Ocular lens development entails epithelial to fiber cell differentiation, defects in which cause congenital cataracts. We report the first single-cell multiomic atlas of lens development, leveraging snRNA-seq, snATAC-seq and CUT&RUN-seq to discover previously unreported mechanisms of cell fate determination and cataract-linked regulatory networks. A comprehensive profile of cis- and trans-regulatory interactions, including for the cataract-linked transcription factor MAF, is established across a temporal trajectory of fiber cell differentiation. Furthermore, we identify an epigenetic paradigm of cellular differentiation, defined by progressive loss of the H3K27 methylation writer Polycomb repressive complex 2 (PRC2). PRC2 localizes to heterochromatin domains across master-regulator transcription factor gene bodies, suggesting it safeguards epithelial cell fate. Moreover, we demonstrate that FGF hyper-stimulation in vivo leads to MAF network activation and the emergence of novel lens cell states. Collectively, these data depict a comprehensive portrait of lens fiber cell differentiation, while defining regulatory effectors of cell identity and cataract formation.

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in a fibroblast growth factor 2 (FGF2)-dependent manner. To better explore the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from (1) intact RPE, (2) transiently reprogrammed RPE (t-rRPE) 6 h post-retinectomy, and (3) reprogrammed RPE (rRPE) 6 h post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for mitogen-activated protein kinase (MAPK)-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis was used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) associated factors, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

Lens epithelial explants are comprised of lens epithelial cells cultured in vitro on their native basement membrane, the lens capsule. Biologists have used lens epithelial explants to study many different cellular processes including lens fiber cell differentiation. In these studies, fiber differentiation is typically measured by cellular elongation and the expression of a few proteins characteristically expressed by lens fiber cells in situ. Chromatin and RNA was collected from lens epithelial explants cultured in either un-supplemented media or media containing 50% bovine vitreous humor for one or five days. Chromatin for ATAC-sequencing and RNA for RNA-sequencing was prepared from explants to assess regions of accessible chromatin and to quantitatively measure gene expression, respectively. Vitreous humor increased chromatin accessibility in promoter regions of genes associated with fiber differentiation and, surprisingly, an immune response, and this was associated with increased transcript levels for these genes. In contrast, vitreous had little effect on the accessibility of the genes highly expressed in the lens epithelium despite dramatic reductions in their mRNA transcripts. An unbiased analysis of differentially accessible regions revealed an enrichment of cis-regulatory motifs for RUNX, SOX and TEAD transcription factors that may drive differential gene expression in response to vitreous.

Genomic sequences residing within introns of few genes have been shown to act as enhancers affecting expression of neighboring genes. We studied an autosomal recessive phenotypic continuum of microphthalmia, anophthalmia and ocular coloboma, with no apparent coding-region disease-causing mutation. Homozygosity mapping of several affected Jewish Iranian families, combined with whole genome sequence analysis, identified a 0.5 Mb disease-associated chromosome 2q35 locus (maximal LOD score 6.8) harboring an intronic founder variant in NHEJ1, not predicted to affect NHEJ1. The human NHEJ1 intronic variant lies within a known specifically limb-development enhancer of a neighboring gene, Indian hedgehog (Ihh), known to be involved in eye development in mice and chickens. Through mouse and chicken molecular development studies, we demonstrated that this variant is within an Ihh enhancer that drives gene expression in the developing eye and that the identified variant affects this eye-specific enhancer activity. We thus delineate an Ihh enhancer active in mammalian eye development whose variant causes human microphthalmia, anophthalmia and ocular coloboma. The findings highlight disease causation by an intronic variant affecting the expression of a neighboring gene, delineating molecular pathways of eye development.

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in an FGF2-dependent manner. To characterize the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from 1) intact RPE, 2) transiently reprogrammed RPE (t-rRPE) 6 hours post-retinectomy, and 3) reprogrammed RPE (rRPE) 6 hours post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for MAPK-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis were used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) regulators, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

Amacrine cells (ACs) comprise a heterogeneous class of inhibitory neurons in the vertebrate retina, exhibiting morphological and functional complexity rivaling that of cortical interneurons. Here, we integrate single-cell and single-nucleus transcriptomic atlases from 24 vertebrate species to reconstruct the evolutionary origins of this extreme diversity. We identify 42 orthologous AC types (oACs), most of which exhibit a one-to-one correspondence across amniotes and, in many cases, across vertebrates. While core molecular identities are conserved, AC types vary in abundance and gene expression across species, likely reflecting adaptations to distinct visual ecologies. AC diversity scales with that of retinal ganglion cells (RGCs), indicative of co-evolution. Finally, we suggest that ACs arose from an AC-RGC hybrid precursor, with glycinergic ACs diverging early in vertebrate evolution, followed by a bifurcation between RGCs and GABAergic ACs. Together, these findings establish a unified evolutionary framework for understanding the diversity, development, and function of a class of inhibitory neurons across vertebrates.