Functional basis of deep brain VAL-opsin photoreceptors in the zebrafish

Biological impacts of light beyond the vision, i.e. non-visual functions of light, signify the need to better understand light detection (or photoreception) systems in vertebrates. Photopigments, which comprise light-absorbing chromophores bound to different G-protein coupled receptor opsins, are responsible for visual and non-visual photoreception. Non-visual opsins in the retina of mammals and extra-retinal tissues of non-mammals play an important role in the non-visual functions of light, e.g. biological rhythm and seasonal reproduction. However, little is known about individual functions of non-visual opsins in extra-retinal tissues such as the deep brain of non-mammalian vertebrates including fish (reviewed in Chapter 1). This thesis focused on deep brain vertebrate ancient long (VAL)-opsins, which are encoded by the val-opsinA (valopa) and val-opsinB (valopb) isoform genes, in the zebrafish. The thesis is built upon research questions—first, which are VAL-opsin photoreceptor neurons located in the brain; second, how val-opsin genes are regulated; and third, what would be their physiological functions in the zebrafish—through which it aims to better understand the non-visual photoreception in vertebrates. The research questions are addressed in three experimental chapters. Firstly, in Chapter 2, I examined the distribution and neurochemical nature of valop isoform-expressing cells in the brain of adult zebrafish. In situ hybridization localized a major valop cell group in the thalamus (co-expressing both isoforms), and several minor valop cell groups in the midbrain or the hindbrain (single isoform); and determined their neurochemical properties. Thus, the VAL-opsin photoreceptors in different brain areas should be involved in multiple physiological functions. Secondly, in Chapter 3, I examined regulation of the valop isoform genes in zebrafish. Real-time quantitative PCR demonstrated differential regulation of the valop isoform genes, which depends on time-of-day, light conditions, and brain areas. Thus, the VAL-opsin photoreceptors are likely to mediate the control of time- and light-dependent physiology. Thirdly, in Chapter 4, I created valop mutant zebrafish to examine possible physiological functions of the VAL-opsin isoforms. Introduction of mutations to the valop genes resulted in non-functional VAL-opsins, virtually. Abnormal eggs or embryos, regardless of wild-type or mutant, resulted from F0 valop mutant breeders. The parental effects of valop knockout suggest a reproductive function of VAL-opsin photoreceptors in zebrafish. This thesis provides the functional basis of VAL-opsin photoreceptors in the zebrafish. In a broader perspective, it introduces the neuronal organization of deep brain opsin photoreceptors in adult fish and research directions for functional studies (as discussed in Chapter 5). Thus, the findings from this thesis advance our knowledge about the non-visual photoreception in vertebrates.