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Masaya Fukushima, Toshiro Iwagawa, Makoto Aihara, Sumiko Watanabe; Fbxl11 is indispensable for rod photoreceptor development. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3843.
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© ARVO (1962-2015); The Authors (2016-present)
Epigenetic regulation of gene expression is of critical importance in retinal development. Fbxl11 is a histone demethylase of dimethylated and monomethylated histone H3 at lysine 36. The purpose of our research is to investigate the function of Fbxl11 in retinal development.
Retina-specific Fbxl11 knockout mice were generated by mating Dkk3-Cre males with Fbxl11flox females. The retinas of the Fbxl11 conditional knockout (Fbxl11-CKO) mice and its wildtype (WT) control were analyzed by immunohistochemistry (IHC), qPCR, and RNA-seq in multiple developmental stages. The RNA-seq data were utilized for differential expression analysis, gene ontology (GO) analysis, and alternative splicing analysis.
In WT retinas, Fbxl11 was expressed ubiquitously at E14.5 and P1, while its expression was confined in inner nuclear layer at P7 and in outer nuclear layer (ONL) at P17. Fbxl11-CKO retinas showed pseudorosette formation in ONL at P7 and markedly thinner ONL at P17. Transcriptomic analysis at P7 using RNA-seq detected 1066 upregulated and 1259 downregulated genes in Fbxl11-CKO retinas, respectively (q < 0.05). GO analysis performed with these gene sets found enriched terms including cell cycle, mitosis, and alternative splicing for the upregulated genes, and vision for downregulated genes, respectively (q < 0.001). Differential alternative splicing event analysis detected increase of intron retention in as many as 1002 genes in Fbxl11-CKO retinas (q < 0.05). IHC at P1 revealed Fbxl11-CKO retinas had thinner Ki67+ layer (p = 0.0007), fewer PNR+ cells (p = 0.006), and increased AC3+ cells (p = 0.006), while the number of Hu+ cells and Brn3b+ cells did not change. AC3+ cells in Fbxl11-CKO retinas partially overlapped with Ki67+ and PNR+ cells. mRNA expression of rod photoreceptor genes including Otx2, Crx, and Nrl were significantly downregulated in Fbxl11-CKO retinas at P1 (p < 0.05), whereas expression of cone photoreceptor gene Rxrg and progenitor genes Hes1, Lhx2, and Rax did not change.
Fbxl11-CKO retinas had already manifested developmental abnormality at P1, predominantly in rod photoreceptors. Our results suggest Fbxl11 plays an indispensable role in photoreceptor differentiation or its maintenance.Further research on genomic Fbxl11 binding sites and histone methylation status will be necessary to fully understand the function of Fbxl11 in retinal development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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