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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].