Night/day changes in pineal expression of >600 genes: central role of adrenergic/cAMP signaling

Abstract

The pineal gland plays an essential role in vertebrate chronobiology by converting time into a hormonal signal, melatonin, which is always elevated at night. Here we have analyzed the rodent pineal transcriptome using Affymetrix GeneChip(R) technology to obtain a more complete description of pineal cell biology. The effort revealed that 604 genes (1,268 probe sets) with Entrez Gene identifiers are differentially expressed greater than 2-fold between midnight and mid-day (false discovery rate <0.20). Expression is greater at night in approximately 70%. These findings were supported by the results of radiochemical in situ hybridization histology and quantitative real time-PCR studies. We also found that the regulatory mechanism controlling the night/day changes in the expression of most genes involves norepinephrine-cyclic AMP signaling. Comparison of the pineal gene expression profile with that in other tissues identified 334 genes (496 probe sets) that are expressed greater than 8-fold higher in the pineal gland relative to other tissues. Of these genes, 17% are expressed at similar levels in the retina, consistent with a common evolutionary origin of these tissues. Functional categorization of the highly expressed and/or night/day differentially expressed genes identified clusters that are markers of specialized functions, including the immune/inflammation response, melatonin synthesis, photodetection, thyroid hormone signaling, and diverse aspects of cellular signaling and cell biology. These studies produce a paradigm shift in our understanding of the 24-h dynamics of the pineal gland from one focused on melatonin synthesis to one including many cellular processes.

FIGURE 1.

Pineal rEx versus retina rEx. This figure demonstrates that a subset of genes is predominantly expressed in the pineal gland or retina or both. Data are based on results obtained in experiment C. The probe sets for the entire microarray are represented by 5% density contours (95% of probe sets are within the outermost contour line). Only symbols representing probe sets with rEx values greater than 8 for either the pineal gland or retina (∼1% of 31,099 probe sets on the Rat230_2 microarray) are shown; the remaining probe sets are not highly expressed in either tissue relative to other tissues. The plotted probe sets fall into three sectors as follows: Pineal, in which 218 genes (256 probe sets) are highly expressed primarily in the pineal gland; Pineal, Retina, in which 55 genes (63 probe sets) are highly expressed at similar levels in both the pineal gland and retina; and Retina, in which 93 genes (109 probe sets) are highly expressed primarily in the retina. The genes represented by these probe sets are listed in Table 4; supplemental Table S5 contains a detailed description of these genes and other unannotated probe sets. An interactive version of this figure that identifies each symbol is available at sne.nichd.nih.gov.

FIGURE 2.

Radiochemical in situ hybridization images. Each panel contains autoradiographs prepared from sections of rat brains through the pineal gland. The sections on the left are from animals killed during the day and those on the right are from animals killed during the night. The sections were incubated with antisense probes identified in the bottom left-hand corner of the Day image. Probes are detailed in supplemental Table S1. The results of quantitation of the signal strength of the pineal labeling appear in Table 5. For further details see “Experimental Procedures.” Hab, habenula; ic, inferior colliculus; mhn, medial habenular nucleus; Raphe, dorsal raphe nucleus; sc, superior colliculus. These figures are available in high resolution at sne.nichd.nih.gov.

FIGURE 3.

Staining of pinealocytes and endothelial cells in the pineal gland. A, image presenting Aanat-labeling of pinealocytes. Dark field photomicrograph of radiochemical in situ hybridization of a part of the superficial pineal gland of the rat with an antisense probe binding to mRNA encoding AANAT. The section was dipped in a photographic emulsion and developed after exposure; the grains are seen using dark field visualization as white dots in the emulsion. Dense labeling is seen above the pineal parenchyma with no labeling of the perivascular space (per vasc). B, same section shown in A in transmitted light. Arrows indicate the perivascular space, which is not labeled in A. Cresyl violet counterstaining is used. C, labeling of the perivascular spaces; endothelial labeling image. Dark field photomicrograph of radiochemical in situ hybridization of the superficial pineal with an antisense probe binding to mRNA encoding Esm1. Note the labeling of the perivascular spaces (arrows). D, high power photomicrograph taken in transmitted light of part of the superficial pineal showing Esm1 labeling of the perivascular cells and only few grains above the pinealocytes. Cresyl violet counterstaining. Bars, A, B and D = 20 μm. C = 50 μm. The probes used are described in supplemental Table S1.

FIGURE 4.

qRT-PCR analysis of transcripts that are night/day differentially expressed or have high rEx values or both. The lighting cycle is represented at the bottom of each column. Transcripts are identified by gene symbol. Each value is the mean ± S.E. of three determinations. Values were normalized to Actb, Gapdh, Hrpt1, and Rnr1. A single asterisk identifies statistically significant rhythmic patterns of gene expression (p < 0.01) based on log transformed raw values analyzed by one-way analysis of variance in JMP. For technical details, see “Experimental Procedures” and the supplemental material.