Contribution of the eye and of opn4xa function to circadian photoentrainment in the diurnal zebrafish.

The eye is instrumental for controlling circadian rhythms in mice and human. Here, we address the conservation of this function in the zebrafish, a diurnal vertebrate. Using lakritz (lak) mutant larvae, which lack retinal ganglion cells (RGCs), we show that while a functional eye contributes to masking, it is largely dispensable for the establishment of circadian rhythms of locomotor activity. Furthermore, the eye is dispensable for the induction of a phase delay following a pulse of white light at CT 16 but contributes to the induction of a phase advance upon a pulse of white light at CT21. Melanopsin photopigments are important mediators of photoentrainment, as shown in nocturnal mammals. One of the zebrafish melanopsin genes, opn4xa, is expressed in RGCs but also in photosensitive projection neurons in the pineal gland. Pineal opn4xa+ projection neurons function in a LIGHT ON manner in contrast to other projection neurons which function in a LIGHT OFF mode. We generated an opn4xa mutant in which the pineal LIGHT ON response is impaired. This mutation has no effect on masking and circadian rhythms of locomotor activity, or for the induction of phase shifts, but slightly modifies period length when larvae are subjected to constant light. Finally, analysis of opn4xa;lak double mutant larvae did not reveal redundancy between the function of the eye and opn4xa in the pineal for the control of phase shifts after light pulses. Our results support the idea that the eye is not the sole mediator of light influences on circadian rhythms of locomotor activity and highlight differences in the circadian system and photoentrainment of behaviour between different animal models.

Functional heterogeneity in the pineal projection neurons of zebrafish.

The zebrafish pineal organ is a photoreceptive structure containing two main neuronal populations (photoreceptors and projections neurons). Here we describe a subpopulation of projection neurons that expresses the melanopsin gene, opn4xa. This new pineal cell type, that displays characteristics of both projection neurons and photoreceptors, share a similar dependency for BMP and Notch signalling pathways with classical non-photosensitive projection neurons (PN). Functionally, however, whereas classical, opn4xa-negative PNs display an achromatic LIGHT OFF response, the novel cell type we describe exhibit a LIGHT ON character that is elicited by green and blue light. Taken together, our data suggest a previously unanticipated heterogeneity in the projection neuron population in the zebrafish pineal organ raising the question of the importance of these differences in pineal function.