Transcriptomic Analysis of the Ocular Posterior Segment Completes a Cell Atlas of the Human Eye

Abstract

Although the visual system extends through the brain, most vision loss originates from defects in the eye. Its central element is the neural retina, which senses light, processes visual signals, and transmits them to the rest of the brain through the optic nerve (ON). Surrounding the retina are numerous other structures, conventionally divided into anterior and posterior segments. Here we used high-throughput single nucleus RNA sequencing (snRNA-seq) to classify and characterize cells in the extraretinal components of the posterior segment: ON, optic nerve head (ONH), peripheral sclera, peripapillary sclera (PPS), choroid, and retinal pigment epithelium (RPE). Defects in each of these tissues are associated with blinding diseases, for example, glaucoma (ONH and PPS), optic neuritis (ON), retinitis pigmentosa (RPE), and age-related macular degeneration (RPE and choroid). From ~151,000 single nuclei, we identified 37 transcriptomically distinct cell types, including multiple types of astrocytes, oligodendrocytes, fibroblasts, and vascular endothelial cells. Our analyses revealed a differential distribution of many cell types among distinct structures. Together with our previous analyses of the anterior segment and retina, the new data complete a Version 1 cell atlas of the human eye. We used this atlas to map the expression of >180 genes associated with the risk of developing glaucoma, which is known to involve ocular tissues in both anterior and posterior segments as well as neural retina. Similar methods can be used to investigate numerous additional ocular diseases, many of which are currently untreatable.

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

“…The identification of PAX8 in particular as a retinal astrocyte marker suggests one potential explanation, as during development (including in the CNS) PAX2 and PAX8 are expressed as part of a signaling axis with the related transcription factor PAX5 (Bouchard et al, 2010;Stoykova & Gruss, 1994;Urbanek et al, 1997). Although PAX5 transcripts were not detected in any of our mouse samples, PAX5 expression has been detected in some (but not all) clusters of astrocytes in the human eye, raising the possibility that PAX5 is a marker of a subpopulation not found in mice (Monavarfeshani et al, 2023). Given that PAX8 expression was likewise not detected ubiquitously in single-cell studies, it may be that PAX8 and PAX5 are markers of distinct subpopulations of human retinal astrocytes.…”

“…Key questions about the role of PAX8 itself, both in retinal astrocytes and more broadly, remain unanswered. A recent single-cell 'atlas' study of the human eye identified PAX8/PAX8 as an astrocyte marker, and while this was reported in the context of the ONH region, a previous study of human retinal cells by the same group is searchable and reveals that PAX8 was detected in a significant fraction of human retinal astrocytes (https://singlecell.broadinstitute.org/single_ cell/study/SCP839/cell-atlas-of-the-human-fovea-and-peripheral-retina) (Monavarfeshani et al, 2023;Yan et al, 2020). Whether the restriction of expression to a subset of astrocytes reflects transcriptional heterogeneity in human retinal astrocytes or an experimental artifact is unclear.…”

“…Likewise, astrocyte-specific Cre-lines, such as Aldh1l1-Cre and Gfap-Cre, are insufficiently specific to separate these populations, as Müller cells constitutively express ALDH1L1 and upregulate GFAP in response to most experimental manipulations (Dyer & Cepko, 2000;Zhou et al, 2022). Although recent advances in single cell sequencing have enabled 'atlas' studies of the retina and eye that provide a window into the retinal astrocyte transcriptome, such studies have not extensively characterized these astrocytes (Lukowski et al, 2019;Monavarfeshani et al, 2023;Yan et al, 2020). Our microdissection approach allows us to bypass previous constraints by directly isolating astrocytes from their niche at the retinal surface, preserving their morphology (Figure 1a) while largely excluding other retinal cell types, including Müller cells .…”

Multiparametric grading of glaucoma severity by histopathology can enable post-mortem substratification of disease state

“…The explosion of highly multiplexed transcriptomics and proteomics platforms is enabling greater resolution of molecular markers in human tissues. For example, a recent study [30] identified 37 clusters and subclusters of distinct cell types in the optic nerve, optic nerve head, retinal pigmented epithelium and peripapillary sclera of non-glaucomatous donor globes. These findings offer insights into expression profiles that define cell subtypes, and computational approaches can be applied to these datasets to infer cell-cell interactions.…”

Transcriptomic profiling of Schlemm’s canal cells reveals a lymphatic-biased identity and three major cell states

“…Single-cell RNA sequencing (scRNA-seq) is revealing the cellular heterogeneity and transcriptomic profiles of various ocular cell types, including the anterior segment from several species (14)(15)(16)(17)(18). These important studies included SECs, but were underpowered and limited for SC due to the low representation of SECs in the studied tissues and the very small numbers of SECs successfully sequenced.…”