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Hélène M Léger, Daniel P. Beiting, William A Beltran, Gustavo D Aguirre, Francis Luca; NDR kinases regulate retinal development, homeostasis and gene expression. Invest. Ophthalmol. Vis. Sci. 2017;58(8):353.
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© ARVO (1962-2015); The Authors (2016-present)
Recent studies suggest that NDR2 protein kinase is important for retina maintenance, although its precise retinal functions are unknown. Notably, an Ndr2 loss-of-function allele causes early retina degeneration (erd) in dogs, characterized by opsin mislocalization and concurrent increases in photoreceptor apoptosis and proliferation. Separate studies indicate that NDR2 and its paralog NDR1 regulate cell proliferation, morphogenesis and gene expression in other tissues. To address how NDR1 and NDR2 regulate retina development and homeostasis, we generated Ndr1 and Ndr2 knock-out (KO) mice and characterized their retinal phenotypes.
Ndr1 KO mice were generated by introducing frame shift mutations into Ndr1 exons 4 and 6, utilizing CRISPR-Cas9 methods. Ndr2 KO mice were obtained by crossing Ndr2-floxed mice (Ndr2 exon 7 flanked by loxP sites; obtained from KOMP, UC Davis) to Actin-cre mice. To analyze retinal phenotypes, eyes were fixed in 4% paraformaldehyde, OCT embedded, sectioned and probed with antibodies to opsin and mitotic markers via immunofluorescence microscopy. To investigate how Ndr2 deletion affects gene expression, cDNA was prepared from triplicate pools of Ndr2 KO and WT retinas from ~1 month old mice and analyzed by RNA-seq. Data normalization and differential gene expression analysis was performed Limma packages (Bioconductor). Transcriptome analysis was performed using Gene Ontology Consortium and Database for Annotation, Visualization and Integrated Discovery (DAVID).
Both Ndr1 KO and Ndr2 KO mice display similar retinal degenerative phenotypes as canine erd, including opsin mislocalization to the ONL. In addition, Ndr KO mouse retinas exhibit increased cell proliferation in the INL (Ndr1 and Ndr2), ONL (Ndr1) and neuroblast (Ndr2). RNA-seq analysis reveals that Ndr2 deletion increases expression of numerous genes associated with neuronal stress and decreases expression of many genes involved in synapse organization.
These data support the hypotheses that retinal NDR1 and NDR2: a) function similarly and b) regulate retinal development and homeostasis via modulating gene expression. Further analyses of retinal NDR mechanisms may influence the development of therapeutic interventions for retinal degenerations and methods to stimulate photoreceptor regeneration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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