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, of synaptogenesis, of ganglion cell diversity and axonal migration (optic disc formation). Results
 1. Epigenetics regulation of neurogenesis in the retina
 Chromatin remodeling is a cell intrinsic molecular mechanism that affects gene expression. DNA methylation is suggested to be actively involved in establishing retinal progenitor cell competence (e.g., hypometylation modulate IRBP gene activation during photoreceptor genesis). However, little is known about the influence of methylation on retinal cell fate. A family of three known DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, partially cooperate to establish and maintain genomic DNA methylation patterns. We have investigated the expression of Dnmt1, Dnmt3a, and Dnmt3b in the developing and adult mouse retina. All three Dnmt genes are highly expressed in the embryonic retina and downregulate after birth. They are differentially distributed in retinal progenitors and post-mitotic retinal neurons and display distinct subcellular localization. Robust nuclear localization of DNMTs in cones compared to rods suggests a potential role of DNA methylation in cell-specific remodeling of chromatin in these two specialized neurons and a potential role in the establishment and maturation of photoreceptor cell phenotypes in mouse. To investigate the role of DNA methylation in retinal differentiation, retina-specific conditional knockout mice were generated in which Dnmt1, Dnmt3a, or Dnmt3b flanked by loxP sites are ablated at subsequent stages of retinal development by either Six3-Cre, Rx-Cre, or Crx-Cre transgenes. Dnmt1 2lox/Rx-Cre mice showed major abnormalities in the retinal pigment epithelium (RPE) and outer segment biogenesis, in addition to alteration in the inner retina. S-cone differentiation was also severely affected. We are exploring the role of RPE-mediated hedgehog signaling in controlling photoreceptor maturation and function. 2. Establishment of photoreceptor cell polarity
 Prickle (Pk)1 and Pk2 are tissue polarity genes that are necessary for the establishment of planar cell polarity (PCP) in Drosophila. We hypothesize that the two genes play essential roles in initiation and maintenance of photoreceptor polarity in mammals. We observed expression of Pk1 and Pk2 at different stages of retinal development by in situ hybridization and immunohistochemistry. When we knocked down Pk1 by in vivo electroporation, photoreceptor migration and proliferation were abnormal, suggesting that interfering with polarity genes can change photoreceptor plasticity. To further investigate the role of Pk1 and Pk2 in photoreceptor development and homeostasis, we are generating conditional Pk1 knockout and Pk2 conditional knockdown mice. 3. Molecular mechanisms of ciliogenesis Knockdown in zebrafish of the membrane protein Reep6, involved in intracellular membrane trafficking, affected eye development. Fluorescently tagged Reep6 localized in the ER by live imaging and immunohistochemistry in zebrafish and/or mouse. The role of Reep6 in neurite outgrowth has been demonstrated in hippocampal neurons. Over expression in zebrafish or in vivo electroporation in mice of Reep6 reduced neurite outgrowth whereas shRNAi against it increased neurite length and branching. To further investigate Reep6 gene function, Reep6-knockout mice have been generated and genetic crosses are underway to obtain homozygous animals. The possible role of Reep6 in rhodopsin transport in photoreceptors is being explored in collaboration with Dr. Rodriguez-Boulan (Cornell University Medical School), who did his sabbatical with us at NIH during this year. 4. 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 investigated Fz8 retinal expression and phenotyped Fz8 mutant. We generated Fz5 and Fz8 double knockout mice to gain insight into the role of these genes in retinal neurogenesis. We showed 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. Fz receptors are also critical for the maintenance of adult axonal integrity. Significance
 Our investigations are defining the role of specific genes and pathways in establishing photoreceptor function during retinal development. Furthermore, understanding Fz5 and Fz8 function will help understand the mechanisms of micophthalmia and retinal coloboma diseases. This project complements our efforts to engineer stem cells into photoreceptors and functionally integrate them into the disrupted architecture of degenerating retinas after transplantation (also see project EY000473-02). We are also initiating 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 #
1ZIAEY000451-03
Application #
8149185
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2010
Total Cost
$2,403,660
Indirect Cost
Name
U.S. National Eye Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Ng, Lily; Lu, Ailing; Swaroop, Alok et al. (2011) Two transcription factors can direct three photoreceptor outcomes from rod precursor cells in mouse retinal development. J Neurosci 31:11118-25
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
Ghosh, Amiya K; Murga-Zamalloa, Carlos A; Chan, Lansze et al. (2010) Human retinopathy-associated ciliary protein retinitis pigmentosa GTPase regulator mediates cilia-dependent vertebrate development. Hum Mol Genet 19:90-8
Hackler Jr, Laszlo; Wan, Jun; Swaroop, Anand et al. (2010) MicroRNA profile of the developing mouse retina. Invest Ophthalmol Vis Sci 51:1823-31
Swaroop, Anand; Kim, Douglas; Forrest, Douglas (2010) Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina. Nat Rev Neurosci 11:563-76
Hurd, Toby; Zhou, Weibin; Jenkins, Paul et al. (2010) The retinitis pigmentosa protein RP2 interacts with polycystin 2 and regulates cilia-mediated vertebrate development. Hum Mol Genet 19:4330-44
Parapuram, Sunil K; Cojocaru, Radu I; Chang, Jessica R et al. (2010) Distinct signature of altered homeostasis in aging rod photoreceptors: implications for retinal diseases. PLoS One 5:e13885
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