Advancing Photoreceptor Specific Labeling of Green Cones and Rods in the Zebrafish Retina: Transgenic and Lectin-Based Strategies

Abstract

Accurate identification and differentiation of photoreceptor cells is essential for studying the development, structure, and function of the retina. The zebrafish (Danio rerio) serves as an excellent model organism for such studies. However, despite its widespread use, a reliable method for selectively staining rod photoreceptors in tissue samples remains unavailable. Currently, the only commercially available antibody for identifying rod photoreceptors in zebrafish immunohistochemistry is zpr-3, which also binds to green cone cells. This overlap complicates the distinction between rods and green cones, particularly in zebrafish larvae, whose retinas have not yet developed the layered structure typical of adults, where rods and cones reside in separate layers. This study explored two approaches for distinguishing rods from the zebrafish retina: generation of transgenic zebrafish individuals with endogenous fluorescent labeling and the use of lectins as labels in cryosections.

The first objective of this thesis was to generate transgenic zebrafish in which rods endogenously express a fluorescent protein, enabling their visualization separately from other retinal cell types using confocal microscopy. The method involved microinjection of the pTol2pA-Xops-membrane-TdTomato plasmid into one-cell stage zebrafish embryos. Larvae expressing the fluorescent protein were screened at five days post-fertilization (dpf) and raised to 7–11 dpf for further analysis. During development, it was observed that although the plasmid successfully induced endogenous fluorescence in some of the rod cells, the expression pattern was sparse, with only a small subset of rods expressing the fluorescent protein. At 7 dpf, mosaic expression of fluorescent rods was visible in the retina, but at 8 dpf, signs of reduced vitality of fish and early retinal degeneration were evident. Only few fish survived to 11 dpf, at which point retinal degeneration was pronounced, and the morphology of the fluorescent rods was no longer microscopically identifiable due to cellular disintegration. While the plasmid enables endogenous fluorescence in rods, its strong expression pattern leads to progressive retinal degeneration, possibly beginning in the rods.

To address this limitation, the second objective of this study was to explore lectin-based labeling as an alternative approach for distinguishing photoreceptor subtypes. Lectins are plant-derived molecules known to bind specific glycan structures in the extracellular matrix (ECM) between photoreceptors and the retinal pigment epithelium. Because the ECM composition differs between rods and cones, lectins may offer subtype-specific labeling. This study specifically examined whether PNA and WGA, two commonly used lectins, could be used to differentiate photoreceptor types in cryosections of zebrafish larvae and adults. In mammalian models, PNA binds to cone-associated matrix, while WGA binds to rod-associated matrix.

Surprisingly, the results showed that in zebrafish, PNA binds to UV, blue, and red cone cell ECM, confirming its cone specificity. However, WGA was found to bind not only to rod-associated matrix, but also to the matrix surrounding green cone cells. Interestingly, PNA does not bind to the green cone ECM. This is a novel finding, as WGA was previously thought to label only rod photoreceptors in zebrafish and mammals. These results suggest that rods and green cones may share molecular features also in their surrounding matrix.