Visual System Homeobox 2 (Vsx2) is a transcription factor expressed in the developing retina that regulates tissue identity, growth, and fate determination. Several mutations in the Vsx2 gene exist in mice, including a spontaneous nonsense mutation and two targeted missense mutations originally identified in humans. Here, we expand the genetic repertoire to include a LacZ reporter allele (Vsx2 LacZ ) designed to express beta-Galactosidase (b-GAL) and simultaneously disrupt Vsx2 function (knock-in/knock-out). The retinal expression pattern of b-GAL is concordant with VSX2, and the mutant allele is recessive. Vsx2 LacZ homozygous mice have congenital bilateral microphthalmia accompanied by defects in retinal development including ectopic expression of non-retinal genes, reduced proliferation, delayed neurogenesis, aberrant tissue morphology, and an absence of bipolar interneurons - all hallmarks of Vsx2 loss-of-function. Unexpectedly, the mutant VSX2 protein is stably expressed, and there are subtle differences in eye size and early retinal neurogenesis when compared to the null mutant, ocular retardation J. We propose that b-GAL expression from the Vsx2 LacZ allele is a reliable reporter of VSX2 expression and that the allele exhibits loss-of-function characteristics. However, the perdurance of the mutant VSX2 protein combined with subtle deviations from the null phenotype leaves open the possibility that Vsx2 LacZ allele is not a complete knock-out. The Vsx2 LacZ allele adds to the genetic toolkit for understanding Vsx2 function.

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.

Vsx2 is a transcription factor essential for retinal proliferation and bipolar cell differentiation, but the molecular mechanisms underlying its developmental roles are unclear. Here, we have profiled VSX2 genomic occupancy during mouse retinogenesis, revealing extensive retinal genetic programs associated with VSX2 during development. VSX2 binds and transactivates its enhancer in association with the transcription factor PAX6. Mice harboring deletions in the Vsx2 regulatory landscape exhibit specific abnormalities in retinal proliferation and in bipolar cell differentiation. In one of those deletions, a complete loss of bipolar cells is associated with a bias towards photoreceptor production. VSX2 occupies cis-regulatory elements nearby genes associated with photoreceptor differentiation and homeostasis in the adult mouse and human retina, including a conserved region nearby Prdm1, a factor implicated in the specification of rod photoreceptors and suppression of bipolar cell fate. VSX2 interacts with the transcription factor OTX2 and can act to suppress OTX2-dependent enhancer transactivation of the Prdm1 enhancer. Taken together, our analyses indicate that Vsx2 expression can be temporally and spatially uncoupled at the enhancer level, and they illuminate important mechanistic insights into how VSX2 is engaged with gene regulatory networks that are essential for retinal proliferation and cell fate acquisition.

Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires spatiotemporal gene expression patterns and disease phenotypes can differ significantly between humans and animal models, with patient-associated mutations causing embryonic lethality reported in some animal models. The emergence of human induced pluripotent stem cell (hiPSC) technology has provided a new resource for dissecting the complex nature of early eye morphogenesis through the generation of three-dimensional (3D) cellular models. By using patient-specific hiPSCs to generate in vitro optic vesicle-like models, we can enhance the understanding of early developmental eye disorders and provide a pre-clinical platform for disease modelling and therapeutics testing. A major challenge of in vitro optic vesicle generation is the low efficiency of differentiation in 3D cultures. To address this, we adapted a previously published protocol of retinal organoid differentiation to improve embryoid body formation using a microwell plate. Established morphology, upregulated transcript levels of known early eye-field transcription factors and protein expression of standard retinal progenitor markers confirmed the optic vesicle/presumptive optic cup identity of in vitro models between day 20 and 50 of culture. This adapted protocol is relevant to researchers seeking a physiologically relevant model of early human ocular development and disease with a view to replacing animal models.

Approximately one-third of childhood blindness is attributed to developmental eye disorders, of which 80% have a genetic cause. Eye morphogenesis is tightly regulated by a highly conserved network of transcription factors when disrupted by genetic mutations can result in severe ocular malformation. Human-induced pluripotent stem cells (hiPSCs) are an attractive tool to study early eye development as they are more physiologically relevant than animal models, can be patient-specific and their use does not elicit the ethical concerns associated with human embryonic stem cells. The generation of self-organizing hiPSC-derived optic cups is a major advancement to understanding mechanisms of ocular development and disease. Their development in vitro has been found to mirror that of the human eye and these early organoids have been used to effectively model microphthalmia caused by a VSX2 variant. hiPSC-derived optic cups, retina, and cornea organoids are powerful tools for future modeling of disease phenotypes and will enable a greater understanding of the pathophysiology of many other developmental eye disorders. These models will also provide an effective platform for identifying molecular therapeutic targets and for future clinical applications.

Early in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retinal progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist.
CRISPR/Cas9-mediated homology-directed repair (HDR) in hiPSCs facilitated the replacement of the VSX2 (Progenitor), BRN3b (Ganglion), and RCVRN (Photoreceptor) stop codons with sequences encoding a viral P2A peptide fused to Cerulean, green fluorescent protein, and mCherry reporter genes, respectively, to generate a triple transgenic reporter hiPSC line called PGP1. This was accomplished by co-electroporating HDR templates and sgRNA/Cas9 vectors into hiPSCs followed by antibiotic selection. Functional validation of the PGP1 hiPSC line included the ability to generate retinal organoids, with all major retinal cell types, displaying the expression of the three fluorescent reporters consistent with the onset of target gene expression. Disaggregated organoids were also analyzed by fluorescence-activated cell sorting and fluorescent populations were tested for the expression of the targeted gene.
Retinal organoids formed from the PGP1 line expressed appropriate fluorescent proteins consistent with the differentiation of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types.
The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.

The transcription factors Prdm1 (Blimp1) and Vsx2 (Chx10) work downstream of Otx2 to regulate photoreceptor and bipolar cell fates in the developing retina. Mice that lack Vsx2 fail to form bipolar cells while Prdm1 mutants form excess bipolars at the direct expense of photoreceptors. Excess bipolars in Prdm1 mutants appear to derive from rods, suggesting that photoreceptor fate remains mutable for some time after cells become specified. Here we tested whether bipolar cell fate is also plastic during development. To do this, we created a system to conditionally misexpress Prdm1 at different stages of bipolar cell development. We found that Prdm1 blocks bipolar cell formation if expressed before the fate choice decision occurred. When we misexpressed Prdm1 just after the decision to become a bipolar cell was made, some cells were reprogrammed into photoreceptors. In contrast, Prdm1 misexpression in mature bipolar cells did not affect cell fate. We also provide evidence that sustained misexpression of Prdm1 was selectively toxic to photoreceptors. Our data show that bipolar fate is malleable, but only for a short temporal window following fate specification. Prdm1 and Vsx2 act by stabilizing photoreceptor and bipolar fates in developing OTX2+ cells of the retina.

The transcription factor OTX2 is required for photoreceptor and bipolar cell formation in the retina. It directly activates the transcription factors Prdm1 and Vsx2 through cell type-specific enhancers. PRDM1 and VSX2 work in opposition, such that PRDM1 promotes photoreceptor fate and VSX2 bipolar cell fate. To determine how OTX2+ cell fates are regulated in mice, we deleted Prdm1 and Vsx2 or their cell type-specific enhancers simultaneously using a CRISPR/Cas9 in vivo retina electroporation strategy. Double gene or enhancer targeting effectively removed PRDM1 and VSX2 protein expression. However, double enhancer targeting favored bipolar fate outcomes, whereas double gene targeting favored photoreceptor fate. Both conditions generated excess amacrine cells. Combined, these fate changes suggest that photoreceptors are a default fate outcome in OTX2+ cells and that VSX2 must be present in a narrow temporal window to drive bipolar cell formation. Prdm1 and Vsx2 also appear to redundantly restrict the competence of OTX2+ cells, preventing amacrine cell formation. By taking a combinatorial deletion approach of both coding sequences and enhancers, our work provides new insights into the complex regulatory mechanisms that control cell fate choice.

Anophthalmia and microphthalmia (A/M) are rare distinct phenotypes that represent a continuum of structural developmental eye defects. Here, we describe three probands from an Egyptian population with various forms of A/M: two patients with bilateral anophthalmia and one with bilateral microphthalmia that were investigated using whole exome sequencing (WES). We identified three causative mutations in three different genes. A new homozygous frameshift mutation c.[422delA];[422delA], p.[N141Ifs∗19];[N141Ifs∗19] in VSX2 was identified in a patient showing bilateral anophthalmia. A previously reported SOX2 deletion c.[70_89del20] p.[N24Rfs∗65];[=] was found in one subject with bilateral anophthalmia. A novel homozygous in-frame mutation c.[431_433delACT];[431_433delACT], p.[Y144del]; [Y144del]) in FOXE3 was identified in a patient with severe bilateral microphthalmia and anterior segment dysgenesis. This study shows that whole exome sequencing (WES) is a reliable and effective strategy for the molecular diagnosis of A/M. Our results expand its allelic heterogeneity and highlight the need for the testing of patient with this developmental anomaly.

More than 8,000 genes are turned on or off as progenitor cells produce the 7 classes of retinal cell types during development. Thousands of enhancers are also active in the developing retinae, many having features of cell- and developmental stage-specific activity. We studied dynamic changes in the 3D chromatin landscape important for precisely orchestrated changes in gene expression during retinal development by ultra-deep in situ Hi-C analysis on murine retinae. We identified developmental-stage-specific changes in chromatin compartments and enhancer-promoter interactions. We developed a machine learning-based algorithm to map euchromatin and heterochromatin domains genome-wide and overlaid it with chromatin compartments identified by Hi-C. Single-cell ATAC-seq and RNA-seq were integrated with our Hi-C and previous ChIP-seq data to identify cell- and developmental-stage-specific super-enhancers (SEs). We identified a bipolar neuron-specific core regulatory circuit SE upstream of Vsx2, whose deletion in mice led to the loss of bipolar neurons.