Transcriptional profiling of retinal astrocytes identifies a specific marker and points to functional specialization

Astrocyte heterogeneity is an increasingly prominent research topic, and studies in the brain have demonstrated substantial variation in astrocyte form and function, both between and within regions. In contrast, retinal astrocytes are not well understood and remain incompletely characterized. Along with optic nerve astrocytes, they are responsible for supporting retinal ganglion cell axons and an improved understanding of their role is required. We have used a combination of microdissection and Ribotag immunoprecipitation to isolate ribosome-associated mRNA from retinal astrocytes and investigate their transcriptome, which we also compared to astrocyte populations in the optic nerve. Astrocytes from these regions are transcriptionally distinct, and we identified retina-specific astrocyte genes and pathways. Moreover, although they share much of the ‘classical’ gene expression patterns of astrocytes, we uncovered unexpected variation, including in genes related to core astrocyte functions. We additionally identified the transcription factor Pax8 as a highly specific marker of retinal astrocytes and demonstrated that these astrocytes populate not only the retinal surface, but also the prelaminar region at the optic nerve head. These findings are likely to contribute to a revised understanding of the role of astrocytes in the retina. We investigated the transcriptional profile of retinal astrocytes by analyzing differential gene expression between highly enriched IP (immunoprecipitated) RNA from these cells and RNA from Input controls. We also compared gene expression with immunoprecipitated RNA from optic nerve head (ONH) and myelinated optic nerve proper (ONP) astrocytes, which form separate populations. Retinal astrocyte RNA from IP samples and Input controls was isolated and used for RNA-seq, then analyzed alongside our existing RNA-seq data for ONH and ONP astrocytes from a prior study; a pair of adapter samples (one each for ONH and ONP astrocytes) were prepared and sequenced alongside the retinal IP and Input samples to control for potential batch effects.