Download ARVO 2015 Annual Meeting Abstracts 410 Lens development

ARVO 2015 Annual Meeting Abstracts
410 Lens development
Wednesday, May 06, 2015 8:30 AM–10:15 AM
605/607 Paper Session
Program #/Board # Range: 4005–4011
Organizing Section: Lens
Contributing Section(s): Lens
Program Number: 4005
Presentation Time: 8:30 AM–8:45 AM
Transcriptome Profiling of Developing Murine Lens through
RNA Sequencing
Shahid Y. Khan1, Sean F. Hackett1, Mei-Chong W. Lee2, C. Conover
Talbot3, Nader Pourmand2, S Amer Riazuddin1. 1The Wilmer Eye
Institute, Johns Hopkins University School of Medicine, Baltimore,
MD; 2Department of Biomolecular Engineering, University
of California Santa Cruz, Santa Cruz, CA; 3Institute for Basic
Biomedical Sciences, Johns Hopkins University School of Medicine,
Baltimore, MD.
Purpose: Transcriptome is the entire repertoire of all transcripts
present in a cell at any particular time. We undertook next-generation
whole transcriptome sequencing approach to gain insight of the
transcriptional landscape of developing mouse lens.
Methods: We ascertained mice lenses at six developmental time
points including two embryonic (E15 and E18) and four postnatal
stages (P0, P3, P6, and P9). The ocular tissue at each time point was
maintained as two distinct pools serving as biological replicates for
each developmental stage. The mRNA and small RNA libraries were
paired-end sequenced on Illumina HiSeq 2000 and subsequently
analyzed using different bioinformatics platforms.
Results: The mapping of mRNA and small RNA libraries generated
187.56 and 154.22 million paired-end reads, respectively. We
detected a total of 14,465 genes in the mouse ocular lens. Of these,
46 genes exhibited 40-fold differential expression compared to
transcriptional levels at E15. Likewise, small RNA profiling identified
379 microRNAs (miRNAs) expressed in mouse lens. Of these, 49
miRNAs manifested an 8-fold or higher differential expression when
compared, as above to the microRNA expression at E15.
Conclusions: In conclusion, we report the first comprehensive profile
of developing murine lens transcriptome including both mRNA and
miRNA through next-generation RNA sequencing. These data will
add to our understanding of lens development and elucidate processes
essential for maintenance of lens transparency.
Commercial Relationships: Shahid Y. Khan, None; Sean F.
Hackett, None; Mei-Chong W. Lee, None; C. Conover Talbot,
None; Nader Pourmand, None; S Amer Riazuddin, None
Support: R01EY022714
Program Number: 4006
Presentation Time: 8:45 AM–9:00 AM
Celf1 mediates post-transcription control of gene expression in
lens development
Archana D. Siddam1, Carole Gautier- Courteille3, Vincent
Legagneux3, Agnes Mereau3, Justine Viet3, Linette Perez-Campos4,
David C Beebe5, Jeffrey M. Gross4, Luc Paillard3, Salil A. Lachke1,
2 1
. Biological Sciences, University of Delaware, Hockessin, DE;
2
Center for Bioinformatics & Computational Biology, University of
Delaware, Newark, DE; 3Institut de Génétique et Développement
de Rennes, Université de Rennes 1, Rennes, France; 4Department of
Molecular Biosciences, University of Texas, Austin, TX; 5Department
of Ophthalmology and Visual Sciences, Washington University, St.
Louis, MO.
Purpose: While the significance of signaling and transcriptional
control mechanisms is well appreciated in lens development, that of
post-transcriptional regulation remains unexplored. To address this
deficit, we applied the lens gene discovery tool iSyTE to identify the
RNA binding protein (RBP) Celf (Cugbp1) and found that its deletion
in mouse or knockdown in fish disrupts lens development and causes
cataract. Here, we report on elucidation of the novel mechanistic
basis of Celf1-mediated control of important regulators in lens
development.
Methods: Immunostaining and qRT-PCR were used to test the
expression of targets in Celf1 conditional knockout (cKO) mouse
mutant lens. RNA immunoprecipitation (RIP) with Celf1 antibody
tested the direct interaction of Celf1 with RNA targets. Celf1mediated translational control was tested by cloning the 5’UTR
of p27 mRNA into reporter plasmid upstream of firefly luciferase.
Transient transfections introduced reporter vector into control or
Celf1-knockdown (KD) mouse lens epithelial cell line 21EM15.
Results: While in normal E16.5 lens p27 protein is downregulated in
centrally located fiber cells, Celf1 cKO mutant lens exhibits ectopic
and up-regulated presence of this protein in this region. RIP analysis
indicates that Celf1 directly binds to p27 mRNA and functional
analysis demonstrates elevated luciferase expression in Celf1-KD
cells, indicating that Celf1 potentially directly inhibits p27 translation
in differentiating lens fiber cells. Interestingly, Prox1 protein is
significantly up-regulated in Celf1 cKO lens epithelium, while other
mRNAs are either up-regulated (p21) or down-regulated (Dnase2b)
in Celf1 cKO lens. These data indicate that Celf1 functions to finetune gene expression by controlling translation or mRNA stability in
both lens epithelial and fiber cells.
Conclusions: In past, based on the fiber cell-specific expression
pattern of the RBP Tdrd7, we had anticipated a relatively
straightforward model that different epithelium-specific or fiberspecific RBPs may function to control post-transcriptional gene
expression in these cells. However, these new data demonstrate that
a single RBP Celf1 expressed in both the epithelium and fiber cells
can control their respective proteomes by distinct post-transcriptional
mechanisms such as RNA stability or translational control, thus
highlighting a previously unanticipated mechanistic sophistication in
lens gene regulation.
Commercial Relationships: Archana D. Siddam, None; Carole
Gautier- Courteille, None; Vincent Legagneux, None; Agnes
Mereau, None; Justine Viet, None; Linette Perez-Campos, None;
David C Beebe, None; Jeffrey M. Gross, None; Luc Paillard,
None; Salil A. Lachke, None
Support: NIH/NEI R01 EY021505, Fight for Sight Summer Student
Fellowship
Program Number: 4007
Presentation Time: 9:00 AM–9:15 AM
A disintegrin and metalloproteinase 10 (ADAM10) is a regulator
of lens development
D J. Sidjanin, Joseph Toonen. Cell Biology, Anatomy &
Neurobiology, Human and Molecular Genetics Center, Medical
College of Wisconsin, Milwaukee, WI.
Purpose: ADAM10 and ADAM17 are two closely related members
of the disintegrin and metalloproteinase (ADAM) family with a role
in ectodomain cleavage of membrane bound proteins. Both ADAM10
and ADAM17 have been implicated in proteolytic cleavage of
EGRF ligands as well as NOTCH receptors. Our previous studies
have shown that ADAM17 solely transactivates EGFR signaling.
Therefore, we proceeded to evaluate the role of ADAM10 during lens
development as a potential regulator of NOTCH signaling. To test
this hypothesis, we set out to generate Adam10 conditional knockout
mice (CKO).
©2015, 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 2015 Annual Meeting Abstracts
Methods: Conditional lens-specific ADAM10-CKO mice where
generated following outcrossing of Adam10floxed/floxed mice to the LeCre
transgenic mice that express the Cre recombinase under the control
of the Pax6 promoter. Adam10-CKO and control mice were further
characterized at different developmental time points using slit lamp
biomicroscopy, histological, EdU, TUNEL, and immunohistological
analyses.
Results: Adam10-CKO mice appeared anophthalmic with hair loss
around the eyes extending to the snout region. Histological analysis
of Adam10-CKO adult eyes revealed absence of the anterior chamber
and poor separation between the cornea, iris, and lens tissues,
thickened iris, and severe microphakia-characterized by the absence
of epithelial and fiber cell structures and the presence of swollen cells
at the periphery. First morphological lens abnormalities in Adam10CKO lenses were noted at E13.5 characterized by reduced number of
lens cells, thinned epithelium, and disorganized fiber cells. By 16.5
the Adam10-CKO lenses have undergone degeneration precluding
further evaluation. Immunohistochemical evaluation revealed that
at E13.5 Adam10-CKO lenses exhibit abberrant lens epithelial
cell proliferation, aberrant lens fiber cell differentiation, without a
significant increase in apoptosis.
Conclusions: Our results demonstrate that ADAM10 is indispensable
for proper lens development. ADAM10 has been previously proposed
to function as a regulator of NOTCH1 signaling. In the lens NOTCH
signaling is mediated by NOTCH2 and lens-specific ablation of
Notch2 or RBP-J results in premature epithelial cell cycle exit and
precocious lens fiber cell differentiation. The identified Adam10CKO lens phenotypes suggest disrupted NOTCH signaling, although
studies confirming disrupted NOTCH2 signaling are currently under
way.
Commercial Relationships: D J. Sidjanin, None; Joseph Toonen,
None
Support: EY18872
Program Number: 4008
Presentation Time: 9:15 AM–9:30 AM
The Endocytic Recycling Regulatory Protein EHD1 Is Required
for Ocular Lens Development
priyanka arya1, 2, Mark Rainey3, Sohinee Bhattacharyya4, 2,
Bhopal Mohapatra5, 2, Murali Kuracha3, Vimla Band1, 2, venkatesh
govindarajan3, Hamid Band1, 2. 1Genetics, Cell Biology and Anatomy,
University of Nebraska Medical Center, Omaha, NE; 2University
of Nebraska Medical Center, Fred & Pamela Buffett Cancer
Center, Omaha, NE; 3Department of Surgery, Creighton University,
Omaha, NE; 4Pathology and Microbiology, University of Nebraska
Medical Center, Omaha, NE; 5Biochemistry and Molecular Biology,
University of Nebraska Medical Center, Omaha, NE.
Purpose: The C-terminal Eps15 homology domain-containing (EHD)
proteins play a key role in endocytic recycling, a fundamental cellular
process that ensures the return of endocytosed membrane components
and receptors back to the cell surface. Towards understanding the
biological roles of mammalian EHD family proteins, we and others
have begun to use mouse gene deletion models.
Here, we provide evidence for a critical requirement of EHD1 for
development of ocular lens and cornea. Our studies show that EHD1/mice display pleiotropic ocular phenotypes, including anophthalmia,
microphthalmia and congenital cataracts.
Methods: EHD1 CKO were generated by crossing EHD1 floxed
mice with Le-Cre transgenic mice. For timed-pregnancy experiments,
the noon of vaginal plug detection was considered E0.5. Pregnant
dams were euthanized, embryos collected, fixed in 10% formalin,
transferred to 70% ethanol prior to paraffin embedding. For antibody
staining, rehydrated tissues were boiled in antigen unmasking
solution, blocked, incubated with primary antibodies, followed by
dectection with flurosecent secondary antibodies.
Results: Histological analyses showed pleiotropic ocular defects in
EHD1-/- mice that included a smaller or absent lens, persistence of
lens stalk and the hyaloid vasculature, and deformed optic cup. To
investigate whether these profound ocular defects resulted from a
role in lens versus optic vesicle, we deleted EHD1 selectively in the
presumptive lens ectoderm using Le-Cre. EHD1 deletion in the lens
resulted in developmental defects that included thinner epithelial
layer with reduced cell count, smaller lenses and absence of corneal
endothelium. Lens epithelium-specific EHD1 mutants exhibited
reduced proliferation and cell survival, and downregulation of
junctional proteins such as E-cadherin and ZO-1.
Conclusions: The ocular phenotypes caused by the loss of a single
regulator of endocytic recycling, EHD1, provide a novel model
system to elucidate mechanistic links between surface receptor
recycling and the cellular processes that control orderly development
of the compartments of mammalian eye. Taken together, these
data reveal a unique cell-autonomous role for EHD1 in early lens
development and suggest a previously unknown link between
endocytic recycling pathway and regulation of key developmental
processes events including proliferation, differentiation and
morphogenesis.
Commercial Relationships: priyanka arya, None; Mark Rainey,
None; Sohinee Bhattacharyya, None; Bhopal Mohapatra,
None; Murali Kuracha, None; Vimla Band, None; venkatesh
govindarajan, None; Hamid Band, None
Support: Graduate fellowship through a Program of Excellence
Graduate Assistantships from University of Nebraska Medical Center
(2012-2015)
Program Number: 4009
Presentation Time: 9:30 AM–9:45 AM
Role of ltbp2 in eye development
Steven Bassnett1, 2, Alicia De Maria1, Yanrong Shi1, Robert Mecham2.
1
Ophthal & Vis Science, Washington Univ Sch of Med, Saint Louis,
MO; 2Cell Biol & Physiol, Washington Univ Sch of Med, St. Louis,
MO.
Purpose: Latent transforming growth factor beta binding protein
2 (ltbp2) is a member of the fibrillin/ltbp superfamily. In humans,
mutations in LTBP2 are known to underlie autosomal recessive WeillMarchesani syndrome, a condition characterized by ectopia lentis and
microspherophakia.
Methods: To better understand the role of ltbp2 in eye development,
its expression in the developing mouse eye was visualized by in
situ hybridization and immunofluorescence. Ltbp2-null mice were
generated by homologous recombination and their ocular phenotype
assessed by confocal microscopy.
Results: In situ hybridization and immunofluorescence analysis
suggested that, in mice, ltbp2 was not expressed until the end of
the first postnatal week, when transcripts were first observed in the
non-pigmented ciliary epithelium (NPCE). Microfibrils containing
ltbp2 were detected on the surface of the NPCE and subsequently
incorporated into the developing ciliary zonule. In ltbp2-null mice
the ciliary zonule appeared to form normally but disintegrated at later
time points leading to ectopia lentis. Lenses from ltbp2-null mice
were transparent but slightly smaller than those from age-matched
littermates.
Conclusions: Ltbp2 is a component of the postnatal ciliary zonule
where it appears to contribute to long-term zonule stability. Lens
growth defects and progressive lens detachment in ltbp2-null mice
are reminiscent of the ocular phenotype of patients with autosomal
recessive Weill-Marchesani syndrome. Ltbp2-/- mice may thus
©2015, 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 2015 Annual Meeting Abstracts
represent a useful model to study the ocular complications associated
with this condition.
Commercial Relationships: Steven Bassnett, None; Alicia De
Maria, None; Yanrong Shi, None; Robert Mecham, None
Support: R01 EY024607, R01 EY09852, P30 EY02687, RPB and
National Marfan Foundation
Program Number: 4010
Presentation Time: 9:45 AM–10:00 AM
Generation of lentoid bodies from human urinary cell derived
induced pluripotent stem cells
Qiuli FU1, 2, Zhenwei QIN1, 2, Zhi FANG1, 2, Junfeng JI3, Ke YAO1, 2.
1
Eye Center, the 2nd Affiliated Hospital, Medical College of Zhejiang
University, Hangzhou, China; 2Zhejiang Provincial Key Lab of
Ophthalmology, Hangzhou, China; 3Center for stem cell and tissue
engineering, Medical college of Zhejiang University, Hangzhou,
China.
Purpose: Cataract, opacification of lens, is a complex disease
involving genetic and environmental factors, which is responsible
for more than half of blindness worldwide. Lack of appropriate
animal and cell models in vitro made it difficult to understand its
pathological mechanisms, therefore limit the process of curing the
disease. This study is aim to establish an in vitro system in which
lens development and cataract process could be mimic using human
induced pluripotent stem cells (hiPSCs).
Methods: Human exfoliated renal epithelial cells which present in
urine were collected and cultured. Then, the isolated urinary cells
were infected with four Yamanaka factors to generate urinary hiPSCs
(UiPSCs) whose characteristics were examined by immunostaining
and teratoma experiment. To establish a strategy to generate lentoid
bodies (LBs) from UiPSCs, we adapted the 3-stage system of LBs
formation from human embryonic stem cells (hESCs) (Yang et al,
FASEB J, 2010). Expression of lens-specific markers was examined
by real-time PCR, immunostaining and western blot. Structure of LBs
was investigated using transmission electron microscropy (TEM).
Results: Small colonies of urinary cells were observed at d4 and used
for retroviruses infection after expansion. Three weeks after infection,
UiPSCs were generated as evidenced by AP staining and expression
of stem cells specific markers (NANOG, SSEA4, SOX2 and TRA181), as well as teratoma formation. 28 days after induction, LBs
showing sphere-like shape and transparent structure were obtained
(Fig.1) and expressed lens-specific markers including αA, αB, β and
γ-crystallins, and MIP/aquaporin 0. More interestingly, transmission
electron microscropy (TEM) revealed the presence of lens epithelial
cells adjacent to the lens capsule, followed by unmature fiberlike cells
with degenerating nucleus and organelles, and mature fiberlike cells
without nucleus and organelles which located in the center of LBs.
To further analyze whether LBs have the function of human lens, we
examined their magnifying ability and approximately 1.7X of the
magnification factor were found.
Conclusions: Our study established an in vitro system through an
invasive procedure which can be utilized to studies of human lens
development and cataractogenesis using patient specific UiPSCs.
Fig.1 Representative pictures of LBs from UiPSCs
Commercial Relationships: Qiuli FU, None; Zhenwei QIN, None;
Zhi FANG, None; Junfeng JI, None; Ke YAO, None
Program Number: 4011
Presentation Time: 10:00 AM–10:15 AM
Molecular characterization of human lens epithelial cell lines
HLE-B3 and SRA01/04 and their utility to model lens biology
Anne M. Terrell, Deepti Anand, Salil A. Lachke. Biology, University
of Delaware, Newark, DE.
Purpose: Insights into human lens development, homeostasis and
pathology have been largely gained from the identification of genetic
alterations in human cataract patients and by the in vivo investigation
of lens biology using various animal models like mouse, chicken,
frog and fish. In vitro models such as culture of primary or permanent
lens epithelial-derived cells are important to gain mechanistic insights
and have been well utilized to characterize the molecular biology of
lens cells. However, gene expression in established lens epithelial cell
(LEC) lines, particularly with reference to their extent to model lens
biology, has not been comprehensively investigated. Therefore, we
molecularly characterized two LEC lines HLE-B3 and SRA01/04,
which are commonly used in lens studies, for their expression of
genes important to lens biology.
Methods: The human LEC line HLE-B3 was obtained from ATCC
(CRL-11421), while the LEC SRA01/04 was obtained directly from
Dr. Venkat Reddy who originally derived this line. These LECs were
validated for their human origin by human short tandem repeats
(STRs) as recommended by the eye research community. Illumina
Human-HT12 Expression BeadChip microarrays was used for whole
genome expression profiling of HLE-B3 and SRA01/04. Based
on analysis of the literature and the web-based lens and cataract
gene discovery tool iSyTE, genes significant to lens biology were
selected for further validation in LECs by RT-PCR analysis and were
compared to a non-LEC line NIH3T3.
Results: Microarray analysis indicated that both HLE-B3 and
SRA01/04 significantly express several genes known to be important
to lens biology or cataracts. These genes identified by microarrays
are: PAX6, ZEB2, PVRL3, SPARC, COL4A1, SLC16A12, LAMC1,
©2015, 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 2015 Annual Meeting Abstracts
PXDN, ALDH7A1, LEPREL1, ETV5, DKK3, CRYAB, CRYBB2,
TDRD7, STX3, DHX32, SFRP1, ALDH1A3, CRIM1, STRA6, ETV6,
EPHA2, ADAMTSL4, MAFG, CAPRIN2, and LAMA1. Significantly,
as previously reported by Andley et al. (IOVS 1994 35:3094-3102)
microarray data validates that the HLE-B3 line expresses CRYBB2.
Moreover, compared to NIH3T3 cells, both LECs exhibited enriched
expression of PAX6, FOXE3, PROX1, ALDH1A1 and DKK3.
Conclusions: In sum, human LECs HLEB3 and SRA01/04 retain
the expression of lens epithelium-enriched genes including PAX6,
FOXE3, PVRL3, ZEB2 and SPARC, while also expressing certain
transition zone and fiber cell-enriched genes.
Commercial Relationships: Anne M. Terrell, None; Deepti Anand,
None; Salil A. Lachke, None
Support: NIH/NEI R01 EY021505
©2015, 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].