Abstract
Purpose :
We have previously shown that deletion of AP-2β transcription factor in neural crest cells contributing to the periocular mesenchyme (POM) of the murine eye, (AP-2β NCC KO mutant) resulted in anterior segment defects that were accompanied by raised intraocular pressure. In the following study we leverage the AP-2β NCC KO model as a means to identify the critical AP-2β-dependent gene regulatory mechanisms responsible for the observed defects, using single cell RNA sequencing (scRNA-Seq).
Methods :
Wnt1Cre+/-/Tfap2b+/- mice were bred with Tfap2blox/lox mice to generate control mice and Wnt1Cre+/-/Tfap2b-/lox mice with Tfap2b, encoding AP-2β, deleted in the neural crest. Angle tissue, cornea, and iris were dissected from postnatal day 7 mice and scRNA-Seq was performed on this mixture. 10,000 cells of each genotype were sequenced at a depth of 100,000 reads/cell. Cellranger 2.2 toolkit (10X Genomics) was used for de-multiplexing, alignment with the mm10 genome and counting unique molecular identifiers (UMI). UMI count matrix was processed with Seurat R package.
Results :
ScRNAseq analysis of the control sample defined about 20 different cell populations associated with the cornea, iris and angle tissue. Alongside known tissues such as corneal endothelium and stroma, and cells within the iris and ciliary body, there were additional populations revealing the complexity of tissues within the angle. Next, comparison of the mutant and control samples revealed several important findings concerning not only cell populations but also gene expression patterns that are disrupted by loss of AP-2β. Thus, the corneal endothelium cell population was missing in the mutant, confirming our earlier histological findings. We also detected multiple gene expression changes related to the properties of the corneal stroma that potentially reflect loss of angiogenic privilege in this layer and additional changes in the angle tissue correlating with the closed angle phenotype. In situ analyses are being used to identify and map the cell populations present in the control and verify the gene expression changes observed in the mutant.
Conclusions :
Our studies have uncovered the complexity of cell populations present in the developing anterior segment of the normal eye, revealing critical gene expression signatures within each population. We have further identified how loss of AP-2β impacts these cell populations and gene expression programs.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.