Numerical data related to Fig 5.

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.

眼睛在调控小鼠与人类的昼夜节律过程中发挥关键作用。本研究聚焦于该功能在昼行性脊椎动物斑马鱼中的保守性。本研究使用缺乏视网膜神经节细胞（RGCs）的lakritz（lak）突变斑马鱼幼体开展实验，结果显示：尽管具备功能的眼睛可参与光掩蔽效应，但对于运动活性的昼夜节律建立而言，眼睛在很大程度上并非必需。此外，在昼夜时间（Circadian Time, CT）16时给予白光脉冲后，眼睛对于相位延迟的诱导并非必需；但在CT21时给予白光脉冲后，眼睛则参与相位提前的诱导。正如夜行性哺乳动物中的相关研究所示，黑视蛋白是光同步化过程的重要介导因子。斑马鱼的黑视蛋白基因之一opn4xa，不仅在视网膜神经节细胞（RGCs）中表达，也在松果体的光敏投射神经元中表达。与以光照关闭模式发挥功能的其他投射神经元不同，松果体中opn4xa阳性的投射神经元以光照开启模式行使功能。本研究构建了opn4xa突变体，该突变体的松果体光照开启响应受到损伤。该突变对光掩蔽效应、运动活性的昼夜节律以及相位移位的诱导均无影响，但当幼体处于持续光照条件下时，会轻微改变其节律周期长度。最后，对opn4xa;lak双突变斑马鱼幼体的分析显示，在光脉冲后的相位移位调控中，眼睛与松果体opn4xa的功能并不存在冗余。本研究结果支持以下观点：眼睛并非光信号影响运动活性昼夜节律的唯一介导因子，同时也凸显了不同动物模型在昼夜节律系统与行为光同步化方面存在的差异。