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Polina Lyuboslavsky, S. Anna Sargsyan, P. Michael Iuvone; Daily oscillation of EGR-1 expression in wild type and dopamine D4 receptor-knock-out mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2252.
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Early-growth response-1 (EGR-1) is a transcription factor, which controls neuronal cell differentiation, eye development, and influences the expression of circadian clock genes. This study was conducted to determine if EGR-1 expression is rhythmic in the retina of C3H/f+/+ mice, a strain of mouse with a wild type rd allele. Dopamine and dopamine D4 receptors (D4Rs) have been implicated in circadian regulation of retinal functions. To investigate the possible role of D4Rs in the regulation of EGR-1 expression, we examined the daily expression pattern of Egr1 transcript in retinas of D4R knock-out (Drd4-/ -) mice on the C3H/f+/+ background.
Mice were housed on a 12 h light / 12 h dark cycle. For Western blotting, retinas were lysed, centrifuged, and supernatant fraction proteins separated on 10% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes and probed with either primary mouse anti-EGR-1 IgG antibody (Sigma) or mouse anti-actin IgG antibody (Sigma), and the secondary horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Santa Cruz Biotechnology). Blots were developed using enhanced chemiluminescence (ECL) The ImageJ gel analysis tool was utilized to obtain band density values for EGR-1 and actin. Quantitative real-time PCR (qRT-PCR) was performed on cDNA generated from total retinal RNA or total RNA from retinal layers isolated by laser capture microdissection. The relative level of expression of Egr1 was measured from cycle threshold (Ct) values for Egr1 and normalization controls Hprt and 18S rRNA using the 2-(ΔΔCt) method.
Diurnal expression of Egr1 mRNA was detected in C3H/f+/+ wild type mice with a peak in the early dark phase of the light-dark cycle. Using laser capture microdissection to isolate retinal layers, rhythmic Egr1 expression was observed in the photoreceptor layer. EGR-1 protein also showed a daily rhythm with a peak at night. A daily rhythm of Egr1 expression, similar to that in wild type mice, was observed in retinas of Drd4-/- mice.
There is a daily rhythm of EGR-1 in retinal photoreceptors of C3H/f+/+ mice maintained on a light-dark cycle. Our results suggest that this rhythm does not depend on D4Rs.
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