Abstract
Purpose: :
We have generated a double knockout mouse lacking both Nrl and peripherin/Rds (Nrl–/–/Rds–/–) to determine the role of Rds in cone outer segment (OS) morphogenesis. In the absence of Rds, cones develop atypical OS structures and are viable, unlike rods. Such OSs consist of dysmorphic membranous structures devoid of lamellae. Whereas the organization of the Nrl–/– retina is laden with rosettes, the Nrl–/–/Rds–/– retina displays a more undulating morphology with very few rosettes. Our goal is to assess the effect of Rds ablation on the retinal transcriptome to ascertain what molecules support such a novel cone OS morphology.
Methods: :
Retinal RNA from WT, Nrl–/–, Rds–/–, and Nrl–/–/Rds–/– mice at P21 was used to hybridize Affymetrix 430_2.0 GeneChips. Hybridization was repeated with three biological replicates, and analyzed with GC–RMA normalization with a stringent false–discovery rate. Microarray data was confirmed with qRT–PCR and protein levels/localization were assessed using Western blot analysis and immunohistochemistry. The levels of the visual chromophore and other retinoid intermediates were assessed by HPLC.
Results: :
Expression levels of cone–specific phototransduction genes were nearly equal between Nrl–/– and Nrl–/–/Rds–/– retinas. Interestingly expression of genes involved in the retinoid recycling pathway (LRAT, RPE65, RDH5, and RGR) were significantly downregulated in Nrl–/–/Rds–/– retina in comparison to Nrl–/– retina. A corresponding decrease in the levels of retinoids was also detected by HPLC. Several members of the α–, ß–, and Δ–crystallin families also demonstrated lower levels of expression in the Nrl–/–/Rds–/–. Additionally, CRB1 and several other genes involved in cell adhesion, microtubule dynamics, and kinetechore association were upregulated in Nrl–/–/Rds–/– retina as compared to Nrl–/– retina.
Conclusions: :
A number of genes are differentially expressed after the loss of the structural protein Rds in cones, suggesting a feedback signaling pathway from the OS to the nucleus in response to the lack of lamellae formation. These data also imply that decreased retinoids may contribute to the reduction in the functional sensitivity observed in the Nrl–/–/Rds–/–. Furthermore, the upregulation of CRB1 in the Nrl–/–/Rds–/– may explain the enhanced preservation of the outer limiting membrane that results in a significant reduction of rosettes. Finally, as no known degenerative pathways were identified in the Nrl–/–/Rds–/–, these data support a generic therapeutic strategy for the cone–specific silencing of Rds associated with human cone dystrophies.
Keywords: photoreceptors • retinoids/retinoid binding proteins • gene microarray