Spatial and Single-cell Transcriptomic Characterization of WIF1-induced Normal-tension Glaucoma [Single Cell Multiome ATAC + Gene Expression]
To explore the potential roles of WIF1 upregulation in glaucoma pathogenesis, we performed WIF1 overexpression in mouse retina via AAV delivery and then assessed its impact on IOP, retinal visual function, and RGC survival. We found it causes visual function defects and RGC loss, especially the RGCs from periphery retina. Our integrative analyses from single cell RNA-seq, ATAC-seq, and spatial transcriptomics have further uncovered the dynamic signaling changes from various retinal cells to RGCs. And our spatial transcriptomics results have also revealed the various changes of central and peripheral RGCs after WIF1 overexpression. Overall design: Two different AAV2/2 were generated for Wif1 overexpressing, CMV-Wif1-HA-IRES-GFP, and CMV-Wif1-HA, whereas the AAV carrying CMV-IRES-GFP was used as a control in this study. To further investigate the mechanism underlying the WIF1-mediated glaucoma, we established the single-cell (sc) omics blueprint of the WIF1-overexpressing retina. We harvested fresh retinas from six mouse eyes, two from the wild-type control group (Ctrl), two at 2 weeks AVI (WIF1-2w), and two at 4 weeks AVI (WIF1-4w), to isolate single nuclei for simultaneous scRNA-seq and scATAC-seq using the 10x Genomics Chromium. And we also performed 10x Visium spatial transcriptomics (ST) on the Ctrl, WIF1-2w, and WIF1-4w retinas on order to gain new insights into the spatial communication between RGCs and other cell populations in the WIF1-overexpressing retina. Then integrated analysis was performed to visualize the temporal spatial cell composition changes, TCF4, WNT signaling pathway regulator, mediated target gene activity changes, cell-cell interaction changes and center vs periphery RGC changes after WIF1 overexpression.