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Sem Genini, Kristin Gardiner, Gustavo Aguirre; Expression and localization of cell cycle and proliferation genes in early retinal degeneration models. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5951.
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© ARVO (1962-2015); The Authors (2016-present)
Terminally differentiated photoreceptors (PR) in the erd-model undergo concurrent apoptosis or sustained proliferation generating new hybrid rod/S-cones (Berta et al. 2011). We now analyzed the expression of selected genes involved in cell cycle regulation (CCNA1, CCNA2, CCND1), eye and PR development/maintenance (GRK1, NDR1, NRL, NR2E3, PAX6, PCNA, RBP3, RCVRN, RDS), or belonging to the Hippo signaling pathway (LATS1, LATS2, MOB1A) to examine proliferation pathways in dogs affected with rcd1 (PDE6B), xlpra2 (RPGRORF15), and erd (STK38L).
qRT-PCR comparisons between mutant and normal retinas (3/group/age) were performed at disease-relevant ages (3, 5, 6-10, and 12-16 wks). The ddCt method using GAPDH for normalization and an unpaired t-test (p<0.05 and fold change >1.5) were applied to identify differentially expressed (DE) genes. Immunohistochemistry (IHC) was used to localize a subset of proteins to expand the mRNA data.
The results showed up-regulation in all 3 diseases of CCNA2 at 7 wks, and CCNA1 and NDR1 at 16 wks. Disease-specific at the later age was up-regulation of PCNA and the Hippo pathway genes LATS1 and MOB1A only in erd, and up-regulation of PAX6 and down-regulation of RBP3 in rcd1. IHC results demonstrated that LATS1 was highly expressed in PR outer segments and co-localized with both RHO and OS2 in erd-mutants, but not in other retinas. Using the mitosis marker phospho-histone H3 (PHH3) we identified proliferating cells in the outer nuclear layer (ONL) in all 3 diseases to varying extents up to 20 wks, but not in normals after 2 wks. Double-immunolabeling of PHH3 with RHO indicated that the proliferating cells were PR and, using CD18 and glutamine synthetase, excluded that they were either microglia/macrophages or Müller cells.
Our results indicate minimal gene expression differences at disease onset (3 wks) and early phases (5 wks) of degeneration. At 7 and 16 wks, an increased number of DE genes was observed, including those that were the same for the 3 mutants. These data suggest that early PR proliferation is a common feature of all 3 disease models, with concurrent dysregulation of critical cell cycle, Hippo pathway, and PR function and cell fate specification genes.
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