Background

We aim to gain a better understanding of retinogenesis by studying epigenetic regulation of neuronal differentiation and by dissecting the key developmental events that establish the mature tissue architecture and functional organization of the retina: establishment of cell polarity, synaptogenesis, ganglion cell diversity and axonal migration (optic disc formation). Results 1. Epigenetics regulation of neurogenesis in the retina

 1.1 DNA methylation is an instructive epigenetic DNA modification that mediates chromatin conformation changes and impacts patterns of gene expression. DNA methyltransferases play fundamental roles in development but their contributions to retinogenesis have not been delineated. To delineate systematically the role of DNA methylation in retinogenesis, we first performed a comprehensive temporal and spatial expression study of Dnmt1, Dnmt3a, and Dnmt3b in developing and mature retina. We find a differential pattern of nuclear Dnmt1, 3a, and 3b distribution in cone versus rod photoreceptors and suggest their involvement in the establishment of cell fate. Furthermore, the observed dynamic patterns of Dnmt1, 3a, and 3b expression may be an indication of chromatin remodeling and reflect patterns of gene expression during retinogenesis. This potential novel role(s) of Dnmts in retinal development constitute the foundation for our current efforts to dissect cell-type specific function(s) of DNA methylation during fate specification and/or homeostasis (1). Cre-lox mediated excision of DNA methytransferase 1 (Dnmt1) Exons 4-5 in neural retina (NR) and retinal pigment epithelium (RPE) drastically disrupts RPE polarization and results in deficient elongation of photoreceptor (PR) outer segments, mostly affecting S-cones. An identical phenotype is observed after Dnmt1 targeted excision in RPE but not after Dnmt1 excision only in PR. These data support a causal relationship between RPE polarization and PR terminal differentiation and highlight a key role of Dnmt1 in controlling maturation of PR and RPE in mice. 1.2 The promoters of many developmentally regulated genes in Drosophila appear to have both activating (trimethyl histone H3 lysine 4) and repressive (trimethyl histone H3 lysine 27) histone modifications. These bivalent promoters are inactive and are primed to have active or repressive marks as cell fate is restricted. Similarly, RNA polymerase II has been found to pause immediately downstream of the transcription start site of key developmental regulatory genes. It is likely that these two mechanisms prime the promoters for future expression for rapid and more controlled gene activation. Whether these mechanisms also apply to rod photoreceptor specification is not known. We are examining the genome wide distribution of multiple histone methylations and RNA polymerase II by ChIP-seq (chromatin immunoprecipitation followed by next-generation sequencing). Gene expression profile data obtained with microarray hybridization and RNA-seq will be overlaid with histone modification profiles and binding of RNA polymerase II to obtain a comprehensive analysis of genetic and epigenetic control of retinal differentiation, with the aim to understand the interplay between transcription factors and epigenetic marks. 2. Establishment of photoreceptor cell polarity
 Prickle (Pk)1 and Pk2 are tissue polarity genes necessary for the establishment of planar cell polarity (PCP) in Drosophila. We hypothesize that Pk1 and Pk2 play essential roles in initiation and maintenance of photoreceptor polarity in mammals and aim to understand how polarity genes interact with neural development to affect fate determination and plasticity. Pk1 and Pk2 are expressed during murine retinal development. Knock down of Pk1 by in vivo electroporation results in abnormal photoreceptor migration and proliferation, suggesting changes photoreceptor plasticity. Pk1 conditional knockout mice show relatively short statures, develop watery eye phenotype and are hypersensitive. Brain and eye morphogenesis are being investigated. The phenotype of Pk2 knockout mice (obtained from Dr. Bassuk, University of Iowa) is currently under investigation. 3. Molecular mechanisms of ciliogenesis BBS9/PTHB1 is one of the seven core proteins that form a stable complex called BBSome, implicated in trafficking of proteins to primary cilia, and is among fifteen known BBS-associated genes. By knocking down bbs9/pthb1 in zebrafish with antisense morpholinos we detect developmental abnormalities including hydrocephaly, retina and brain defects, consistent with the core phenotypes observed in syndromic ciliopathies. In addition, many morphants reveal heart edema, sluggish heartbeat, and no visible signs of blood circulation. Knockdown of bbs9/pthb1 also causes reduced number and length of cilia in Kupffer's vesicle. Similarly, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of primary cilia. These data suggest a key role of BBS9 in biogenesis and/or function of primary cilia in zebrafish and mammals (see EY000473-03). 4. Synaptogenesis We are investigating the mechanism and molecules involved in circuit formation between photoreceptors and second-order neurons using two approaches: 1) by comparing synapse formation in retinas from Nrl-/- (cone-only) and Crxp-Nrl (rod-only) mice;and 2) by testing, using siRNA delivery, the involvement in synaptic connection development of candidate genes identified through gene profiling of rod photoreceptors. Preliminary data suggest that Nrl-/- photoreceptors form either chimeric or underdeveloped synapses and that in Nrl-/- retina, connections between photoreceptor and rod bipolar cell decrease before eye opening while in wild type they increase. We hypothesize that synaptogenesis between rod and rod bipolar neurons is initiated by intrinsic molecular cues, rather than by activity before eye opening. 5. Optic disc formation
 Based on protein structure similarity and on expression pattern in the optic disc region, we hypothesized that Frizzled (Fz)5 and 8 orchestrate Wnt-Frizzled signaling and that their disruption leads to coloboma.
We generated Fz5 and Fz8 double knockout mice to gain insight into the role of these genes in retinal neurogenesis. We show that Fz receptors are involved in retinal neurogenesis via maintaining retinal cell polarity. They act in a dosage-dependent way and cross talk with other signaling pathways, including Shh and Notch. Furthermore, they are critical for the maintenance of adult axonal integrity. For the first time we show that Frizzled signaling pathway is involved in retinal neurogenesis, likely through non-canonical Frizzled signaling that affects neuroblast organization and apical-basal polarity. Significance
 Our investigations are defining the role of specific genes and epigenetic signals in establishing photoreceptor function during retinal development. This project complements our efforts to engineer stem cells into photoreceptors and functionally integrate them into the disrupted architecture of degenerating retinas after transplantation (see EY000474-03). We have also initiated studies on neurons in the inner retina with Dr. Tudor Badea (a newly-recruited tenure-track investigator in N-NRL). These investigations will assist in developing novel paradigms for treatment of retinal and macular degenerative diseases and glaucoma.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAEY000451-04
Application #
8339785
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2011
Total Cost
$3,753,100
Indirect Cost
Name
U.S. National Eye Institute
Department
Type
DUNS #
City
State
Country
Zip Code
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Nasonkin, Igor O; Lazo, Kevin; Hambright, Dustin et al. (2011) Distinct nuclear localization patterns of DNA methyltransferases in developing and mature mammalian retina. J Comp Neurol 519:1914-30
Hao, Hong; Tummala, Padmaja; Guzman, Eduardo et al. (2011) The transcription factor neural retina leucine zipper (NRL) controls photoreceptor-specific expression of myocyte enhancer factor Mef2c from an alternative promoter. J Biol Chem 286:34893-902
Cheng, Hong; Khan, Naheed W; Roger, Jerome E et al. (2011) Excess cones in the retinal degeneration rd7 mouse, caused by the loss of function of orphan nuclear receptor Nr2e3, originate from early-born photoreceptor precursors. Hum Mol Genet 20:4102-15
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Jia, Li; Oh, Edwin C T; Ng, Lily et al. (2009) Retinoid-related orphan nuclear receptor RORbeta is an early-acting factor in rod photoreceptor development. Proc Natl Acad Sci U S A 106:17534-9