Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals

Abstract

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. We have found that the two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high level in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead us to propose that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock.

Figure 1

Northern blot analysis of mCry1 and mCry2 mRNAs in various tissues. Mouse multiple tissue Northern blots (CLONTECH) were hybridized to random-primed ³²P-labeled probes.

Figure 2

Expression of mCry1 and mCry2 in mouse retina. In situ hybridization was performed with ³⁵S-labeled antisense RNA of the appropriate genes. (A) Dark-field microgram showing uniform expression of both mCry1 and mCry2 in the GCL and INL from macular (M) region to peripheral retina. Silver grains detected in the outer segment (OS) are due to absorption of light by residual pigment epithelium that remained in these retinal preparations. (Bar = 200 μm.) (B) Bright-field microgram. The mRNA locations of CRY1, CRY2, and opsin are compared. Note that mCry1 and mCry2 are expressed in the GCL and INL only. Arrows indicate clusters of ganglion cells that express Cry1 and Cry2. In contrast, opsin is highly expressed in the inner segment (IS) and the outer nuclear layer (ONL) and is not detected in GCL and INL. The other layers of the retina are also indicated as follows: IPL, inner plexiform layer; OPL, outer plexiform layer. (Bar = 30 μm.)

Figure 3

Distribution of mCry1 and mCry2 in the mouse brain. Mouse coronal brain sections (made at zeitgeber time = ZT6) passing through the SCN were probed with mCry1 and mCry2 antisense RNA. Both Cry1 and Cry2 are abundant in all cerebral cortical layers but are particularly abundant in the pyramidal cell layer of the hippocampus (H), the granular cell layer of dentate gyrus (DG), and the pyramidal cell layer of the piriform cortex (PFC). Note that the strongest signal of Cry1 was observed in the SCN where the Cry2 signal is marginal. (Bar = 1 mm.)

Figure 4

Circadian expression of Cry1 and Cry2 mRNA in the mouse SCN and retina. Mice kept under a 12-hr light/12-he dark cycle were sacrificed every 4 hr, and retinal sections and coronal brain sections encompassing the SCN were prepared for in situ hybridization using ³⁵S-labeled mCry1 and mCry2 RNA probes. Only the results of mCry1 hybridization to SCN are shown because the SCN signal with mCry2 was very weak at all Zeitgeber time (ZT) points and not amenable to quantitative analysis. (A) Expression of mCry1 in SCN. In situ hybridization of mCry1 with coronal brain sections passing through the SCN region are shown. Seven sections (20 μm) were made for each point and the one with the strongest signal for each point are shown in these micrograms. The double arrows indicate the SCN. (B) Expression of mCry1 and mCry2 in the retina. Retinal sections from the central part of the retina were hybridized with mCry1 and mCry2. The GCL and the INL are indicated. (C) Quantitative analyses of Cryptochrome expression in the SCN and the retina as a function of light/dark cycles (34). Grain density from autoradiograms of all section containing SCN were measured and the total grain density for each time point is expressed relative to zeitgeber time (ZT) = 0 value, which represents the values just before turning on the light (at 0800 hr). For retina, for each time point, the grain density was measured in the GCL and INL of the entire retinal region present in the autoradiogram and normalized for the length of the retina present in each section. The intensity for mCry1 and mCry2 are expressed relative to the values of mCry1 and mCry2 at ZT = 0, respectively. In absolute terms, the grain density with mCry2 probe was about 3-fold higher than that obtained with the mCry1 probe at all times.