June 2020

Yan W, Peng Y-R, van Zyl T, Regev A, Shekhar K, Juric D, Sanes JR. Cell Atlas of The Human Fovea and Peripheral Retina. Sci Rep 2020;10(1):9802.Abstract
Most irreversible blindness results from retinal disease. To advance our understanding of the etiology of blinding diseases, we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptomes of ~85,000 cells from the fovea and peripheral retina of seven adult human donors. Utilizing computational methods, we identified 58 cell types within 6 classes: photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and non-neuronal cells. Nearly all types are shared between the two retinal regions, but there are notable differences in gene expression and proportions between foveal and peripheral cohorts of shared types. We then used the human retinal atlas to map expression of 636 genes implicated as causes of or risk factors for blinding diseases. Many are expressed in striking cell class-, type-, or region-specific patterns. Finally, we compared gene expression signatures of cell types between human and the cynomolgus macaque monkey, Macaca fascicularis. We show that over 90% of human types correspond transcriptomically to those previously identified in macaque, and that expression of disease-related genes is largely conserved between the two species. These results validate the use of the macaque for modeling blinding disease, and provide a foundation for investigating molecular mechanisms underlying visual processing.
Zampaglione E, Kinde B, Place EM, Navarro-Gomez D, Maher M, Jamshidi F, Nassiri S, Mazzone AJ, Finn C, Schlegel D, Comander J, Pierce EA, Bujakowska KM. Copy-number variation contributes 9% of pathogenicity in the inherited retinal degenerations. Genet Med 2020;22(6):1079-1087.Abstract
PURPOSE: Current sequencing strategies can genetically solve 55-60% of inherited retinal degeneration (IRD) cases, despite recent progress in sequencing. This can partially be attributed to elusive pathogenic variants (PVs) in known IRD genes, including copy-number variations (CNVs), which have been shown as major contributors to unsolved IRD cases. METHODS: Five hundred IRD patients were analyzed with targeted next-generation sequencing (NGS). The NGS data were used to detect CNVs with ExomeDepth and gCNV and the results were compared with CNV detection with a single-nucleotide polymorphism (SNP) array. Likely causal CNV predictions were validated by quantitative polymerase chain reaction (qPCR). RESULTS: Likely disease-causing single-nucleotide variants (SNVs) and small indels were found in 55.6% of subjects. PVs in USH2A (11.6%), RPGR (4%), and EYS (4%) were the most common. Likely causal CNVs were found in an additional 8.8% of patients. Of the three CNV detection methods, gCNV showed the highest accuracy. Approximately 30% of unsolved subjects had a single likely PV in a recessive IRD gene. CONCLUSION: CNV detection using NGS-based algorithms is a reliable method that greatly increases the genetic diagnostic rate of IRDs. Experimentally validating CNVs helps estimate the rate at which IRDs might be solved by a CNV plus a more elusive variant.
Zieske JD, Hutcheon AEK, Guo X. Extracellular Vesicles and Cell-Cell Communication in the Cornea. Anat Rec (Hoboken) 2020;303(6):1727-1734.Abstract
One question that has intrigued cell biologists for many years is, "How do cells interact to influence one another's activity?" The discovery of extracellular vesicles (EVs) and the fact that they carry cargo, which directs cells to undergo changes in morphology and gene expression, has revolutionized this field of research. Little is known regarding the role of EVs in the cornea; however, we have demonstrated that EVs isolated from corneal epithelial cells direct corneal keratocytes to initiate fibrosis. Intriguingly, our data suggest that EVs do not penetrate epithelial basement membrane (BM), perhaps providing a mechanism explaining the importance of BM in the lack of scarring in scrape wounds. Since over 100-million people worldwide suffer from visual impairment as a result of corneal scarring, the role of EVs may be vital to understanding the mechanisms of wound repair. Therefore, we investigated EVs in ex vivo and in vivo-like three-dimensional cultures of human corneal cells using transmission electron microscopy. Some of the major findings were all three major cell types (epithelial, fibroblast, and endothelial cells) appear to release EVs, EVs can be identified using TEM, and EVs appeared to be involved in cell-cell communication. Interestingly, while our previous publication suggests that EVs do not penetrate the epithelial BM, it appears that EVs penetrate the much thicker endothelial BM (Descemet's membrane). These findings indicate the huge potential of EV research in the cornea and wound healing, and suggest that during homeostasis the endothelium and stromal cells are in communication. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.