From blue light to clock genes in zebrafish ZEM-2S cells

Abstract

Melanopsin has been implicated in the mammalian photoentrainment by blue light. This photopigment, which maximally absorbs light at wavelengths between 470 and 480 nm depending on the species, is found in the retina of all classes of vertebrates so far studied. In mammals, melanopsin activation triggers a signaling pathway which resets the circadian clock in the suprachiasmatic nucleus (SCN). Unlike mammals, Drosophila melanogaster and Danio rerio do not rely only on their eyes to perceive light, in fact their whole body may be capable of detecting light and entraining their circadian clock. Melanopsin, teleost multiple tissue (tmt) opsin and others such as neuropsin and va-opsin, are found in the peripheral tissues of Danio rerio, however, there are limited data concerning the photopigment/s or the signaling pathway/s directly involved in light detection. Here, we demonstrate that melanopsin is a strong candidate to mediate synchronization of zebrafish cells. The deduced amino acid sequence of melanopsin, although being a vertebrate opsin, is more similar to invertebrate than vertebrate photopigments, and melanopsin photostimulation triggers the phosphoinositide pathway through activation of a G(q/11)-type G protein. We stimulated cultured ZEM-2S cells with blue light at wavelengths consistent with melanopsin maximal absorption, and evaluated the time course expression of per1b, cry1b, per2 and cry1a. Using quantitative PCR, we showed that blue light is capable of slightly modulating per1b and cry1b genes, and drastically increasing per2 and cry1a expression. Pharmacological assays indicated that per2 and cry1a responses to blue light are evoked through the activation of the phosphoinositide pathway, which crosstalks with nitric oxide (NO) and mitogen activated protein MAP kinase (MAPK) to activate the clock genes. Our results suggest that melanopsin may be important in mediating the photoresponse in Danio rerio ZEM-2S cells, and provide new insights about the modulation of clock genes in peripheral clocks.

Figure 1. Quantitative PCR of per1b, cry1b, per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 10 min, and total RNA was extracted 1, 2, 6 and 12 h after the stimulus. ‘a’ is significantly different from ‘b’ and ‘b’ is significantly different from ‘c’ (p<0.05). In this and in figures 2 to 9, values are the mean ± Standard Error of Mean (n = 4–9).

Figure 2. Quantitative PCR of per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 10 min, in the presence or absence of the PLC inhibitor, U-73122, at 100 nM, and total RNA was extracted 2 h after the stimulus. In this and the following figures, the asterisk means statistically significant differences from all other groups (p<0.05).

Figure 3. Quantitative PCR of per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 10 min, in the presence or absence of the calcium chelator, BAPTA-AM, at 1 µM, and total RNA was extracted 2 h after the stimulus.

Figure 4. Quantitative PCR of per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 10 min, in the presence or absence of the PKC inhibitor, Ro 31-8220, at 100 nM and total RNA was extracted 2 h after the stimulus.

Figure 5. Quantitative PCR of per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 10 min, in the presence or absence of the CAMK II inhibitor, KN-93, at 1 µM, and total RNA was extracted 2 h after the stimulus.

Figure 6. Quantitative PCR of per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 10 min, in the presence or absence of the NOS inhibitor, L-NAME, at 1 mM and total RNA was extracted 2 h after the stimulus.

Figure 7. Quantitative PCR of per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with the guanylyl cyclase activator, Y-C1, at 40 µM in DD, and total RNA was extracted after 2 h.

Figure 8. Quantitative PCR of per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 10 min, in the presence or absence of the MEK inhibitor, PD-98059 at 40 µM, and total RNA was extracted 2 h after the stimulus.

Figure 9. Quantitative PCR of per2 and cry1a in a Danio rerio embryonic cell line ZEM-2S.

The cells (2×10∧6) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 10 min, in the presence or absence of the adenylyl cyclase inhibitor, SQ-22536, at 20 µM, and total RNA was extracted 2 h after the stimulus.

Figure 10. Quantification of cAMP in a Danio rerio embryonic cell line ZEM-2S.

The cells (8×10∧4) were stimulated with blue light (450–475 nm, 87.85 to 95.17 µwatts/cm²) for 1, 5 or 10 min, and cAMP was measured immediately after each light pulse. Forskolin at 10 µM was used as a positive control in DD. Values are the mean ± SEM (n = 3).