PLAE Web App Enables Powerful Searching and Multiple Visualizations Across One Million Unified Single-Cell Ocular Transcriptomes.

Purpose: To create a high-performance reactive web application to query single-cell gene expression data across cell type, species, study, and other factors. Methods: We updated the content and structure of the underlying data (single cell Eye in a Disk [scEiaD]) and wrote the web application PLAE (https://plae.nei.nih.gov) to visualize and explore the data. Results: The new portal provides quick visualization of over a million individual cells from vertebrate eye and body transcriptomes encompassing four species, 60 cell types, six ocular tissues, and 23 body tissues across 35 publications. To demonstrate the value of this unified pan-eye dataset, we replicated known neurogenic and cone macula markers in addition to proposing six new cone human region markers. Conclusions: The PLAE web application offers the eye community a powerful and quick means to test hypotheses related to gene expression across a highly diverse, community-derived database. Translational Relevance: The PLAE resource enables any researcher or clinician to study and research gene expression patterning across a wide variety of curated ocular cell types with a responsive web app.

Building the mega single-cell transcriptome ocular meta-atlas.

BACKGROUND: The development of highly scalable single-cell transcriptome technology has resulted in the creation of thousands of datasets, >30 in the retina alone. Analyzing the transcriptomes between different projects is highly desirable because this would allow for better assessment of which biological effects are consistent across independent studies. However it is difficult to compare and contrast data across different projects because there are substantial batch effects from computational processing, single-cell technology utilized, and the natural biological variation. While many single-cell transcriptome-specific batch correction methods purport to remove the technical noise, it is difficult to ascertain which method functions best. RESULTS: We developed a lightweight R package (scPOP, single-cell Pick Optimal Parameters) that brings in batch integration methods and uses a simple heuristic to balance batch merging and cell type/cluster purity. We use this package along with a Snakefile-based workflow system to demonstrate how to optimally merge 766,615 cells from 33 retina datsets and 3 species to create a massive ocular single-cell transcriptome meta-atlas. CONCLUSIONS: This provides a model for how to efficiently create meta-atlases for tissues and cells of interest.

Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion.

The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.

Punctiform and Polychromatic Pre-Descemet Corneal Dystrophy: Clinical Evaluation and Identification of the Genetic Basis.

PURPOSE: This study reports the clinical features and genetic bases of 3 previously unreported families with punctiform and polychromatic pre-Descemet corneal dystrophy (PPPCD). DESIGN: Observational case series. METHODS: Full ophthalmic assessment was performed for members of 3 unreported families with PPPCD. Structural and biomechanical alterations of the cornea were screened. Whole exome sequencing (WES) was performed in the first family. Novel or rare variants that segregated with the affected status were screened in the other 2 families using Sanger sequencing. Identified variants that segregated with the affected status in all families were characterized by using in silico prediction tools and/or in vitro splice assays. Additionally, 2 previously reported PPPCD families were screened for variants identified in the 3 unreported PPPCD families. RESULTS: PPPCD was diagnosed in 12 of the 21 examined members of the 3 unreported families. The only refractive, topographic, or biomechanical abnormality associated with PPPCD was a significantly increased corneal stiffness. WES and Sanger sequencing identified 2 variants that segregated with the affected status in all 3 families: a rare intronic PDZD8 c.872+10A>T variant and a novel missense PRDX3 c.568G>C (p.Asp190His) variant. The same PRDX3 variant was identified in the previously reported PPPCD family expressing the common PPPCD phenotype and was predicted by in silico prediction tools to be damaging to protein function. CONCLUSIONS: PPPCD is associated with an alteration of corneal biomechanics and a novel missense variant in PRDX3. Screening of additional families will determine whether all families demonstrate a PRDX3 variant or whether locus heterogeneity may exist for PPPCD.

ZEB1 insufficiency causes corneal endothelial cell state transition and altered cellular processing.

The zinc finger e-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT), and of the reverse mesenchymal to epithelial transition (MET) processes. ZEB1 plays an integral role in mediating cell state transitions during cell lineage specification, wound healing and disease. EMT/MET are characterized by distinct changes in molecular and cellular phenotype that are generally context-independent. Posterior polymorphous corneal dystrophy (PPCD), associated with ZEB1 insufficiency, provides a new biological context in which to understand and evaluate the classic EMT/MET paradigm. PPCD is characterized by a cadherin-switch and transition to an epithelial-like transcriptomic and cellular phenotype, which we study in a cell-based model of PPCD generated using CRISPR-Cas9-mediated ZEB1 knockout in corneal endothelial cells (CEnCs). Transcriptomic and functional studies support the hypothesis that CEnC undergo a MET-like transition in PPCD, termed endothelial to epithelial transition (EnET), and lead to the conclusion that EnET may be considered a corollary to the classic EMT/MET paradigm.

Alternative polyadenylation factors link cell cycle to migration.

BACKGROUND: In response to a wound, fibroblasts are activated to migrate toward the wound, to proliferate and to contribute to the wound healing process. We hypothesize that changes in pre-mRNA processing occurring as fibroblasts enter the proliferative cell cycle are also important for promoting their migration. RESULTS: RNA sequencing of fibroblasts induced into quiescence by contact inhibition reveals downregulation of genes involved in mRNA processing, including splicing and cleavage and polyadenylation factors. These genes also show differential exon use, especially increased intron retention in quiescent fibroblasts compared to proliferating fibroblasts. Mapping the 3' ends of transcripts reveals that longer transcripts from distal polyadenylation sites are more prevalent in quiescent fibroblasts and are associated with increased expression and transcript stabilization based on genome-wide transcript decay analysis. Analysis of dermal excisional wounds in mice reveals that proliferating cells adjacent to wounds express higher levels of cleavage and polyadenylation factors than quiescent fibroblasts in unwounded skin. Quiescent fibroblasts contain reduced levels of the cleavage and polyadenylation factor CstF-64. CstF-64 knockdown recapitulates changes in isoform selection and gene expression associated with quiescence, and results in slower migration. CONCLUSIONS: Our findings support cleavage and polyadenylation factors as a link between cellular proliferation state and migration.