Functional and Genomic Study of the Novel (Orphan) Opsin Profile of the Zebrafish

The Retina is a thin layer of interconnected nerve cells that covers the inside surface of the back of the eyeball. The whole process of Vision itself begins with a small group of proteins (known as opsins) which perform the unique task of absorbing light and translating it into an electrical/biological signal. In the retina, it was traditionally assumed that these opsins were confined to the rod and cone cells, known collectively as photoreceptors. These photoreceptor cells are the functional equivalent of the pixels in the CCD array of a digital camera. It has more recently been discovered that there is actually a rich diversity of additional candidate opsins produced in alternative cells in the retina, however their function remains largely unexplored. Furthermore, these same light sensitive proteins are also found in the brain in places like the pineal organ. Collectively these observations indicate that the whole process of light detection is far more complex than originally thought. Here in the current program we propose a systematic study of these potentially light sensing proteins by looking at the whole diverse opsin photopigment family in the zebrafish, a common model organism. Initially, we will insert the DNA that codes for these proteins into host cells grown in culture conditions. In this way we can measure and compare their functional properties as well as studying how they might interact with one another. This will allow us to define which of these possible photopigments can confer photosensitivity in cells which normally do not respond to light. Finally, it is very hard to measure the real function of these light sensitive proteins in the retina or brain because they are only found in relatively few cells and as a result they are simply very hard to find. We will therefore use new techniques to genetically modify zebrafish, so as to generate animals where the cells that normally produce these photopigments are modified to glow with a fluorescent marker. These approaches will help us explore and understand the whole range of new light detection mechanisms present in the eye and brain.

In this proposal we seek to place the family of zebrafish novel opsins into a functional context. It is clear that the retina of vertebrates contain a whole family of non-rod/non-cone photopigments. Structurally these proteins resemble the known opsin photopigments but their function remains largely unexplored. Thus we propose to transiently express vertebrate ancient (VA) opsin; melanopsin (Opn4); encephalopsin (Opn3); neuropsin (Opn5); and teleost multiple tissue (TMT) opsin in our established N2A cell expression system allowing us to characterise the photopigment and/or photoisomerase potential of each of these candidate photopigments. If any of the candidate opsins fails to express photopigment characteristics we will examine their expression in other host cell lines, or in the presence of promiscuous G-protein binding partners. We will express these proteins as 6xHis-tagged opsins allowing us to use them in proteomic pull down assays to probe binding partners associated with signalling cascades; an approach we have adopted in studies of human-OPN4. We will use laser capture microdissection to examine the expression profile and co-expression propensity of the native pigments in the zebrafish retina using Q-PCR and cross correlation analysis. We already have evidence that some of the novel opsins are co-expressed in a sub-population of retinal neurones. We will therefore examine the extent of synergistic interaction in co-expression studies. Finally, we will generate transgenic animals where expression of EGFP under the control of specific opsin promoter sequences. These reporter animals will then be utilized to study the light driven responses of opsin expressing cells in both retinal and pineal preparations, where EGFP fluorescence will be used to guide recording electrodes. This will allow us to study the photopigment potential of this gene family in the native retinal and pineal environment and provide a functional profile for each photopigment candidate.