Pinilla Lozano5, 6, Kyle Wallace1, Eric Clark1, Joe
Phillips1, David M. Gamm3, 4. 1University of Wisconsin-Madison,
Madison, WI; 2Morgridge Institute for Research, Madison, WI;
3
Department of Ophthalmology and Visual Sciences, University
of Wisconsin, Madison, WI; 4McPherson Eye Research Institute,
Madison, WI; 5Ophthalmology, University Hospital Lozano Blesa,
Zaragoza, Spain; 6IIS Aragon, Aragon Health Sciences Institute,
Zaragoza, Spain.
Purpose: Loss of MITF expression results in microphthalmia and
RPE defects in rodent models, but its role in human retinogenesis
remains unknown. We created a MITF double knockout (dKO) hESC
line to examine the consequences of its absence during early retinal
development.
Methods: The second common exon of MITF was targeted by two
rounds of BAC-mediated homologous recombination to inactivate
both alleles in WA09 hESCs. Correct targeting and gene inactivation
were confirmed by genomic PCR, RT-PCR, and Western blots. Light
microscopy, immunocytochemistry (ICC), RT-PCR, and RT-qPCR
were then performed at different stages of retinal cell development
to examine the impact of MITF loss on the production, proliferation,
and maturation of early neuroretinal cells and RPE.
Results: RT-PCR and ICC analyses confirmed the absence of MITF
transcripts and protein in dKO-MITF hESCs. Upon differentiation,
ICC revealed that dKO-MITF hESCs sequentially adopted anterior
neuroectoderm and early eye field fates in a manner similar to
isogenic control WA09 hESCs. However, optic vesicle-like structures
(OVs) generated from dKO-MITF hESCs were significantly
smaller than control OVs (>60% reduction; p<0.0001). In addition,
expression levels of PAX6, RX, and SIX6 were reduced in dKO-MITF
vs. control OVs (54, 76, and 79% reductions, respectively), as was
the number of proliferating cells (Ki67+ cells: 15.4% vs. 23.4%; p≤
0.01). Following onset of VSX2 expression, cell proliferation within
dKO-MITF OVs matched that of control OVs. Furthermore, dKOMITF OVs produced CRX/Recoverin+ precursors in a temporal and
spatial pattern indistinguishable from control OVs. However, RPE
generated from dKO-MITF hESCs failed to fully mature; instead,
dKO-MITF RPE remained unpigmented and lacked typical RPE
organization even after several months in culture.
Conclusions: Using a genetically engineered hESC line, we showed
that MITF is specifically involved in human OV proliferation and
RPE maturation. Such information may lead to improvements in
retinal cell production in vitro and/or provide insight into retinal
development at stages previously inaccessible in humans.
Commercial Relationships: Anna Petelinsek, None; Elizabeth E.
Capowski, None; Sara Howden, None; Lynda S. Wright, None;
Isabel Pinilla Lozano, None; Kyle Wallace, None; Eric Clark,
None; Joe Phillips, None; David M. Gamm, None
Support: Foundation Fighting Blindness Wynn-Gund Research
Acceleration Award; NIH RO1EY21218 and P30HD03352; E.
Matilda Ziegler Foundation for the Blind;Retina Research Foundation
(Kathryn and Latimer Murfee and Emmett A. Humble Chairs);
McPherson Eye Research Institute (Sandra Lemke Trout Chair)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
Program Number: 698 Poster Board Number: C0241
Presentation Time: 1:30 PM–3:15 PM
Prenatal and Postnatal Nutrient Effects in Neonatal Rat Growth
and Retinal Development
Yuta Saito, Emi Ozawa, Haruo Takahashi. Ophthalmology, Showa
University, Tokyo, Japan.
Purpose: In preterm human infants, risk of retinopathy of
prematurity (ROP) has been linked to small for gestational age, low
circulating levels of insulin-like growth factor-1 (IGF-1) and slow
postnatal weight gain. To prevent severe ROP, postnatal nutrition in
preterm infants is very important.
The aim of this study is to investigate prenatal and postnatal nutrient
effects on body weight gain, retinal vascularization and IGF-1 in
plasma in neonatal rat pups.
Methods: Sprague-Dawley rat dams were fed either an unrestricted,
isocaloric normal protein (20%) or low protein (10%) diet to cause
pup growth restriction from 7 days before gestation. The same diet
was continued after the birth of pups. Neonatal rat pups were divided
in two groups after birth, smaller litters (7 rats) and larger litters (14
rats) to cause postnatal growth retardation. On day 8, after measuring
body weight, the rat pups were sacrificed and blood samples
were collected. Retinas were dissected, stained with adenosine
diphosphatase and flat-mounted. The total retinal area (TRA) and
vascularized retinal areas (VA) were measured. Concentration of
IGF-1 in plasma was measured with ELISA. These results were
compared in 20%-7rats, 20%-14rats, 10%-7rats and 10%-14rats
groups. Statistical analyses were performed with Mann-Whitney’s
U test and Kruskal-Wallis test. P value <0.05 was considered
significantly.
Results: The pups from dams fed the low protein diet weighted
significantly lighter at birth (5.8±0.1g vs. 6.8±0.2g P<0.001). The
results on day 8 were shown in table.
Conclusions: Prenatal and postnatal undernutrition may delay
postnatal retinal vascularization and retinal development, and cause
low concentration of IGF-1 in Plasma.
Commercial Relationships: Yuta Saito, None; Emi Ozawa, None;
Haruo Takahashi, None
Support: KAKENHI Number 25870737
Program Number: 699 Poster Board Number: C0242
Presentation Time: 1:30 PM–3:15 PM
Cc2d2a is utilized in assembling subdistal appendages required
for ciliogenesis
Shobi Veleri. Neurobiol-Neurodegen & Repair Lab, NEI, Bethesda,
MD.
Purpose: Meckel syndrome is a lethal form of syndromic ciliopathy
with pleiotropic clinical features like retinal dystrophy, mental
retardation, polydactyly. The patients with mutations in CC2D2A
(Coiled-Coil & C2 Domain containing 2A) displayed Meckel
syndrome. CC2D2A protein is localized to the cilia basal body. The
patient derived fibroblasts have basal body, however, they failed to
develop cilia indicating a mechanistic disconnect in the basal body
due to CC2D2A mutation. To examine this disconnect we have
generated Cc2d2a loss of function diseases models in the mouse.
Methods: The Cc2d2a-knockout (Cc2d2a-/-) mouse was generated
by targeted deletion of exons 6 to 8 relying on homologous
recombination. Immunochemistry and electron microscopy were
employed to examine the basal body from mice fibroblasts.
Results: The Cc2d2a-/- mouse is embryonic lethal. It displays
developmental defects like situs inversus, heterotaxy, polydactyly,
anophthalmia, hydrocephalus and liver fibrosis. Occasionally,
the Cc2d2a-/- mouse survived for a month but was with severe
hydrocephalus and retinal dystrophy. Analysis of the embryonic
node, embryonic fibroblasts, and kidney tubules showed that cilia
biogenesis was disrupted in the Cc2d2a-/- mouse. Embryonic
fibroblasts (MEFs) isolated from Cc2d2a-/- mice lack cilia
even though contain mother centriole (MC or basal body) and
pericentriolar proteins. In addition, MEFs have reduced levels of
Odf2 (associated with subdistal appendages (SDA)) and ninein.
Transmission electron microscopy revealed lack of SDAs in
Cc2d2a-/- MEFs and consistent with this observation immuno-EM
showed Cc2d2a localization onto SDA.
Conclusions: Our studies show that Cc2d2a function is critical for
embryonic development. We conclude that Cc2d2a is essential for the
assembly of SDA, which is anchoring cytoplasmic microtubules and
prime MC for axoneme biogenesis.
Commercial Relationships: Shobi Veleri, None
Program Number: 700 Poster Board Number: C0243
Presentation Time: 1:30 PM–3:15 PM
Fishing for evolutionarily conserved Pax6 eye enhancers in mice
Jena Chojnowski1, Jorn Lakowski1, 2, Kenji Johnson1, James D.
Lauderdale1. 1Cellular Biology, University of Georgia, Athens,
GA; 2Neurosciences & Mental Health, University College London,
London, United Kingdom.
Purpose: To identify evolutionarily conserved cis-regulatory
elements that mediate different aspects of PAX6 expression in
the developing vertebrate eye. The PAX6 transcription factor
plays several roles in eye development, including control of cell
proliferation, maintenance of the retinogenic potential of progenitor
cells, and cell fate specification. These roles in turn are mediated by
modular cis-regulatory elements that are widely spaced within the
Pax6 locus. Although a number of elements have been identified,
the regulatory mechanisms governing several aspects of PAX6
expression remain poorly understood.
Methods: We have taken a comparative approach using mouse and
zebrafish to investigate Pax6 transcriptional regulation. Whereas
mouse has a single Pax6 transcript unit, zebrafish have two Pax6
genes (Pax6a and Pax6b) and Pax6 regulatory elements have
been partitioned between them. Mice harboring either Pax6a or
Pax6b bacterial artificial chromosome (BAC) reporter transgenes
were generated. Reporter gene expression was assessed in eyes of
developing mouse embryos and compared to endogenous PAX6
expression.
Results: The Pax6a and Pax6b BAC reporter transgenes exhibit
overlapping and distinct patterns of expression in the developing
mouse eye. Pax6a expression more closely replicates that of the
mouse gene, and is expressed in the surface ectoderm, lens, retina,
and retinal pigmented epithelium of the developing optic cup. As
development progresses, Pax6a transgene expression is detected in
retinal ganglion cells, amacrine cells, horizontal cells, and Müller
glia of the mature retina and also in anterior structures of the
eye. Interestingly, the Pax6b transgene exhibits a more restricted
spatiotemporal expression pattern in the developing eye suggesting
the action of distinct cis-regulatory modules.
Conclusions: Many if not most of the cis-acting regulatory sequences
governing Pax6 transcription in both mice and zebrafish have
been functionally conserved throughout evolution. This functional
conservation in combination with comparative genomic data provides
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
a robust framework for the identification of novel PAX6 regulatory
elements and a deeper understanding of Pax6 regulatory mechanisms.
Commercial Relationships: Jena Chojnowski, None; Jorn
Lakowski, None; Kenji Johnson, None; James D. Lauderdale,
None
Program Number: 701 Poster Board Number: C0244
Presentation Time: 1:30 PM–3:15 PM
The Effect of Titanium Dioxide Nanoparticles on the Developing
Retina
Yuhao Li1, Yajie Wang1, Zizi He1, Yangwu Fang1, Yang Xu2, Yanan
Chen1. 1Department of Pathology, Nankai University School of
Medicine, Tianjin, China; 2State Key Laboratory of Medicinal
Chemical Biology, College of Chemistry, Nankai University, Tianjin,
China.
Purpose: To investigate whether TiO2 NPs was toxic on zebrafish
embryos and developing retina via aqueous exposure.
Methods: Embryonic zebrafish were exposed to TiO2 NPs at a
concentration of 1mg/L from 1-4 cell stage to 72 hpf. The mortality
and hatching rate were calculated at 24 hpf and 48 hpf respcetively.
An mRNA probe for atonal homolog 7 (atoh7) was used as a
marker to explore whether the neurogenesis initiated on time.
Differentiation of ganglion cells, cones and rods was determined
using immunohistochemistry at 72hpf with Zn12, Zpr1 and Zpr3
antibodies respectively. Microglia in the brain and retina were labeled
using an mRNA probe for fms.
Results: TiO2 NPs exposure did not induce any embryonic
developmental malformations. The onset of neurogenesis and
neuronal differentiation were not delayed following TiO2 exposure.
The location and number of microglia didn’t have a significant
difference between the TiO2 NPs exposed and control group.
Conclusions: TiO2 NPs exposure at 1mg/L doesn’t disrupt the
development of zebrafish embryos and retinal neurogenesis.
Commercial Relationships: Yuhao Li, None; Yajie Wang, None;
Zizi He, None; Yangwu Fang, None; Yang Xu, None; Yanan Chen,
None
Support: Chinese National Natural Science Foundation (81250017,
81301080), National Key Technology R&D Program of China
(2012BAI08B06)
Program Number: 703 Poster Board Number: C0246
Presentation Time: 1:30 PM–3:15 PM
Transcriptional Activation Differs Significantly Between the Two
Isoforms of Isl1
Amanda G. Kautzman, Irene E. Whitney, Benjamin E. Reese.
Neuroscience Research Institute, University of California Santa
Barbara, Santa Barbara, CA.
Purpose: Islet1 (Isl1) is a LIM homeodomain transcription factor
that plays an essential role in retinal development. It forms a well
characterized transcriptional complex with two other proteins, Lhx3
and Ldb1 (Isl1:Lhx3:Ldb1). It has, however, a splicing site that
generates two isoforms, Isl1α and Isl1 β, and the Isl1β isoform lacks
a critical portion of a protein-binding domain with which Isl1 binds
to Lhx3. We recently demonstrated that the two isoforms of Isl1 are
expressed in developing retinal tissue, and that some classes of retinal
ganglion cell express only the β isoform. The present study sought
direct evidence that these two isoforms function distinctly in their
ability to regulate gene expression, using a luciferase reporter with
Isl1:Lhx3:Ldb1 DNA recognition elements in the promoter region to
assay transcriptional activation.
Methods: HEK293T cells, which endogenously express Ldb1,
were transiently transfected with multiple combinations of plasmids
designed to express Lhx3, Isl1α, Isl1β, or GFP. Cells were also
co-transfected with a luciferase reporter that has been previously
shown to be activated specifically by the Isl1:Lhx3:Ldb1 complex.
Cell lysates were assayed for luciferase activity, in three biological
replicates, each experiment being performed in triplicate.
Results: The activation of luciferase by Isl1α:Lhx3:Ldb1 was 5
fold greater than Isl1β:Lhx3:Ldb1. A one-way ANOVA and posthoc comparisons showed the luciferase activation in Lhx3+Islα
overexpressing cells to be significantly different from Lhx3+Isl1β
and all other conditions, while activation by Lhx3+Isl1β was
indistinguishable from Lhx3 alone.
Conclusions: These results demonstrate a functional difference
between the two isoforms of Isl1; furthermore, they suggest that the
β isoform may not be capable of forming a complex with Lhx3 and
Ldb1. This may suggest that Isl1β containing-complexes have unique
gene targets from Isl1α. Given our previous finding that Isl1α and
Isl1β are differentially expressed in subsets of retinal ganglion cells,
these results further suggest that these isoforms likely play distinct
roles in neuronal differentiation during development.
Commercial Relationships: Amanda G. Kautzman, None; Irene
E. Whitney, None; Benjamin E. Reese, None
Support: NIH Grant EY-19968
Program Number: 704 Poster Board Number: C0247
Presentation Time: 1:30 PM–3:15 PM
Primary Cilium Regulates iPS Cell Derived RPE Maturation
Juliet Hartford, Helen May-Simera, Jason Silver, Janine Davis,
Kiyoharu J. Miyagishima, Vladimir Khristov, Omar Memon, Andrea
Li, Sheldon S. Miller, Kapil Bharti. National Eye Institute, National
Institutes of Health, Bethesda, MD.
Purpose: The retinal pigment epithelium (RPE) is a ciliated
monolayer of cells situated adjacent to retinal photoreceptors.
The RPE is critical for maintaining the health and integrity of
photoreceptors. Ciliopathies are a class of disorders that affect cilia
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
formation or functioning and lead to photoreceptor degeneration.
The role of cilia proteins in photoreceptor development is well
understood, however, its function in the RPE is not known. The goal
of this study is to understand the role of the primary cilium in RPE
development and function in mouse and human models.
Methods: RPE cells in WT and two different cilia mutant mice
(Bbs8-/- and Mkks-/-) were analyzed using immunohistochemistry,
scanning electron microscopy, and qPCR. Primary cilium in human
induced pluripotent stem (iPS) cell derived RPE was manipulated
using cilia inducers and inhibitors. Immunohistochemistry,
scanning and transmission electron microscopy, gene expression,
electrophysiology, intracellular calcium measurements, and fluid
transport were used to determine the maturity of iPS cell derived
RPE.
Results: Ciliopathy mutant mice displayed persistent upregulation
of developmental transcription factors MITF and PAX6 in the RPE.
This was consistent with incomplete maturation of RPE in mutant
mice. iPS cell derived RPE treated with primary cilia agonists and
antagonists reinforced this result. Induction of primary cilium in
iPS cell derived RPE improved melanogenesis, induction of apical
processes, barrier resistance across the monolayer, and ability to
transport fluid. All these properties are consistent with improved
maturation of RPE cells. In contrast, suppression of cilium function
dramatically reduced RPE maturation.
Conclusions: Disrupted cilia function results in delayed/improper
RPE maturation that could contribute to retinal degeneration in
ciliopathy patients. Cilium induction in iPS cell derived RPE leads
to physiologically and phenotypically stable RPE cells. We propose
that the primary cilium helps mature RPE cells through modulation
of RPE developmental pathways and that manipulation of these
pathways can help generate mature RPE cells that serve as more
effective disease and cell-therapy models.
Commercial Relationships: Juliet Hartford, None; Helen MaySimera, None; Jason Silver, None; Janine Davis, None; Kiyoharu
J. Miyagishima, None; Vladimir Khristov, None; Omar Memon,
None; Andrea Li, None; Sheldon S. Miller, None; Kapil Bharti,
None
Support: NIH Intramural Grant
Program Number: 705 Poster Board Number: C0248
Presentation Time: 1:30 PM–3:15 PM
Midkine-a protein localization in the embryonic and adult retina
of the zebrafish
Travis D’Cruz, Esther Gramage, Peter F. Hitchcock. Ophthalmology
and Visual Sciences, University of Michigan, Ann Arbor, MI.
Purpose: Midkine-a (Mdka) is a retinoic acid-induced, heparinbinding growth factor with multiple functions in neural development
and repair. In the zebrafish retina, actively dividing retinal progenitor
cells and Müller glia express mdka mRNA during development,
while in the adult, mdka expression is restricted to the horizontal cells
and is robustly regulated by the circadian rhythm. The purpose of
this study was to determine the protein localization of Mdka in the
developing and adult retinas of zebrafish.
Methods: Whole embryos and larvae and eye cups from adults were
fixed with 4% paraformaldehyde and embedded in OCT. Sections
were obtained from embryos at 30 and 48 hours post fertilization
(hpf), larvae at 72, 96 and 120 hpf, and adult eyes. Fluorescence
immunohistochemistry using polyclonal antibodies raised against
Mdka, cell type-specific antibody markers and confocal microscopy
were used to determine the localization of Mdka. Specificity
of the antibody was determined by the selective loss of Mdka
immunolabeling in sections and Western blots from embryos injected
with morpholino oligonucleotides targeted to mdka.
Results: At 30 hpf, Mdka is localized throughout the basal processes
of neuroepithelial cells in the undifferentiated retina. At 48 and
72 hpf, it becomes restricted to the presumptive inner and outer
plexiform layers. At 96 and 120 hpf, when the larval retina is fully
differentiated and mdka expression is restricted to Müller glia and
horizontal cells, Mdka protein is in the outer nuclear layer, but does
not co-localize with cone-specific markers or the Müller glia marker
glutamine synthetase. At the same time, Mdka forms a single small
plaque of protein at the apical surface of each horizontal cell nucleus.
In adults, the horizontal cell staining persists and is regulated by the
circadian rhythm.
Conclusions: Matching the complex pattern of gene regulation,
Mdka protein localization follows a complex temporal and spatial
pattern in the zebrafish retina. Our results suggest that Mdka protein
may be secreted by Müller glia into the extracellular space of the
outer nuclear layer. However, for horizontal cells, the protein is
accumulated in a specific location inside the cell and regulated by the
circadian rhythm.
Commercial Relationships: Travis D’Cruz, None; Esther
Gramage, None; Peter F. Hitchcock, None
Support: NIH Grant EY007060
Program Number: 706 Poster Board Number: C0249
Presentation Time: 1:30 PM–3:15 PM
Regulation of RPE phenotype by Annexin A8 and Wnt signalling
Katharina Lueck, John Greenwood, Stephen E. Moss. Cell Biology,
UCL Institute of Ophthalmology, London, United Kingdom.
Purpose: Fenretinide (FR), a retinoic acid derivative, is capable
of trans-differentiating retinal pigment epithelial (RPE) cells into
a neuronal-like phenotype in culture. Microarray analysis pre- and
post-FR treatment revealed down-regulation of Annexin (Anx)
A8 and various proteins involved in Wnt signalling in transdifferentiated cells. AnxA8, a member of a superfamily of calciumdependent phospholipid-binding proteins, is expressed in RPE cells
and involved in membrane and cytoskeletal organisation and cell
proliferation. The purpose of this study was to analyse the role of
AnxA8 and its relationship with Wnt signalling in epithelial transdifferentiation.
Methods: At 10% confluence, human RPE cells were treated with
3% charcoal dextran-treated foetal bovine serum (FBS) for 24 h. 3
mM FR or vehicle (0.1% dimethylsulfoxide) was added to the cells
every day for 7 days. As a second approach, AnxA8 was suppressed
in RPE cells using short interfering RNA (siRNA). Cells were then
analysed for expression of AnxA8, neuronal markers (Calbindin,
Calretinin) and Wnt signalling proteins (β-Catenin, Frizzled-1,
Frizzled-4, Wnt2b, Wnt3a) using immunofluorescence staining,
qPCR and western blot analysis.
Results: FR and AnxA8 siRNA treatment both induced a decrease
in AnxA8 expression and inhibited cell proliferation. FR also led
to trans-differentiation of ARPE-19 cells into neuron-like cells and
a concomitant up-regulation of neuronal markers. Additionally,
expression of proteins involved in Wnt signalling was decreased.
The effect of FR was partially reversible by activating Wnt signalling
using recombinant Wnt3a or SB216763, a glycogen synthase kinase3β inhibitor.
Conclusions: These data imply an important role for AnxA8 in
maintaining RPE phenotype. Down-regulation of AnxA8 appears
to be sufficient for neuronal trans-differentiation of RPE cells and
the expression of neuronal markers. Further, the interdependence of
AnxA8 and Wnt proteins suggests that AnxA8 might be an important
regulator in Wnt signalling.
Commercial Relationships: Katharina Lueck, None; John
Greenwood, None; Stephen E. Moss, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
Support: BBSRC
Program Number: 707 Poster Board Number: C0250
Presentation Time: 1:30 PM–3:15 PM
ICK ciliary kinase is essential for ciliogenesis in retinal/neuronal
progenitors and regulation of ciliary protein transport at the
ciliary tip
Takahisa Furukawa, Taro Chaya, Yoshihiro Omori. Molecular
and Developmental Biology, Inst for Protein Rsrch, Osaka & JST,
CREST, Osaka, Japan.
Purpose: We previously showed that Mak is required for ciliary
length regulation in retinal photoreceptor cells. Recent reports
showed that mutations in human MAK cause retinitis pigmentosa.
By contrast to cell type-specific expression of Mak, another murine
ortholog of Chlamydomonas LF4, Intestinal Cell Kinase (ICK),
shows ubiquitous expression including in the developing CNS.
However, the exact biological functions of ICK have not yet been
elucidated.
Methods: We generated and analyzed retina- or brain-specific ICKdeficient mutant mice as well as conventional ICK-deficient mutant
mice.
Results: In ICK-null mutant mice, we observed some phenotypes
of defective Hh signaling including polydactyly, shortened leg
bones, and developmental defects of the cerebellum, hippocampus
and retina. At the cellular level, we found that ICK is essential for
ciliogenesis at least in retinal and neural progenitor cells and MEFs.
Conclusions: Our results show that ICK controls protein transport at
the ciliary tip and plays an essential role in ciliogenesis in retinal and
neuronal progenitors but not in mature neurons, proposing a role for
ICK in the regulation of IFT.
Commercial Relationships: Takahisa Furukawa, None; Taro
Chaya, None; Yoshihiro Omori, None
Support: This work was supported by CREST from Japan Science
and Technology Agency, Grant-in-Aid for Scientific Research and
Specially Designated Research Promotion and Scientific Research
on Innovative Areas “Intracellular Logistics” from the Ministry of
Education, Culture, Sports and Technology of Japan .
Program Number: 708 Poster Board Number: C0251
Presentation Time: 1:30 PM–3:15 PM
The role of interleukin-33 expression in retinal tissue
Jobu Sugita1, Yosuke Asada1, Hiroyuki Kawano2, Nobuyuki Ebihara1,
Akira Murakami2, Susumu Nakae3, Akira Matsuda1. 1ophthalmology,
Laboratory of Ocular Atopic Diseases, Juntendo University Graduate
School of Medicine, Tokyo, Japan; 2ophthalmology, Department of
Ophthalmology, Juntendo University Graduate School of Medicine,
Tokyo, Japan; 3ophthalmology, Frontier Research Initiative, Institute
of Medical Science, University of Tokyo, Tokyo, Japan.
Purpose: Interleukin-33 (IL-33) is a IL-1 family cytokine, known to
have pro-fibrotic function as other Th2 cytokines. It is also proposed
that IL-33 work as an alarmin in response to cellular injury. In this
study, we investigated IL-33 expression in retinal tissue and its role
for retinal fibrotic responses.
Methods: Immunohistochemical analysis and Western blotting
analysis of mouse retinal tissue (BALB/C strain) were carried out
with anti-mouse IL-33 antibody (R&D systems). Organ culture of
mouse retinal tissue was carried out using OPTI-MEM medium
(Invitrogen) in 96 well culture dishes, and IL-33 concentration in the
supernatant was quantified by mouse IL-33 ELISA kit (e-bioscience).
IL-33 expression after needle injury of the retina was evaluated
with realtime PCR. The effect of recombinant IL-33 injection to the
vitreous cavity was analyzed using IL-33 knockout mouse.
Results: Immunohistological analysis showed that IL-33 expression
was observed in the nucleus of Muller cells of retinal tissue. Western
blotting analysis of retinal tissue confirmed IL-33 protein expression.
IL-33 protein produced from organ culture of retinal tissue increased
from 403pg/ml at 1 hour to 1761pg/ml at 24 hour. Statistically
significant IL-33 mRNA upregulation was observed 24 hour after
needle injury. Recombinant IL-33 injection to the vitreous induced
TGF-beta1, TIMP-1 and COL1A1 expression in the retinal tissue.
Conclusions: Profibrotic cytokine IL-33 is expressed in mouse
retinal tissue and may have some roles for fibrotic responses of the
eye.
Commercial Relationships: Jobu Sugita, None; Yosuke Asada,
None; Hiroyuki Kawano, None; Nobuyuki Ebihara, None; Akira
Murakami, None; Susumu Nakae, None; Akira Matsuda, None
Program Number: 709 Poster Board Number: C0252
Presentation Time: 1:30 PM–3:15 PM
Ectopic BMP4 alters Neural Retina and Retinal Pigmented
Epithelium Specification
Vijay K. Kalaskar1, James D. Lauderdale2. 1Biomed & Health
Sciences Inst, University of Georgia, Athens, GA; 2Cellular Biology,
University of Georgia, Athens, GA.
Purpose: To evaluate the role of BMP4 signaling in neural retina and
retinal pigmented epithelium (RPE) development by ectopic Bmp4
expression in a mouse whole embryo culture system.
Methods: Mouse embryos were obtained from our breeding colony
with noon on the day of plug discovery designated as embryonic
day 0.5 (E0.5). Wild-type (WT) embryos of CD1-C57BL/6J genetic
background were implanted with affi-gel agarose beads treated either
with recombinant BMP4 or Noggin proteins in the eye region of
embryos at E10.5 and cultured in a standardized serum free medium.
Contralateral eyes implanted with BSA protein treated beads were
used as control. Eye tissues from these embryos cultured for 10
– 18 hours were analyzed for development and differential gene
expression using immunofluorescence, western blot, qRT-PCR and in
situ hybridization.
Results: Ectopic expression of Bmp4 in the ocular region altered
neural retinal specification and affected the development of RPE
pigmentation. The neural retina showed significant down-regulation
of specific markers such as Vsx2 (Chx10) and Pax6. While in the
RPE, the pigmentation was affected in a stage-dependent manner.
When the ocular tissue was exposed to BMP4 before the stage of
visible pigmentation (~30-32 somite stage (ss)), the development of
pigmentation was inhibited and when exposed after the initiation of
pigmentation (~34-35 ss), the RPE showed decreased pigmentation.
Further evaluation of the ocular tissue revealed significant changes
in the expression of early genes such as Rx, Six3 in the retina and
Wnt13, Otx2, Mitf and other downstream pigmentation genes in the
RPE in the BMP4 treated eyes compared to the BSA treated eyes.
Conclusions: BMP4 alters the expression of early genes important
in neural retinal specification and RPE pigmentation indicating a
potential role for BMP4 in RPE and retinal development.
Commercial Relationships: Vijay K. Kalaskar, None; James D.
Lauderdale, None
Support: 1) Children’s Glaucoma Foundation, 2) Sharon Stewart
Aniridia Research Trust
Program Number: 710 Poster Board Number: C0253
Presentation Time: 1:30 PM–3:15 PM
Hedgehog signaling regulates cell movements underlying choroid
fissure formation
Kristen Kwan, Emily Wirick. Human Genetics, University of Utah,
Salt Lake City, UT.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
Purpose: The choroid fissure is a transient, yet critical structure
through which retinal axons exit and vasculature enters the eye.
Disruption of choroid fissure formation or fusion results in uveal
coloboma. Loss-of-function mutations in the Hedgehog (Hh)
receptor patched2 result in colobomata in humans and zebrafish.
These mutations result in overactive Hh signaling, yet the specific
mechanisms by which overactive signaling leads to colobomata are
unknown. Previous work demonstrated that Hh signaling plays a
key role in patterning the dorsal-ventral axis of the eye, however,
it is unknown whether it plays a more direct role controlling cell
movements during eye morphogenesis.
Methods: We developed 4-dimensional live imaging techniques
for visualizing and tracking cell movements during optic cup
morphogenesis. For cell tracking, embryos are labeled for membranes
(EGFP-CAAX) and nuclei (H2A-mCherry); 4D datasets are acquired
via laser scanning confocal microscopy. Cell tracking is performed
using custom LongTracker manual tracking software, and results
visualized using FluoRender. The photoactivatable fluorophore
Kaede is used to quantify movements of specific cell populations
contributing to the ventroanterior retina, including the anterior margin
of the choroid fissure. Overactivation of Hh signaling is achieved via
expression of activated Smoothened or use of the zebrafish patched2
(blowout) mutant.
Results: We find that cells moving into the optic vesicle during
a specific time period that we term late evagination populate the
ventroanterior retina, including the anterior margin of the choroid
fissure. We hypothesized that activated Hh signaling would affect
late evagination, thereby disrupting choroid fissure formation. We
find that activated Hh signaling, either via expression of activated
Smoothened or using the zebrafish patched2 mutant, inhibits late
evagination cell movements: cells do not migrate out of the brain,
and as a result, choroid fissure formation is disrupted, resulting in
colobomata.
Conclusions: We have identified the cellular origin of the anterior
choroid fissure. Overactive Hh signaling affects a very early step of
optic cup and choroid fissure formation, suggesting a direct effect on
morphogenesis and cell behavior. We are currently determining the
effect of overactive Hh signaling on actin-based single-cell behaviors,
as well as whether Hh is acting via canonical or non-canonical
pathways.
Commercial Relationships: Kristen Kwan, None; Emily Wirick,
None
Support: University of Utah Research Foundation, Knights Templar
Eye Foundation
Program Number: 711 Poster Board Number: C0254
Presentation Time: 1:30 PM–3:15 PM
Laminin β2 and γ3 Chains Regulate Retinal Progenitor Cell
Division Polarity
Dmitri Serjanov1, 2, Galina Bachay1, 2, Dale D. Hunter1, 2, William
J. Brunken1, 2. 1Ophthalmology, SUNY Downstate Medical Center,
Brooklyn, NY; 2SUNY Eye Institute, Brooklyn, NY.
Purpose: Basement membranes are highly organized extracellular
matrices that are important sources of developmental cues. Laminin,
a heterotrimeric molecule, is an indispensable organizational
component of basement membranes. Mutations in laminin genes
lead to defective CNS and ocular development in mice and humans.
This study investigates the role of two constituents of CNS laminins,
laminin β2 and γ3 chains in the regulation of the retinal progenitor
cell polarity as characterized by angles of division, cell cycle
dynamics and the consequent neurogenesis.
Methods: Retinae from P3 WT, laminin β2-/- and laminin γ3-/- mice
were used in this study. Immunohistochemistry was performed using
centrosomal and mitotic markers. Retinal progenitor cell angles of
division were visualized through 3D-reconstruction of the dividing
nuclei. Division angles were calculated in 3D by measuring the angle
between a line joining opposing centrosomes and the plane of the
apical retinal surface.
Results: It has been previously shown that deletion of the laminin
β2 and γ3 chains results in alteration of the cell cycle of retinal
progenitor cells, retinal ganglion cell development, Müller glial
cell polarity and photoreceptor development. Here, we demonstrate
different cell division dynamics between developmentally older
and younger regions of the retina as well as a disruption in laminin
mutants. In WT retinae, cell divisions in younger regions are
preferentially symmetrical and gradually become more asymmetric
as the retina becomes older. In laminin β2-/-retinae, the divisions in
all regions are skewed, with noticeable division abnormalities. In
laminin γ3-/- retinae, younger regions are unaffected, whereas older
regions are more symmetrically oriented than in WT retinae. There
is also a high incidence of centrosomal abnormalities in laminin
β2-/-retinae, such as multipolar cells and centrosomes with multiple
centrioles.
Conclusions: Together with our previous findings, these data suggest
that the ILM provides orientation cues and that laminin deletions lead
to premature progenitor pool depletion, resulting in overproduction of
early-born cells at the expense of later-born ones.
Commercial Relationships: Dmitri Serjanov, None; Galina
Bachay, None; Dale D. Hunter, None; William J. Brunken, None
Support: NEI Grant EY12676, RPB Challenge Grant to Department
of Ophthalmology
Program Number: 712 Poster Board Number: C0255
Presentation Time: 1:30 PM–3:15 PM
Increased dendritic branching in direction selective retinal
ganglion cells in nob1 mice
Hung-Ya Tu1, 5, April Bang5, Adam R. McQuiston4, Chuan-Chin
Chiao2, 3, Ching-Kang J. Chen5. 1Institute of Molecular Medicine,
National Tsing Hua University, Hsinchu, Taiwan; 2Department of
Life Science, National Tsing Hua University, Hsinchu, Taiwan;
3
Institute of Systems Neuroscience, National Tsing Hua University,
Hsinchu, Taiwan; 4Department of Anatomy and Neurobiology,
Virginia Commonwealth University, Richmond, VA; 5Department
of Biochemistry and Molecular Biology, Virginia Commonwealth
University, Richmond, VA.
Purpose: Persistent retinal waves in nob1 mice result in abnormal
ganglion cell projections to the brain. However, it remains unclear
whether dendritic morphology and synaptic connectivity are
also altered in the retina. Therefore, we examined the dendritic
development of a genetically identified retinal ganglion cell in the
nob1 background.
Methods: Transgenic mice expressing GFP under the control of a
thyrotropin-releasing hormone receptor promoter (Gong et al., 2003;
Rivlin-Etzion et al., 2011) were obtained from Dr. William Guido
and crossed into the nob1 background. Intrinsic membrane properties
of GFP-positive ganglion cells were recorded by whole-cell current
clamp with potassium-based internal solution containing biocytin.
The dendritic morphologies of recorded cells were visualized by
dye-conjugated streptavidin. Image stacks were acquired in a Zeiss
LSM-710 at 0.5 μm z-interval and traced in the Neurolucida program
for quantitative morphometric analyses.
Results: The wild type GFP-positive ganglion cells displayed
characteristic intrinsic membrane properties and were bistratified with
dendrites co-fasiculated with cholinergic amacrine cells as previously
reported (Rivlin-Etzion et al. J Neurosci 31:8760-8769, 2011). In
the nob1 background, membrane potentials of these cells oscillated
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
with a characteristic peak frequency of 4-6 Hz, first observed around
postnatal day (P) 15 (4.1±0.1 Hz, n=7) and persisted into adulthood
(5.9±0.2 Hz at P28, n=11; p < 0.001). Cells in heterozygous female
(S+) mutants possessed hyper synaptic activity but not rhythmic
oscillations as observed in homozygous female and mutant male
animals. The numbers of dendritic branching points in adult ganglion
cells in the nob1 and S+ backgrounds were significantly higher than
those of the control animals. We also examined GFP-positive cells in
adult mice lacking Gβ5 and R7 RGS proteins where ERG b-waves
were similarly missing. We found that they oscillated similarly to
nob1 mice (4.9±0.4 Hz; n=11; p > 0.05) but surprisingly did not
display differences in their dendritic morphologies.
Conclusions: The results from the nob1 mice suggest that rhythmic
and arrhythmic hyperactivities in the retina facilitate dendritic
branching in this bistratified ganglion cell type. The lack of such
a phenotype in mice lacking Gβ5 and R7 RGS proteins implies
that these proteins may have a cell-autonomous role in dendritic
development and/or plasticity.
Commercial Relationships: Hung-Ya Tu, None; April Bang, None;
Adam R. McQuiston, None; Chuan-Chin Chiao, None; ChingKang J. Chen, None
Support: NIH Grant EY013811; NIH Grant EY022228
Program Number: 713 Poster Board Number: C0256
Presentation Time: 1:30 PM–3:15 PM
Characterization of Retinal Structure and Function in Mice
Carrying Ezh2 Deficiency Specifically in Retinal Ganglion Cells
Lin Cheng1, 2, Honghua Yu2, Naihong Yan2, 3, Honghao Zhou1,
Dongfeng Chen2. 1Institute of Clinical Pharmacology, Central South
University, Changsha, China; 2Schepens Eye Research Institute,
Department of Ophthalmology, Harvard Medical School, Boston,
MA; 3Department of Ophthalmology and Ophthalmic Laboratories,
West China Hospital, Sichuan University, Chengdu, China.
Purpose: Previously in our lab we detected photoreceptor
degeneration in Chx10-Cre-Ezh2flox/flox mice, suggesting that
histone methylase Ezh2 plays an essential role in the retinal
photoreceptor development and function. In this study, we sought
to investigate the role of Ezh2 in retinal ganglion cell (RGC)
development and function.
Methods: To analyze the role of Ezh2 in RGC development, we
generated RGC specific deficiency of Ezh2 by crossing Math5cre with Ezh2flox/floxt mice. Retinal functions were assessed
by electroretinography (ERG) in animals aged 1- 8 months. The
thickness of the ganglion cell complex (GCC) was measured with
spectrum domain-OCT. RGCs and optic nerve fibers were counted
in anti-βIII-tubulin immunolabeled retinal flat-mounts and optic
nerve cross sections, respectively. The expression levels of various
retinal cell markers, such as photoreceptor (recoverin) and ganglion
cell (β-III-tubulin) markers were quantitatively assessed by both
immunolabeling and real time RT-PCR. cDNA microarray was
performed to determine the gene expression profile in Ezh2-deficient
mice. In addition, purified RGC were cultured and their cell survival
and neurite outgrowth were determined. In the above studies,
Ezh2flox/flox mice were used as control animals.
Results: Math5Cre-Ezh2flox/flox mice showed no significant
difference in ERG a wave and b wave responses and GCC thickness.
The retinal lamination and staining pattern of recoverin+ and β-IIItubulin+ cells were visibly comparable between the knockout and
control mice. Moreover, we did not observe significant differences
in wildtype and Math5-Ezh2-/- mice in RGC survival and neurite
outgrowth ability in culture.Results of the cDNA microarray showed
that 0.3% of genes were upregulated and 0.05% of genes were
downregulated in RGCs.
Conclusions: No apparent developmental or functional defects were
observed in Math5Cre-Ezh2-/- mice, suggesting that Ezh2 may not
play a significant role in RGC development or function.
Commercial Relationships: Lin Cheng, None; Honghua Yu, None;
Naihong Yan, None; Honghao Zhou, None; Dongfeng Chen, None
Support: Department of Veterans Affairs (1I01RX000110),
Department of Defense (W81XWH-09-2-0091), Lion’s Foundation
Grants to D.F.C. and K.S.C., China Scholarship Council (CSC).
Program Number: 714 Poster Board Number: C0257
Presentation Time: 1:30 PM–3:15 PM
Expression and role of classical cadherins in the mammalian
retina
Irina De la Huerta1, 2, Xin Duan2, Masahito Yamagata2, Joshua R.
Sanes2. 1Ophthalmology, University of California San Francisco,
San Francisco, CA; 2Center for Brain Science and Department of
Molecular and Cellular Biology, Harvard University, Cambridge,
MA.
Purpose: The circuitry of the mammalian retina depends on the
formation of correct synapses during development. Classical
cadherins are homophilic adhesion molecules present at synapses in
the central nervous system and known to stabilize neuronal contacts.
In this study we describe the expression of classical cadherins in the
mouse retina and examine the roles of selected cadherins in synaptic
specificity.
Methods: In situ hybridization was used to determine the expression
of 18 classical cadherins in the mouse retina during development.
The cadherins that marked relatively small subsets of retinal ganglion
cells (RGCs), or of bipolar or amacrine interneurons known to
synapse with RGCs, were selected for further study. We used mouse
lines in which a marker was knocked in after the start codon of each
cadherin gene of interest to further characterize the connections made
by cadherin-expressing neurons and to determine the effect of loss of
cadherin expression.
Results: We began by examining the expression of all classical
cadherins in the mouse retina during development. Seven cadherins
were expressed in RGCs and/or in bipolar and amacrine interneurons
that are known to synapse with RGCs in the inner plexiform layer of
the retina (IPL). The IPL is composed of 5 sublayers (S1-5), of which
S1-3 are the OFF sublaminae while S4-5 are the ON sublaminae.
We traced the projections of neurons expressing the seven cadherins
of interest and determined that four cadherins (cdh 4, 8, 13, and 22)
marked predominantly OFF-projecting RGCs while two cadherins
(cdh 6 and 10) were expressed by ON-OFF RGCs. Cadherin 8
also marked a subset of OFF bipolar cells while cadherin 9 was
expressed by a subset of ON bipolar cells. Analysis with previously
validated markers showed that the cdh8- and cdh9-positive bipolar
cells are types 2 and 5 respectively. Patterns of cadherin expression
are established before eye opening and are therefore independent of
visual experience. In ongoing work we are using loss- and gainof-function studies to elucidate the roles of cdh8 and chd9 in the
synaptic specificity of ON- and OFF- bipolar cells.
Conclusions: Classical cadherin expression distinguishes subtypes of
RGCs and interneurons during development. Certain cadherins may
play a role in targeting retinal interneurons to the appropriate synaptic
sublaminae in the IPL.
Commercial Relationships: Irina De la Huerta, None; Xin Duan,
None; Masahito Yamagata, None; Joshua R. Sanes, None
Support: NIH Grants NS29169 and EY019355 and a National
Science and Engineering Research Council of Canada Grant to
I.D.l.H.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
Program Number: 715 Poster Board Number: C0258
Presentation Time: 1:30 PM–3:15 PM
Differential Cell Adhesion in Organization of the Cone Synapse
Peter G. Fuerst. 1Biology, University of Idaho, Moscow, ID;
2
WWAMI Medical Education Program, University of Idaho, Moscow,
ID.
Purpose: Development of the retina’s functional circuitry involves
the coordinated interactions of multiple cell types. Despite the limited
number of cell types in the retina compared to other parts of the
brain the retina contains some of the nervous system’s most complex
synapses. For example, development of cone synapses involves
multiple bipolar and horizontal cells contacting and making synapses
with distinct cone photoreceptors, each of which in turn is contacted
by multiple bipolar cells of different types.
Methods: In this study we assay the localization of cadherin,
protocadherin and IGF superfamily adhesion molecules, as well
as PDZ containing scaffolding molecules and catenins at the cone
synapse. Functional analysis of these molecules was focused on
gain and loss of function of the IGF superfamily gene Dscam
(Down Syndrome Cell Adhesion Molecule). The influence of cell
density, cell death and disorganization of downstream circuitry was
also assayed by utilizing bax, bcl2 and cleaved caspase 3 null mice
and by conditional disorganization of inner retinal circuitry. The
integrity of the cone synapse was assayed by immunohistochemistry,
3-dimensional electron microscopy and electroretinography (ERG)
analysis.
Results: A complex localization pattern of cell adhesion molecules
was observed. For example some bipolar cells make contacts at
both cone and rod synapses and localization of some cadherins
was observed on the bipolar cell dendrites contacting cone but not
rod synapses. Functional analysis of Dscam function in the outer
plexiform layer indicated that this gene is required to provide
heteroneuronal recognition within cell types, while ectopic expression
resulted in the formation of ectopic synapses in the outer nuclear
and photoreceptor layers. A role for cell density and the integrity of
downstream circuitry was found with respect to the contacts between
some bipolar cells and the cone synapse.
Conclusions: Our data are consistent with the differential adhesion
hypothesis of development. As more players underlying adhesionmediated organization of the retina are discovered an elegant
picture of the mechanism underlying retinal synaptic organization is
emerging.
Commercial Relationships: Peter G. Fuerst, None
Support: NIH Grant EY020857
Program Number: 716 Poster Board Number: C0259
Presentation Time: 1:30 PM–3:15 PM
Abnormal synaptic transmission between photoreceptors and
bipolar cells in DHDDSK42E/K42E mice
Rong Wen1, Byron L. Lam1, Ziqiang Guan2, Zhengying Wang1,
Ning Wang1, Yihui Chen1, Yiwen Li1. 1Bascom Palmer Eye Institute,
University of Miami, Miami, FL; 2Biochemistry, Duke University
Medical Center, Durham, NC.
Purpose: We recently identified a single-nucleotide mutation
c.124A>G in the DHDDS gene encoding dehydrodolichol
diphosphate synthase (DHDDS), which changes a highly conserved
Lys42 to Glu and is responsible for 12% of autosomal recessive RP
(arRP) cases in patients of Ashkenazi Jewish (AJ) origin. The present
work characterizes electrophysiological changes in recently created
DHDDSK42E/K42E mice.
Methods: Transgenic mice with DHDDSK42E genotype were created
by the knock-in (KI) technology and bred into homozygosity. Lipids
were extracted from plasma and dolichols were measured by liquid
chromatography-mass spectrometry (LC-MS). Scotopic full-field
ERGs were recorded from 3 month old DHDDSK42E/K42E mice and
compared to age-matched wild-type (wt) animals.
Results: The DHDDSK42E/K42E genotype was confirmed by PCR. A
characteristic shortening of dolichol length distribution was found
in the plasma of DHDDSK42E/K42E mice, similar to what was found
in patients. Dolichol 17 (D17) became the dominant species in the
mutant mice instead of dolichol 18 (D18) in wt animals. As a result,
the DHDDSK42E/K42E mice have much higher plasma D17/D18 ratio.
The ERG a-wave in the DHDDSK42E/K42E mice was smaller than
a-wave of the wt controls, and the b-wave was disproportionally
smaller with the b- to a-wave amplitude ratio being close to 1. In
contrast, the ratio was more than 2 in the wt controls.
Conclusions: These results indicate that abnormal dolichol
biosynthesis by the K42E DHDDS mutation leads to impaired
synaptic transmission between photoreceptors and bipolar cells.
Previous studies using artificial membrane suggest a potential role of
dolichols in facilitating vesicle fusion. Our results provide the first in
vivo evidence supporting a biological function of free dolichols in the
activities of synaptic vesicles.
Commercial Relationships: Rong Wen, None; Byron L. Lam,
None; Ziqiang Guan, None; Zhengying Wang, None; Ning Wang,
None; Yihui Chen, None; Yiwen Li, None
Support: NIH grants R01EY018586, P30-EY014801, LIPID MAPS
Collaborative Grant GM-069338, Department of Defense grant
W81XWH-09-1-0674, Adrienne Arsht Hope for Vision fund, and
Research to Prevent Blindness, Inc.
Program Number: 717 Poster Board Number: C0260
Presentation Time: 1:30 PM–3:15 PM
Identification of early retinal bipolar cell-specific genes
Ko Park1, Joseph A. Brzezinski1, Tatiana Eliseeva1, Kenneth Jones2.
1
Ophthalmology, Universith of Denver, Aurora, CO; 2Biochemistry
and Molecular Genetics, University of Colorado School of Medicine,
Aurora, CO.
Purpose: The mechanisms that control bipolar cell formation
during retinogenesis are incompletely understood. Mice that lack the
transcription factor Blimp1 (Prdm1) form bipolar cells at the expense
of photoreceptors shortly after birth. To discover early bipolar cell
regulators, we identified genes that were precociously upregulated in
Blimp1 mutant retinas.
Methods: Postnatal day (P) 2 retinas from five Blimp1 conditional
null mice and five controls were processed for high throughput
RNA-sequencing. Samples were sequenced to an average depth of
30 million reads. The reads were mapped to annotated transcripts
and normalized as reads per kilobase exon per million mapped
reads. Genes that were statistically different between conditions and
that changed at least 40% were considered candidate regulators.
Candidates were further evaluated by in situ hybridization and
immunohistochemistry.
Results: RNA-seq comparison between P2 Blimp1 conditional
null and control retinas revealed approximately 84 significantly
upregulated genes. This included several known bipolarspecific genes, such as Vsx1 and Scgn; which we validated by
immunohistochemistry. We also characterized the expression of a
novel upregulated gene, Tmem215, which codes for a transmembrane
protein of unknown function. We first observed Tmem215 expression
by in situ hybridization in the central retinas of P2 mice. Expression
spread to the periphery by P5 and became more robust in adult
retinas. Tmem215 expression was confined to a narrow region within
the inner retina, consistent with bipolar cell localization.
Conclusions: We have identified several potential early bipolarspecific genes by searching for precociously upregulated genes in
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
Blimp1 mutant retinas. The expression of one of these candidates,
Tmem215, closely paralleled the spatial and temporal pattern of
bipolar cell genesis and maturation. It remains unclear what role
Tmem215 plays in bipolar cell development.
Commercial Relationships: Ko Park, None; Joseph A. Brzezinski,
None; Tatiana Eliseeva, None; Kenneth Jones, None
Program Number: 718 Poster Board Number: C0261
Presentation Time: 1:30 PM–3:15 PM
Genomic Control of Horizontal Cell Regularity
Patrick W. Keeley1, 2, Benjamin E. Reese1, 3. 1Neuroscience Research
Institute, Univ of California, Santa Barbara, Santa Barbara, CA;
2
Molecular, Cellular, and Developmental Biology, Univ of California,
Santa Barbara, Santa Barbara, CA; 3Psychological and Brain
Sciences, Univ of California, Santa Barbara, Santa Barbara, CA.
Purpose: Retinal neurons are often arranged in nonrandom mosaics,
as their somata are distributed to minimize proximity to neighboring
cells of the same type. The mosaic of horizontal cells (HC) is an
exemplar of such a distribution, but little is known of molecular
determinants controlling its patterning. We have previously shown
that different strains of mice vary in the regularity of their HC
mosaics, indicating a genetic component to this trait.
Methods: The present study adopted a forward genetic approach to
seek candidate genes controlling nerve cell patterning, quantifying
the regularity of the HC mosaic in 25 genetically diverse recombinant
inbred strains of mice, each of known genotype, being derived from
the C57BL/6J and A/J inbred laboratory strains. For each strain (n ~
3 mice), we sampled the population of horizontal cells at eight retinal
locations in every mouse and calculated four spatial statistics: nearest
neighbor regularity index (NNRI), Voronoi domain regularity index
(VDRI), effective radius (ER), and packing factor (PF).
Results: The regularity indexes varied across the 25 RI strains,
increasing by 24% from the lowest to the highest strain, while the
coefficient of variation (CoV) within each strain showed relatively
little variation (average CoV=0.04). The estimated heritability of
these indexes was ~0.5, indicating a sizeable proportion of the
variation in trait values across all 95 mice could be ascribed to an
effect of genotype. The two regularity indexes correlated highly
with one another across the recombinant inbred strains (r=0.80), and
each was significantly correlated with PF (r=0.78 and 0.81 for NNRI
and VDRI, respectively). These indexes were moderately correlated
with ER (r=0.59 and 0.46), while showing no significant correlation
with cell density (r=-0.27 and -0.15). Using quantitative trait loci
mapping, we identified two genomic loci, on Chromosomes 1 and 14,
that modulate the regularity of the HC mosaic. Together, these two
quantitative trait loci account for ~44% and ~31% of the interstrain
variation in NNRI and VDRI, respectively.
Conclusions: Using the population of horizontal cells, we show
that mosaic regularity and packing are heritable traits, and that these
traits can be mapped to discrete genomic loci. Further interrogation
of these loci should identify candidate gene variants responsible for
this variation in mosaic patterning, ultimately revealing the molecular
mechanisms of mosaic assembly.
Commercial Relationships: Patrick W. Keeley, None; Benjamin
E. Reese, None
Support: NIH Grant EY-19968
Program Number: 719 Poster Board Number: C0262
Presentation Time: 1:30 PM–3:15 PM
The bHLH transcription factors Ascl1a and NeuroD function in
a regulatory feedback loop with the Notch pathway to regulate
proliferation of photoreceptor progenitors
Scott M. Taylor1, Karen Alvarez-Delfin2, Carole Saade2, James M.
Fadool2, Peter F. Hitchcock1. 1Ophthal & Visual Sciences, University
of Michigan, Ann Arbor, MI; 2Biological Science, Florida State
University, Tallahassee, FL.
Purpose: Development of photoreceptors in the vertebrate retina
requires precise regulation of cell cycle entry and exit. In the
retina of the embryonic zebrafish, the bHLH transcription factor
NeuroD mediates exit of photoreceptor progenitors from the cell
cycle (Ochocinska and Hitchcock, 2009). The purpose of this study
was to determine the mechanisms through which NeuroD governs
photoreceptor genesis in the zebrafish retina.
Methods: First, genetic mosaic analysis was performed to determine
if NeuroD functions cell autonomously in the developing retina.
Second, morpholino-induced NeuroD loss-of-function (LOF) was
used in combination with in-situ hybridization and qRT-PCR to
determine which molecules/pathways are regulated by NeuroD.
Third, LOF approaches were used for putative target proteins to
experimentally test the hypothesized relationships with NeuroD and
with each other.
Results: Genetic mosaic analysis revealed that NeuroD functions
non-cell autonomously in the developing retina, and therefore
subsequent experiments were focused on identifying mechanisms
that could mediate this function. In-situ hybridization and qRT-PCR
revealed that expression of notch1a, her4, ascl1a and hes6 increase
following NeuroD LOF. Inhibition of the Notch pathway using
the gamma secretase inhibitor DAPT rescued the NeuroD LOF
phenotype and restored ascl1a expression to normal levels. Ascl1a
LOF resulted in the loss of neuroD expression, increased Notch
pathway activity, and increased ascl1a expression. Both Ascl1a
and NeuroD LOF resulted in increased cyclinD1 and cyclinB1
expression, indicating a shared mechanism of cell cycle regulation.
Conclusions: Taken together, these data indicate that within
photoreceptor progenitors the Notch pathway functions upstream of
Ascl1a, which, in turn, governs the expression of NeuroD, which
provides feedback inhibition onto the Notch pathway and ascl1a. The
data also show that loss of Ascl1a maintains retinal progenitors in a
proliferative, undifferentiated state and that Ascl1a strongly regulates
its own expression.
Commercial Relationships: Scott M. Taylor, None; Karen
Alvarez-Delfin, None; Carole Saade, None; James M. Fadool,
None; Peter F. Hitchcock, None
Support: NIH T32 EY013934, R01 EY07060-22, F32 EY023129-02
Program Number: 720 Poster Board Number: C0263
Presentation Time: 1:30 PM–3:15 PM
The ponli enhancer activates transcription in the zebrafish green,
red and blue cone photoreceptors
Wei Fang1, 2, Xiangyun Wei1, 2. 1Department of Ophthalmology,
University of Pittsburgh School of Medicine, Pittsburgh, PA;
2
Department of Developmental Biology, University of Pittsburgh
School of Medicine, Pittsburgh, PA.
Purpose: The ponli gene encodes a membrane-associated apical
polarity protein. The Ponli protein is restrictively expressed the
inner segment membrane regions of green, red, and blue cone
photoreceptors in zebrafish. Ponli is expected to play a critical role
in the cone mosaic patterning in the zebrafish. It is unknown how
the distinct expression pattern of the ponli gene is established and
maintained in the zebrafish retina. Thus, we set out to identify the cis-
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
regulatory elements of the ponli gene, which we hypothesize should
be conserved among different teleost species that express the ponli
gene.
Methods: The conserved cis-elements were predicted by aligning the
sequences of ponli genes of five teleost species using the ClustalW2
sequence alignment program. The functions of these predicted
cis-elements were investigated via mutation analyses in vivo. The
expression levels and patterns of reporter genes and the endogenous
ponli gene were analyzed by confocal microscopy, real-time PCR,
and Western blotting.
Results: We showed that the promoter of the ponli gene resides
in a 130-bp sequence upstream of its transcription start site and its
enhancer is located in the 5.7-kb first intron. These cis-regulatory
elements drive specific expression of mCherry in green, red, and blue
cones in the Tg(ponli130-bp promter+5.7-kb enhancer:HA-mcherry)pt118b transgenic
fish. The first intron of the ponli gene contains a 200-bp region
that is sequentially and functionally conserved among five teleosts.
This critical region is likely a central part of the ponli enhancer that
determines the cell-type specificity. In addition, the expression level
of the ponli cis-regulatory elements is moderate, thousands times
lower than that of the green opsin promoter.
Conclusions: The ponli enhancer provides us with an entry point
to study how vertebrate cone and rod photoreceptors differ in their
biochemical, structural, and functional properties by expressing
distinct gene profiles. The ponli enhancer may potentially serve as a
handle to reveal specific interactions between cis- and trans-acting
transcriptional regulatory elements in these cones. In addition, the
ponli enhancer provides us with a unique tool to specifically and
moderately express transgenes in the zebrafish green, red, and blue
cones.
Commercial Relationships: Wei Fang, None; Xiangyun Wei, None
Support: NIH Core Grant P30EY008098, NIH R01 Grant
EY016099, RPB Wasserman Merit Award (to X.W.)
Program Number: 721 Poster Board Number: C0264
Presentation Time: 1:30 PM–3:15 PM
Immunocytochemical analysis of misplaced rhodopsin-positive
cells in the developing rodent retina
Klaudia Szabo1, Arnold Szabo1, Anna Enzsoly2, Agoston Szel1, Ákos
Lukáts1. 1Human Morphology & Dev Biol, Semmelweis University,
Budapest, Hungary; 2Department of Ophthalmology, Semmelweis
University, Budapest, Hungary.
Purpose: During the first postnatal weeks of the developing rodent
retina, rhodopsin can be detected in a number of neuron-like cells
in the inner retina. In the present study we aimed to characterize the
morphology, number and staining characteristics of this peculiar
population.
Methods: Misplaced rhodopsin-positive cells (MRCs) were analyzed
on the retinas of four rodent species, labeled with various rhodopsinspecific antibodies. To investigate their possible relation with nonphotoreceptor cells, sections were double-stained against distinct
retinal cell types, while the possibility of photoreception was assessed
by counter staining with cone- and melanopsin specific antibodies
and proteins of the phototransduction cascade. The possibility of
synapse formation and apoptosis were also investigated.
Results: In all species studied, misplaced cells comprised a few
percent of all rhodopsin-positive elements. Their ratio was relatively
constant with a decline from the end of the second week, and
MRCs disappeared near completely from the retina by P24. MRCs
resembled resident neurons of the inner retina; outer segmentlike processes were seen only rarely. MRCs expressed no other
photopigment types and showed no colocalization with any of the
bipolar, horizontal, amacrine and ganglion cell markers used. While
all MRCs colabeled for arrestin and recoverin, other proteins of the
phototransduction cascade were only detectable in a minority of the
population. Only a few MRCs were shown to form synaptic-like
endings.
Conclusions: Our results showed that during development a
significant percentage of the hodopsin-expressing cells are displaced
to the inner retinal layers. Although most MRCs lack morphological
features of photoreceptors, they contain some, but not all elements of
the phototransduction cascade, indicating that they are most probably
misplaced rods that failed to complete differentiation and integrate
into the photoreceptor mosaic.
Commercial Relationships: Klaudia Szabo, None; Arnold Szabo,
None; Anna Enzsoly, None; Agoston Szel, None; Ákos Lukáts,
None
Support: Hungarian Scientific Research Fund (OTKA #73000),
TÁMOP- 4.2.1.B-09/1KMRB2010-0001.
Program Number: 722 Poster Board Number: C0265
Presentation Time: 1:30 PM–3:15 PM
F-Spondin, a Neuronal Guidance Molecule and a Regulator of
Amyloid Beta Generation is Expressed Discretely in a Subset of
Cone Photoreceptors within the Retina
Rupalatha Maddala1, Paulo Ferreira2, Vasanth Rao3.
1
Ophthalmology, Duke University Medical Center, Durham, NC;
2
Opthalmology & Cell Biology, Duke University Medical Center,
Durham, NC; 3Opthalmology & Pharmacology, Duke University
Medical Center, Durham, NC.
Purpose: F-Spondin, a floor plate enriched multidomain extracellular
matrix-associated protein is known to play a critical role in axonal
pathfinding and in neuronal migration, differentiation and survival
by regulating cell adhesive interactions. Additionally, F-spondin is
involved in the regulation of proteolysis of amyloid precursor protein
and ApoER2 and generation of amyloid beta. Here we report the
discrete distribution profile of F-spondin in the photoreceptor layer of
both the mouse and human retina.
Methods: F-Spondin expression and distribution profiles in
the retinas of mice (C57BL6) were determined by immunoblot,
immunofluorescence and RT-PCR analyses. F-spondin colocalization
with rhodopsin, cone arrestin, M-opsin, S-opsin and peanut
agglutinin was determined using mouse retinal sections and flat
mounts. The levels of F-spondin in cone dystrophy and cone-only
mouse models were determined in the Ranbp2 coneless (Tg-HRGPcre:Ranbp2Flox/Flox) and Nrl null mice respectively, using immunoblot
and immunofluorescence analyses.
Results: F-spondin exhibits a mosaic pattern and distributes
discretely to the outer and inner segments of a subset of peanut
agglutinin (PNA) labeled photoreceptor cells (cones) but not to
the rhodopsin presenting cells (rod photoreceptors), based on the
results of immunofluorescence analysis. Unlike PNA, F-spondin is
not localized to the synaptic terminals of photoreceptors. F-spondin
appears to colocalize with S-opsin at both the dorsal and ventral
regions of mouse retina. Developmentally, F-Spondin expression in
the mouse retina coincides with the morphogenesis of photoreceptor
outer segments. Compared to wild-type retinas, the retinas of Ranbp2
coneless and Nrl null mice, the levels of F-spondin were decreased
and increased, respectively.
Conclusions: Taken together, the observation of novel, discrete
and intense co-distribution of F-spondin with cone photoreceptors
suggests a critical role for F-spondin in photoreceptor morphogenesis,
survival or function.
Commercial Relationships: Rupalatha Maddala, None; Paulo
Ferreira, None; Vasanth Rao, None
Support: R01 EY12201, R01EY 18590, P30-EY005722 and RPB.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2014 Annual Meeting Abstracts
Program Number: 723 Poster Board Number: C0266
Presentation Time: 1:30 PM–3:15 PM
An analysis of photoreceptor basal body positioning in zebrafish
with mutations in cytoplasmic Dynein 1 and Dynactin
Joseph Fogerty, Brian D. Perkins. Cole Eye Institute, Cleveland
Clinic, Cleveland, OH.
Purpose: The photoreceptor outer segment is an elaborate primary
cilium specialized for photon detection. Anchoring of the basal
body at the apical membrane is a prerequisite for outer segment
extension, but little is known about the processes that regulate this
event. We hypothesized that the microtubule motor cytoplasmic
dynein 1 is required for basal body localization prior to photoreceptor
ciliogenesis. Cannonball mutant zebrafish carry a nonsense mutation
in the heavy chain subunit of cytoplasmic dynein 1 (dync1h1), and
the mikre oko mutant has a nonsense mutation in the p150Glued
subunit of Dynactin (dctn1a), a key Dynein1 regulatory complex.
Previous studies on these mutants showed inner segment organelle
positioning defects as well as impaired outer segment morphogenesis.
We utilized these mutants to evaluate the requirement of Dynein1
based motility for proper basal body positioning.
Methods: Cannonball and mikre oko mutant fish were crossed
to the Tg(-5actb2:cetn2-GFP) transgenic line, which expresses a
centrin-GFP fusion protein from the beta-actin promoter and labels
basal bodies. Basal body positioning relative to the outer limiting
membrane was assayed at multiple time points in frozen sections
counterstained with phalloidin.
Results: Both cannonball and mikre oko fish underwent a significant
degree of retinal degeneration by 4dpf, with rounded nuclei and
disorganized lamination frequently observed. The apical actin
network was disorganized and often absent in mutant fish, but GFP+
basal bodies were present in areas with an intact outer limiting
membrane. Where basal bodies were observed, they were positioned
at the expected distance from the OLM.
Conclusions: Basal bodies in cannonball and mikre oko fish localize
properly to the apical membrane in those areas of retina that establish
an apical actin network. The patches of retina lacking phalloidin
reactivity have no observable basal bodies and may represent areas
that lose cell polarity after exhaustion of the maternal protein.
Commercial Relationships: Joseph Fogerty, None; Brian D.
Perkins, None
Support: EY017037
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].