Abstract
Purpose :
Retina depends on retinal pigment epithelium (RPE) for its survival. Traditionally, these tissues have been studied separately using samples from different animal models or utilizing in vitro cell cultures, omitting the critical interaction between photoreceptors and RPE. Therefore, there is a high demand for in vivo models allowing genetic or pharmaceutical manipulation and imaging of these tissues to resolve their interactive processes in a living animal. Zebrafish is one of the most accessible animal models to study retinal physiology in vivo and currently the main vertebrate model that is used to image retinal tissue functions in real time. Here, our aim was to extend this approach further to study retina-RPE interactions in living zebrafish by fluorescently tagging photoreceptor and RPE cells for in vivo imaging.
Methods :
We microinjected plasmids encoding membrane targeted fluorescent proteins with photoreceptor or RPE specific promoters to 1-cell stage low-pigmented zebrafish embryos and raised them to 6 days post fertilization. For histological samples, the larvae were fixed with 4% paraformaldehyde and prepared for cryosectioning. The 10 µm thick cryosections were labelled with standard immunohistochemical protocol. Samples were then imaged with a laser scanning confocal microscope. For live in vivo confocal imaging, the larvae were anesthetized and mounted in agarose for immobilization. Larval photoreceptor and RPE cells were then imaged over 30-90 minutes time-lapse.
Results :
Here, we show with immunohistochemical samples how our plasmids correctly target the different photoreceptor cell types and RPE in zebrafish. In vivo, the cells can be visualized with resolution that allows detection of their subcellular details. With optimized sample preparation and handling, time-lapse imaging can be performed to study the RPE and photoreceptor cells and their interaction in a living animal. We present images and time-lapse video examples for different photoreceptor types and RPE cells and show how we can track individual cells and their particles in living zebrafish larvae.
Conclusions :
We have generated zebrafish with fluorescently tagged RPE cells and photoreceptor outer segments using carefully designed, cell type specific reporters. Our time-lapse confocal in vivo imaging approach presents a useful tool to study retinal organization of photoreceptors and their interaction with RPE.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.