Abstract
Purpose::
MicroRNAs (miRNAs) are small, highly conserved molecules which regulate the expression of genes by binding to the 3'-untranslated regions of specific mRNAs. Each miRNA is thought to regulate multiple genes. Dicer, an RNase III endonuclease, is essential for the production and function of mature miRNAs. As miRNAs play essential roles in tissue morphogenesis, ablation of Dicer in mice results in early embryonic death. Here, a conditional KO of Dicer has been created, with enzyme selective inactivation in the retina. We analyze the effects of Dicer removal on the fine structure and synaptic architecture of the retina. This is the first example of miRNA inactivation in an area of the mouse CNS.
Methods::
Eyes of Dicer flox/Dicer flox;chx10-Cre mice (aged 16 days to 11 months) were fixed in 4% paraformaldehyde, infiltrated with sucrose, frozen at -20°C and serially sectioned at a cryostat. Sections were processed by multiple fluorescence immunocytochemistry with cell type-specific antibodies and examined with a Leica spectral confocal microscope. We studied photoreceptors, rod and cone bipolar cells, horizontal cells, cholinergic amacrine cells and Müller glia. We also studied retinal connections with antibodies to synaptic proteins.
Results::
All the expected cell types are represented in the Dicer KO retinas. However, as early as P16, retinal architecture appears altered by numerous rosettes. These are spherical structures initially comprising only inward-pointing photoreceptors and OPL. Later, bipolar and horizontal cells also become displaced toward the rosettes, apparently pulled by the dislocating photoreceptors. Bipolar and horizontal cells sprout profusely toward the outer retina. Concomitantly, synaptic proteins become mislocated up to the proximity of outer limiting membrane. Müller cells overexpress GFAP and participate to the rosette structure. The severity of the phenotype increases with time, ultimately resulting into a retinal degeneration, in which photoreceptors are extensively lost and bipolar cells show abnormal morphology and staining properties.
Conclusions::
This study strongly suggests that Dicer is required for the appropriate retinal layering in the late stage of development. Also, the activity of this enzyme seems necessary for the maintenance of retinal homeostasis throughout life.
Keywords: gene/expression • retina: distal (photoreceptors, horizontal cells, bipolar cells) • retinal connections, networks, circuitry