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Awais Zia, Jacob Nellissery, Jerome Roger, Anand Swaroop, Tiansen Li; NRL Localizes to Discrete Regions of Euchromatin in Rod Photoreceptors of Developing and Adult Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3943.
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© ARVO (1962-2015); The Authors (2016-present)
NRL (neural retina leucine zipper) is a key transcription factor that regulates rod photoreceptor cell fate and homeostasis. Mutations in NRL are associated with retinal degeneration and enhanced S cone syndrome. Previous studies reported NRL localization in human rod photoreceptors, but investigations in mice have been lacking due to technical difficulties and appropriate antibodies for use in mouse immunohistochemistry (IHC). The goal of these studies is to generate and evaluate anti-NRL antibodies and perform subcellular localization in developing and mature mouse retina.
Antibodies were raised against recombinant mouse NRL (aa23-130). IHC was performed using different processing conditions. We used wild-type (WT), Nrl-knockout and two mouse transgenic lines - one carrying a GFP reporter (Nrl-GFP) and the other carrying mNrl (Nrl-mNrl) under the control of 2.5 kb mouse Nrl promoter.
Immunoblot analysis identified a 28 kDa NRL protein in WT but not in Nrl-knockout retina. In adult WT retina, Nrl localizes to peripheral region of rod nuclei. Co-labeling of Nrl with euchromatin marker H3K4Me3 shows that Nrl associates exclusively with euchromatin. Nrl immunostaining appears as discrete puncta dispersed along the euchromatin. Double staining for Nrl with anti-RNA Polymerase II reveals significant colocalization, suggesting that Nrl resides in active transcription regions. Other photoreceptor transcription factors, including CRX, OTX2, NR2E3, and ROR-beta, also colocalize with Nrl in these actively transcribed regions. Co-labeling of Nrl with BrdU pulse in P0 retina shows that Nrl is expressed in post-mitotic cells at least 4 hrs after the BrdU injection. In the Nrl-mNrl transgenic; Nrl-knockout retina, which express Nrl only in a small percentage of cells and at lower than physiological levels, co-localization of Nrl with rhodopsin but not S-opsin, and cone arrestin suggests that Nrl-positive cells commit to rod phenotype and do not reside in a hybrid state. We also show that Nrl staining pattern largely overlaps with GFP-positive cells in Nrl-GFP retina.
NRL associates exclusively with euchromatin of rod nuclei and appears to reside in actively transcribed regions. We confirm that Nrl is expressed in post-mitotic rods and that GFP expression in Nrl-GFP mice broadly reflects native Nrl expression. We also demonstrate that Nrl expression in Nrl-knockout cone photoreceptors can re-transform these cells to acquire rod phenotype.
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