Expression of Osteopontin in M2 and M4 intrinsically photosensitive retinal ganglion cells
PurposeMelanopsin-expressing intrinsically photosensitive (ip) retinal ganglion cells (RGC) can be divided into six different subtypes (M1 - M6). Yet, only for some of these subtypes specific markers exist that could be employed to study of the function of individual subtypes. Osteopontin (Spp1) marks alpha()-RGC, suggesting that, across ipRGC, it would only mark the M4-ipRGC subtype (synonymous to: ON-sustained-RGC). Recent evidence suggests that osteopontin expression could spread to other ipRGC subtypes. Therefore, this study aims to characterise the expression pattern of osteopontin across ipRGC subtypes. MethodsSingle-cell RNA sequencing data from RGC were analysed to identify expression patterns of Spp1 across ipRGC. Immunohistochemistry (IHC) was performed on retinal cryosections and flatmounts from C57BL/6J mice to characterize the localization of osteopontin across ipRGC. Neurite tracing was employed to study dendritic morphology and identify individual ipRGC subtypes. ResultsscRNAseq analysis revealed Spp1 expression in two distinct clusters of ipRGC. IHC confirmed osteopontin co-localization with Smi-32, an established marker for RGC, including M4-ipRGCs. Spp1 immunoreactivity was moreover identified in one additional group of ipRGC. By dendritic morphology and stratification those cells were clearly identified as M2-ipRGC. ConclusionsOur findings demonstrate that osteopontin is expressed in both M2- and M4-ipRGC, challenging the notion of osteopontin as a marker exclusively for RGC. IHC double-labelling for osteopontin and melanopsin provides a novel method to identify and differentiate M2 ipRGC from other subtypes. This will support the study ipRGC physiology in a subtype specific manner and may for instance foster research in the field of optic nerve injury.