Exorh protein is expressed without a significant rhythm (A, B) Embryos were injected with (A) control or (B) exorh atg MO, fixed at 64 hpf, and processed for fluorescent whole mount immunostaining with the anti-bovine Rhodopsin antibody 4D2. (A) Control embryos have robust fluorescent signal in the pineal organ that is (B) severely reduced in Exorh depleted embryos. (C-C′) Embryos injected with beta-galactosidase mRNA have undetectable levels of immunoreactivity with the 4D2 antibody at 8 hpf. (D-D′) In contrast, embryos injected with exorh mRNA show strong antibody staining at 8 hpf. (E, F) Embryos were fixed in a circadian time course and then processed for 4D2 antibody staining. One-way Analysis of Variance (ANOVA) and Tukey′s analysis revealed no significant changes in pineal Exorh protein levels that followed a daily rhythm (ne9 embryos per time point). However, a few time points were significantly different (p ≤ 0.05) from each other including 72 and 108 hpf, 72 and 116 hpf, 76 and 116 hpf, and 80 and 116 hpf. (A-B, and E) are dorsal views, anterior to the top and (C-D′) are lateral views. (C′) and (D′) are higher magnification images of the regions boxed in (C) and (D), respectively. Scale bars = 20 μm for (A-B, C′, D′, E) and 100 μm for (C, D).
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