“Life without the day-night cycle: exploring photoreceptors beyond the eye" (MIUR-DAAD Joint Mobility Program)

A major goal for biology is to understand how the environment influences living systems. In the case of fish, light is known to have a major effect on many aspects of biology ranging from development and growth to sex determination, behaviour and reproduction. Importantly, unlike mammals most fish tissues express photoreceptors and thereby light exposure can directly affect fish physiology independently of the eye. Furthermore, recent discoveries have revealed a surprising complexity of photoreceptors in fish, although the key mechanisms that sense and respond to light remain poorly understood. In this bilateral project we aim to tackle the following basic question: What are the key elements within fish and how has their evolution been influenced by prevailing lighting conditions? We will use a comparative study involving two fish models: i) the zebrafish, Danio rerio representing a “normal” fish, being exposed to the day night cycle and offering a wealth of tools for detailed genetic analysis and ii) the Somalian blind cavefish, Phreatichthys andruzzii, a species that has evolved for millions of years in complete darkness and shows striking adaptations linked with loss of light perception. Notably, this includes complete eye degeneration, loss of entrainment of the circadian clock by white light as well as pseudogenization of many genes involved in photoreception (1,2). Thus effectively, this cavefish represents a “natural mutant” for photoreception. We aim to apply a multidisciplinary approach including a combination of genomic, cellular and behavioural tools that will rely on a close interaction and exchange of personnel between the Italian (I) and German (DE) laboratories: i) We will compare the behaviour controlled by non-visual photoreceptors of P.andruzzii (I) and zebrafish (DE) to provide direct insight into the function of extraocular photoreceptors. Different behavioural activities (activity rhythms, phototactic response, visual motor response) will be recorded in adults and larval stages of both species under different lighting conditions (characterized by duration, irradiance, and wavelength). This will allow us to compare how short and long-term behavioural responses to light have adapted during evolution under the extreme conditions of total darkness. ii) We will identify loss of function mutations in genes involved in photoreception. Specifically we will use bioinformatics tools to compare our P. andruzzii transcriptomes (Heart, Fin, Brain and Fibroblasts) with counterpart transcriptomes from zebrafish (DE and I) to identify genes involved in the phototransduction cascade, with a special focus on opsin genes. iii) We will establish cavefish and zebrafish-derived cell cultures (DE) and study how different wavelengths of light trigger activation of light-regulated signaling cascades by testing the expression of endogenous genes by qPCR as well as transfected light responsive promoter, luciferase reporter constructs (Per2, Cry5, Cry1a). iv) By ectopically expressing the normal zebrafish counterparts of our candidate photoreception genes identified in (ii) in our cavefish cell lines we will test for rescue of normal peripheral tissue photoreception (as assayed in point iii) (I). (1) Cavallari et al. (2011) PLoS Biology,9, e1001142 (2) Calderoni et al. (2016) Heredity (Edinb). 117(5):383-392