Purpose: Nocturnal rhythms of gene expression in the retina are known to be both darkness- and circadian clock-dependent, but their role and cellular location are not well defined. In the present study we have used a new transgenic rat model (early growth response gene 1-destablized, enhanced green fluorescent protein 2; egr-1-d2EGFP) to investigate the rhythmic regulation of darkness-related gene expression.
Methods: Adult transgenic rats were sampled during the light and dark phases of a standard laboratory lighting schedule. The cellular location of transgene expression in retinal sections was detected either via immunohistochemistry for green fluorescent protein (GFP) or via direct microscopy. The GFP expression pattern was compared to endogenous proteins (Egr-1, melanopsin, rhodopsin) via dual fluorophore immunohistochemistry. Day-night changes in GFP and Egr-1 expression were quantified by western blot analysis of retinal protein extracts.
Results: Nocturnal transgene expression was abundant in the outer nuclear layer (ONL) of the retina, recapitulating expression of the endogenous Egr-1 protein. The transgene provided greatly enhanced visualization of the ONL cellular expression pattern, in part due to cellular filling by GFP molecules that pervade rod photoreceptor cells including inner and outer segments. The transgene was also expressed in isolated (Egr-1-positive) cells of the inner nuclear layer and the ganglion cell layer. In the ONL, a marked day-night rhythm in transgene expression was found to be predominantly within an inner zone of this retinal nuclear layer. This concentration of rhythmic GFP/Egr-1 to the inner ONL was not associated with differential localization of rhodopsin.
Conclusions: Analysis of a novel transgenic rat strain has identified subpopulations of rod photoreceptor cells that differ with respect to rhythmic nocturnal expression of egr-1. These studies demonstrate the value of this genetic approach that has provided a model for the functional characterization of retinal rhythms, specifically addressing the role of Egr-1 within nocturnal transcriptional events in a rod photoreceptor population. Because the darkness-dependent induction of Egr-1 is gated by a circadian clock, this model can also provide insights into the cellular mechanisms of circadian regulation in the retina.