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Hannah Fann, Dustin Thad Whitaker, Matthew Brooks, Anand Swaroop; Genetic influences on the nuclear architecture of photoreceptors. Invest. Ophthalmol. Vis. Sci. 2017;58(8):343.
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© ARVO (1962-2015); The Authors (2016-present)
Each cell has an unambiguously organized nuclear architecture involving the two major types of chromatin, heterochromatin and euchromatin. Only rod photoreceptors from nocturnal animals have an “inverted” pattern that features a single, large heterochromatin chromocenter localized to the nuclear interior. Every other cell type, including the closely related cone photoreceptors, exhibit a “conventional” nuclei pattern that features several heterochromatin chromocenters scattered throughout the nuclei. This study investigates genes that may play a role in regulating these nuclear architecture patterns in photoreceptor cells.
An expansive genetic screen was performed to identify abnormal phenotypes concerning nuclear architecture within rod photoreceptors. A candidate list of 720 genes was established to have differential expression between rod and cone photoreceptors, as determined by RNA-sequencing transcriptional profiling data. Selected genes from the target list were knocked down through in vivo electroporation in native retinas using shRNA-mediated gene silencing with a rod-specific fluorescent reporter in postnatal day 0 mice. After maturation, mounted retinas were stained with DAPI and imaged with a Zeiss 700 confocal microscope in order to visualize any alterations present in rod nuclei. Additionally, DNA fluorescence in situ hybridization against a major satellite repeat region of the genome was used to quantify the number of chromocenters within each nuclei.
Approximately 2.5% of the genes from the screen altered the physical characteristics of rod nuclei architecture. Knockdown of these genes increased the number of chromocenters within the rod nuclei and increased euchromatin area distributed throughout the cell body as compared to the control groups. Confirmation that the observed phenotype can be attributed to the knockdown of these genes was achieved by analyzing multiple retinas injected with the original shRNA. Additional validation was achieved by injecting and imaging a second knockdown construct of each gene and by performing rescue experiments.
Through this extensive and ongoing screen, it has been shown that some target genes affect chromatin organization in rod photoreceptor cells, moving away from the inverted pattern. Further characterization is currently underway in order to understand the mechanistic properties of the nuclear arrangement caused by these genes.
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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