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Steve The Luan Huynh, David M Sherry, Preethi Gunaratne, Deborah C Otteson; Reelin and Synaptic Genes are Enriched in GCL Transcriptome during Early Inner Plexiform Layer Development. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Successful vision restoration by retinal ganglion cell regeneration will require appropriate re-expression of genes required for synaptic connectivity. To determine expression of genes involved in early development of retinal circuitry, the retinal ganglion cell layer (GCL) was isolated by laser-capture microdissection (LCM) for whole transcriptome sequencing.
The GCL was isolated using LCM on C57Bl/6 mice, postnatal day 2 (P2) and total RNA pooled (n=4). Whole retinal RNA was isolated without LCM (n=2). cDNA libraries (100ng/sample) were sequenced (Illumina). Genes with fragments/kilobase of transcript/million mapped reads (FPKM) scores >0.75 and GCL vs. whole retina fold-change (FC) enrichment >2 were clustered using DAVID bioinformatics. Cryosections of C57Bl/6 retina (P2, P4, P7, P30) were immunostained for reelin (RELN) and protein kinase C, and imaged with confocal microscopy.
Of 12464 genes detected in the GCL, 2119 were enriched in GCL vs. whole retina (false discovery rate <0.05). Highly enriched gene ontology terms included synapses, cell junctions, and neuronal projections. GCL-enriched neurotransmitter receptor genes included nicotinic cholinergic [Chrna3, a4, a6, a7, b2, b3, g] and glutamate receptors [ionotropic: Gria1-4 (AMPA), Grik2, 5 (kainate), Grin1, 2a, 2b, 2d and 3a (NMDA); metabotropic: Grm1, 2, 3, 4, 5, 8]. Chrng and Grin2b (FC=18.21, 11.45 respectively) were among the 100 most highly expressed GCL genes. Reln was highly expressed and enriched in GCL (FPKM 131.2, FC 10.2). Genes encoding RELN interacting proteins were expressed (Dab1, Vldlr, Lrp8) or enriched (Dab2, Itga3) in the GCL. RELN was detected throughout the inner plexiform layer (IPL) at P2 but was restricted to the ON-sublamina by P30, with some labeling in cells of the inner nuclear layer and GCL at P7 and P30.
Enrichment of genes involved in synaptic activity in the GCL is consistent with the initiation of IPL connectivity at P2. Cholinergic receptor expression in the GCL corresponds to the onset of spontaneous cholinergic retinal waves at P0. Enrichment of glutamate receptor expression in the GCL occurred well before the initiation of glutamatergic waves at P9 or light-driven activity. Reln and Dab2 enrichment and expression of RELN-interacting genes in the GCL, with IPL localization of RELN in the ON-sublamina suggest a potential role in establishing the ON pathway.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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