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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].