Multi ‐ omics studies of eye and lens development

Abstract

Early eye development involves formation of a symmetric pair of optic vesicles from the anterior neuroectoderm and parallel formation of closely apposed lens placodes. Their reciprocal invaginations generate the 3D‐foundation of the optic cups. Embryonic lens development is initiated within the border between the anterior neuroectoderm and naïve ectoderm, called the anterior pre‐placodal region. The earliest stages of lens development require coordinated actions of DNA‐binding transcription factor Pax6, signalling growth factor BMP4 and a pair of chromatin remodelling enzymes p300 and CBP. Studies in model vertebrates postulated a common Pax6+ progenitor cell population to generate both the lens and olfactory placodes. Recent progress in technologies to probe chromatin structure, landscape of transcription factor binding, DNA methylation, coding (mRNA) and noncoding (e.g. eRNAs and ncRNAs) transcriptomes and chromatin conformation have been shown to generate unprecedented mechanistic insights into the earliest cell fate decisions and tissue organization. To examine lens development at E8.5, E9.5 and E10.5 mouse embryos, single cell RNA‐seq (scRNA‐seq) experiments using the 10XGenomics Chromium platform were conducted. E9.5 Pax6 null embryos and controls were also included. Whole genome bisulfite sequencing (WGBS) was performed in E14.5 and P0.5 microdissected mouse lenses to map DNA methylation and integrate the data with corresponding ATAC‐ and RNA‐seq data sets. Open chromatin and DNA methylation changes were defined through identification of differentially accessible regions (DARs), unmethylated/low methylated regions (UMRs/LMRs) coupled with differential gene expression during lens differentiation. To probe 3D‐organization of lens chromatin and to identify temporally and spatially regulated lens‐specific distal enhancers, Hi‐C was used in conjunction with ChIP‐seq analysis of transcription factor CTCF in microdissected newborn lenses. Previous ChIP‐seq data using whole newborn lenses include H3K27ac peaks to identify super‐enhancers. Pax6 proteins were found both in open and closed chromatin domains, including both closed and methylated regions. In vitro binding of Pax6 proteins was not inhibited by 5‐methylcytosines within one or two CpG dinucleotides present in the Pax6 binding sites. In summary, cell fate decisions are governed by complex changes in chromatin landscape controlled by sequence‐specific DNA‐binding transcription factors and their recruitment of chromatin remodelling complexes. A combination of different –omics is a powerful approach to examine molecular mechanisms governing eye development. These studies also reveal additional levels of genetic code complexity and stimulate follow up mechanistic studies of the individual cellular and molecular processes of lens morphogenesis and their underlying GRNs and identification of cataract‐causing mutation in non‐coding regulatory sequences.