Live imaging of photoreceptor outer segment phagocytosis in zebrafish

Abstract

Retina depends on retinal pigment epithelium (RPE) for its survival. To maintain normal functionality, photoreceptors renew 10% of their outer segments every day and this process requires efficient phagocytosis of the aged photoreceptor fragments by the RPE. Phagocytosis is a dynamic process with strong circadian regulation, but detailed understanding on the regulation of the process and differences between photoreceptor types is presently inadequate. Previous studies have been carried out mainly with in vitro models and cell cultures, and therefore, the dynamics of the phagocytosis process in the context of a living animal has remained elusive. Here, we present an approach that utilizes transgenic zebrafish lines with fluorescently tagged photoreceptor and RPE cells for in vivo imaging of their interactive functions in a living animal.Zebrafish is one of the most accessible animal models to study retinal physiology in vivo and currently the only vertebrate model that can be used to image retinal tissue dynamics in real time. By utilizing already existing low‐pigmented zebrafish lines and microinjecting plasmids coding for photoreceptor and RPE cell membrane targeted tags, we have created low‐pigmented fish with fluorescent RPE cells and the outer segments of the different photoreceptor types. Here, we first show with histological sections how the plasmids are expressed in the correct photoreceptor types and RPE cells in larval zebrafish eyes. Then, we present our in vivo imaging approach where we anesthetise and mount the zebrafish to a confocal microscope. Our results using time‐lapse imaging demonstrate how we can follow the interaction between photoreceptors and RPE in live animals in resolution that enables detecting the movement of photoreceptor outer segment fragments in the subretinal space. Taken together, our study introduces a new approach to investigate the dynamics of phagocytosis of different photoreceptor types by the RPE in real time in a living animal.