Human Eye Development Research Articles

Single-cell transcriptomics reveals the molecular basis of human iPS cell differentiation into ectodermal ocular lineages.

The generation of a self-formed, ectodermal, autonomous multi-zone (SEAM) from human induced pluripotent stem cells (hiPSCs) offers a unique perspective to study the dynamics of ocular cell differentiation over time. Here, by utilising single-cell transcriptomics, we have (i) identified, (ii) molecularly characterised and (iii) ascertained the developmental trajectories of ectodermally-derived ocular cell populations which emerge within SEAMs as they form. Our analysis reveals interdependency between tissues of the early eye and delineates the sequential formation and maturation of distinct cell types over a 12-week period. We demonstrate a progression from pluripotency through to tissue specification and differentiation which encompasses both surface ectodermal and neuroectodermal ocular lineages and the generation of iPSC-derived components of the developing cornea, conjunctiva, lens, and retina. Our findings not only advance the understanding of ocular development in a stem cell-based system of human origin, but also establish a robust methodological paradigm for exploring cellular and molecular dynamics during SEAM formation at single-cell resolution and highlight the potential of hiPSC-derived systems as powerful platforms for modelling human eye development and disease.

Retinoic acid signaling regulates spatiotemporal specification of human green and red cones.

Trichromacy is unique to primates among placental mammals, enabled by blue (short/S), green (medium/M), and red (long/L) cones. In humans, great apes, and Old World monkeys, cones make a poorly understood choice between M and L cone subtype fates. To determine mechanisms specifying M and L cones, we developed an approach to visualize expression of the highly similar M- and L-opsin mRNAs. M-opsin was observed before L-opsin expression during early human eye development, suggesting that M cones are generated before L cones. In adult human tissue, the early-developing central retina contained a mix of M and L cones compared to the late-developing peripheral region, which contained a high proportion of L cones. Retinoic acid (RA)-synthesizing enzymes are highly expressed early in retinal development. High RA signaling early was sufficient to promote M cone fate and suppress L cone fate in retinal organoids. Across a human population sample, natural variation in the ratios of M and L cone subtypes was associated with a noncoding polymorphism in the NR2F2 gene, a mediator of RA signaling. Our data suggest that RA promotes M cone fate early in development to generate the pattern of M and L cones across the human retina.

Ethanol Causes Cell Death and Neuronal Differentiation Defect During Initial Neurogenesis of the Neural Retina by Disrupting Calcium Signaling in Human Retinal Organoids.

Fetal Alcohol Syndrome (FAS) affects a significant proportion, exceeding 90%, of afflicted children, leading to severe ocular aberrations such as microphthalmia and optic nerve hypoplasia. During the early stages of pregnancy, the commencement of neural retina neurogenesis represents a critical period for human eye development, concurrently exposing the developing retinal structures to the highest risk of prenatal ethanol exposure due to a lack of awareness. Despite the paramount importance of this period, the precise influence and underlying mechanisms of short-term ethanol exposure on the developmental process of the human neural retina have remained largely elusive. In this study, we utilize the human embryonic stem cells derived retinal organoids (hROs) to recapitulate the initial retinal neurogenesis and find that 1% (v/v) ethanol slows the growth of hROs by inducing robust cell death and retinal ganglion cell differentiation defect. Bulk RNA-seq analysis and two-photon microscope live calcium imaging reveal altered calcium signaling dynamics derived from ethanol-induced down-regulation of RYR1 and CACNA1S. Moreover, the calcium-binding protein RET, one of the downstream effector genes of the calcium signaling pathway, synergistically integrates ethanol and calcium signals to abort neuron differentiation and cause cell death. To sum up, our study illustrates the effect and molecular mechanism of ethanol on the initial neurogenesis of the human embryonic neural retina, providing a novel interpretation of the ocular phenotype of FAS and potentially informing preventative measures for susceptible populations.

Quality assessment of polymer materials for human model eye development.

We developed model eyes using six polymer materials to determine which materials were most appropriate in simulating real human sclera and extraocular muscle (EOM). Five three-dimensional (3-D) printed polymers (FlexFill, PolyFlex, PCTPE, Soft PLA, and NinjaFlex) and one silicone material were systematically tested by board-certified ophthalmologists and senior ophthalmology residents. Material testing included scleral passes with 6-0 Vicryl sutures through each eye model. Participants completed a survey designed to collect demographic data, subjective assessment of each material's accuracy in simulating real human sclera and EOM, and a ranking for each polymer material to identify which would be most suitable for an ophthalmic surgery training tool. The Wilcoxon signed-rank test was conducted to determine if there was a statistically significant difference in the distribution of ranks between the polymer materials. The distribution of ranks for silicone material's "sclera" and "EOM" components were statistically significantly higher than that of all other polymer materials (all p < 0.05). Silicone material received the highest rank for both "sclera" and "EOM" components. Survey results indicated that the silicone material effectively simulated real human tissue. Silicone model eyes performed better than 3-D printed polymers as an educational tool for incorporation into a microsurgical training curriculum. Silicone models provide a low-cost teaching tool that allows for independent practice of microsurgical techniques without requiring a wet-laboratory facility.

PAX6 disease models for aniridia.

Aniridia is a pan-ocular genetic developmental eye disorder characterized by complete or partial iris and foveal hypoplasia, for which there is no treatment currently. Progressive sight loss can arise from cataracts, glaucoma, and aniridia-related keratopathy, which can be managed conservatively or through surgical intervention. The vast majority of patients harbor heterozygous mutations involving the PAX6 gene, which is considered the master transcription factor of early eye development. Over the past decades, several disease models have been investigated to gain a better understanding of the molecular pathophysiology, including several mouse and zebrafish strains and, more recently, human-induced pluripotent stem cells (hiPSCs) derived from aniridia patients. The latter provides a more faithful cellular system to study early human eye development. This review outlines the main aniridia-related animal and cellular models used to study aniridia and highlights the key discoveries that are bringing us closer to a therapy for patients.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

SummaryLeber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

Expression of Cell Cycle Markers and Proliferation Factors during Human Eye Embryogenesis and Tumorigenesis.

The expression pattern of the markers p19, Ki-67, MSX1, MSX2, PDL1, pRB, and CYCLINA2 was quantitatively and semiquantitatively analyzed in histologic sections of the developing and postnatal human eye at week 8, in retinoblastoma, and in various uveal melanomas post hoc studies by double immunofluorescence. The p19 immunoreactivity characterized retinal and/or choroidal cells in healthy and tumor tissues: expression was lower in the postnatal retina than in the developing retina and retinoblastoma, whereas it was high in epithelioid melanomas. Ki67 expression was high in the developing eye, retinoblastoma, and choroidal melanomas. MSX1 and MSX2 expression was similar in the developing eye and retinoblastoma, whereas it was absent in the postnatal eye. Their different expression was evident between epithelioid and myxoid melanomas. Similarly, PDL1 was absent in epithelioid melanomas, whereas it was highly expressed in developing and tumor tissues. Expression of pRB and CYCA2 was characteristic of developing and tumorous eye samples but not of the healthy postnatal eye. The observed expression differences of the analyzed markers correlate with the origin and stage of cell differentiation of the tissue samples. The fine balance of expression could play a role in both human eye development and ocular tumorigenesis. Therefore, understanding their relationship and interplay could open new avenues for potential therapeutic interventions and a better understanding of the mechanisms underlying the developmental plasticity of the eye and the development of neoplasms.

PAX6-positive microglia evolve locally in hiPSC-derived ocular organoids.

SummaryMicroglia are the resident immune cells of the central nervous system (CNS). They govern the immunogenicity of the retina, which is considered to be part of the CNS; however, it is not known how microglia develop in the eye. Here, we studied human-induced pluripotent stem cells (hiPSCs) that had been expanded into a self-formed ectodermal autonomous multi-zone (SEAM) of cells that partially mimics human eye development. Our results indicated that microglia-like cells, which have characteristics of yolk-sac-like linage cells, naturally develop in 2D eye-like SEAM organoids, which lack any vascular components. These cells are unique in that they are paired box protein 6 (PAX6)-positive, yet they possess some characteristics of mesoderm. Collectively, the data support the notion of the existence of an isolated, locally developing immune system in the eye, which is independent of the body’s vasculature and general immune system.

Ocular Disorders and Stem Cell Therapy

Sustenance of visual function is the ultimate focus of ophthalmologists. Failure of complete recovery of visual function and complications that follow conventional treatments have shifted search to a new form of therapy using stem cells. Stem cell progenitors play a major role in replenishing degenerated cells despite being present in low quantity and quiescence in our body. Unlike other tissues and cells, regeneration of new optic cells responsible for visual function is rarely observed. Understanding the transcription factors and genes responsible for optic cells development will assist scientists in formulating a strategy to activate and direct stem cells renewal and differentiation. We review the processes of human eye development and address the strategies that have been exploited in an effort to regain visual function in the preclinical and clinical state. The update of clinical findings of patients receiving stem cell treatment is also presented.

CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids.

Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide “retina-like” structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.

Retinal organoids: a window into human retinal development.

ABSTRACTRetinal development and maturation are orchestrated by a series of interacting signalling networks that drive the morphogenetic transformation of the anterior developing brain. Studies in model organisms continue to elucidate these complex series of events. However, the human retina shows many differences from that of other organisms and the investigation of human eye development now benefits from stem cell-derived organoids. Retinal differentiation methods have progressed from simple 2D adherent cultures to self-organising micro-physiological systems. As models of development, these have collectively offered new insights into the previously unexplored early development of the human retina and informed our knowledge of the key cell fate decisions that govern the specification of light-sensitive photoreceptors. Although the developmental trajectories of other retinal cell types remain more elusive, the collation of omics datasets, combined with advanced culture methodology, will enable modelling of the intricate process of human retinogenesis and retinal disease in vitro.

Molecular diagnostic challenges for non-retinal developmental eye disorders in the United Kingdom.

Overall, approximately one‐quarter of patients with genetic eye diseases will receive a molecular diagnosis. Patients with developmental eye disorders face a number of diagnostic challenges including phenotypic heterogeneity with significant asymmetry, coexisting ocular and systemic disease, limited understanding of human eye development and the associated genetic repertoire, and lack of access to next generation sequencing as regarded not to impact on patient outcomes/management with cost implications. Herein, we report our real world experience from a pediatric ocular genetics service over a 12 month period with 72 consecutive patients from 62 families, and that from a cohort of 322 patients undergoing whole genome sequencing (WGS) through the Genomics England 100,000 Genomes Project; encompassing microphthalmia, anophthalmia, ocular coloboma (MAC), anterior segment dysgenesis anomalies (ASDA), primary congenital glaucoma, congenital cataract, infantile nystagmus, and albinism. Overall molecular diagnostic rates reached 24.9% for those recruited to the 100,000 Genomes Project (73/293 families were solved), but up to 33.9% in the clinic setting (20/59 families). WGS was able to improve genetic diagnosis for MAC patients (15.7%), but not for ASDA (15.0%) and congenital cataracts (44.7%). Increased sample sizes and accurate human phenotype ontology (HPO) terms are required to improve diagnostic accuracy. The significant mixed complex ocular phenotypes distort these rates and lead to missed variants if the correct gene panel is not applied. Increased molecular diagnoses will help to explain the genotype–phenotype relationships of these developmental eye disorders. In turn, this will lead to improved integrated care pathways, understanding of disease, and future therapeutic development.

PAX 6D instructs neural retinal specification from human embryonic stem cell‐derived neuroectoderm

PAX6 is essential for neural retina (NR) and forebrain development but how PAX6 instructs NR versus forebrain specification remains unknown. We found that the paired-less PAX6, PAX6D, is expressed in NR cells during human eye development and along human embryonic stem cell (hESC) specification to retinal cells. hESCs deficient for PAX6D failed to enter NR specification. Induced expression of PAX6D but not PAX6A in a PAX6-null background restored the NR specification capacity. ChIP-Seq, confirmed by functional assays, revealed a set of retinal genes and non-retinal neural genes that are potential targets of PAX6D, including WNT8B. Inhibition of WNTs or knocking down of WNT8B restored the NR specification capacity of neuroepithelia with PAX6D knockout, whereas activation of WNTs blocked NR specification even when PAX6D was induced. Thus, PAX6D specifies neuroepithelia to NR cells via the regulation of WNT8B.

Ocular surface ectoderm instigated by WNT inhibition and BMP4.

We sought to elucidate how and when the ocular surface ectoderm commits to its differentiation into the corneal epithelium in eye development from human induced pluripotent stem cells (hiPSCs) under the influence of WNT signaling and the actions of BMP4. These signals are key drivers ocular surface ectodermal cell fate determination. It was discovered that secreted frizzled related protein-2 (SFRP2) and Dickkopf1 (DKK1), which are expressed in neural ectoderm, are both influential in the differentiation of hiPSCs, where they act as canonical WNT antagonists. BMP4, moreover, was found to simultaneously initiate non-neural ectodermal differentiation into a corneal epithelial lineage. Combined treatment of hiPSCs with exogenous BMP4 aligned to WNT inhibition for the initial four days of differentiation increased the ocular surface ectodermal cell population and induced a corneal epithelial phenotype. Specification of a surface ectodermal lineage and its fate is thus determined by a fine balance of BMP4 exposure and WNT inhibition in the very earliest stages of human eye development.

Three-Dimensional Model of the Human Eye Development based on Computer Tomograph Images.

The objective of this study was to obtain a virtual biomechanical three dimensional model of the human eye though a multidisciplinary collaboration between researchers in various medical and informational fields in order to reach a better understanding of the optical performance of the healthy and diseased eye. Material and Method. In order to obtain the virtual model, we analyzed the CT and MRI images of six patients, aged between 21 and 80 years old, dating from February 2013 until January 2019. These scans totalized 4226 images. We selected to use for the construction of the model the CT images of a male patient of 54 years old. In Vesalius and Geomagic for SolidWorks programs were used. Results. Based on the CT images analysis and using the above mentioned programs, we created a virtual model of the human skull in which the orbit is located, including the eye globe and the extraocular muscles. The SolidWorks virtual model allows the attachment of materials with real properties of the eye tissues. This model can be used in various simulations for the healthy and diseased eye. Conclusions. The biomechanical eye model of the eye was created based on a “in vivo” eye model. As the SolidWorks format enables using materials with identical properties to those of the human eyeball, this virtual model can provide very realistic eye simulations.

The Molecular Basis of Human Anophthalmia and Microphthalmia.

Human eye development is coordinated through an extensive network of genetic signalling pathways. Disruption of key regulatory genes in the early stages of eye development can result in aborted eye formation, resulting in an absent eye (anophthalmia) or a small underdeveloped eye (microphthalmia) phenotype. Anophthalmia and microphthalmia (AM) are part of the same clinical spectrum and have high genetic heterogeneity, with >90 identified associated genes. By understanding the roles of these genes in development, including their temporal expression, the phenotypic variation associated with AM can be better understood, improving diagnosis and management. This review describes the genetic and structural basis of eye development, focusing on the function of key genes known to be associated with AM. In addition, we highlight some promising avenues of research involving multiomic approaches and disease modelling with induced pluripotent stem cell (iPSC) technology, which will aid in developing novel therapies.

Genetic Variation in Human Gene Regulatory Factors Uncovers Regulatory Roles in Local Adaptation and Disease.

Differences in gene regulation have been suggested to play essential roles in the evolution of phenotypic changes. Although DNA changes in cis-regulatory elements affect only the regulation of its corresponding gene, variations in gene regulatory factors (trans) can have a broader effect, because the expression of many target genes might be affected. Aiming to better understand how natural selection may have shaped the diversity of gene regulatory factors in human, we assembled a catalog of all proteins involved in controlling gene expression. We found that at least five DNA-binding transcription factor classes are enriched among genes located in candidate regions for selection, suggesting that they might be relevant for understanding regulatory mechanisms involved in human local adaptation. The class of KRAB-ZNFs, zinc-finger (ZNF) genes with a Krüppel-associated box, stands out by first, having the most genes located on candidate regions for positive selection. Second, displaying most nonsynonymous single nucleotide polymorphisms (SNPs) with high genetic differentiation between populations within these regions. Third, having 27 KRAB-ZNF gene clusters with high extended haplotype homozygosity. Our further characterization of nonsynonymous SNPs in ZNF genes located within candidate regions for selection, suggests regulatory modifications that might influence the expression of target genes at population level. Our detailed investigation of three candidate regions revealed possible explanations for how SNPs may influence the prevalence of schizophrenia, eye development, and fertility in humans, among other phenotypes. The genetic variation we characterized here may be responsible for subtle to rough regulatory changes that could be important for understanding human adaptation.

Effect of arsenic exposure on early eye development in zebrafish (Danio rerio).

Arsenic is a metalloid that contaminates drinking water supplies worldwide. Owing to concerns for human health, the World Health Organization and the US Environmental Protection Agency have established a safe level in drinking water of ≤10ppb. Recently, arsenic exposure has also been linked to lower IQ values in children. The effect of arsenic on neurogenesis, specifically eye development, has not been widely explored. This study aimed to examine the effect of environmentally relevant concentrations of arsenic on early eye development by morphological and molecular analysis. The zebrafish, Danio rerio, was chosen to model the impact of arsenic on retinogenesis because of similarities to human eye development. Arsenic exposure to zebrafish embryos resulted in a significant increase in eye diameter at 14days postfertilization. This was coupled with a trend in thinning of the retinal pigmented epithelium (RPE) layer in embryos exposed to 500ppb arsenic. Reverse transcription-quantitative polymerase chain reaction analysis of genes associated with eye development revealed differential expression of Pax6a, Pax2a, Ngn1, Sox2 and Shha relative to control. Pax6a, Pax2a and Sox2 are important in the formation of the RPE. Proper formation of the RPE is necessary for growth of the sclera, which, in turn, is responsible for maintaining the shape of the eye. This could potentially be explained by the disruption of gene expression under arsenic exposure during critical time points in early eye development. These results provide insight into the effects arsenic may be having on early eye development in children exposed to contaminated drinking water supplies.

Human eye conditions: insights from the fly eye.

The fruit fly Drosophila melanogaster has served as an excellent model to study and understand the genetics of many human diseases from cancer to neurodegeneration. Studying the regulation of growth, determination and differentiation of the compound eyes of this fly, in particular, have provided key insights into a wide range of diseases. Here we review the regulation of the development of fly eyes in light of shared aspects with human eye development. We also show how understanding conserved regulatory pathways in eye development together with the application of tools for genetic screening and functional analyses makes Drosophila a powerful model to diagnose and characterize the genetics underlying many human eye conditions, such as aniridia and retinitis pigmentosa. This further emphasizes the importance and vast potential of basic research to underpin applied research including identifying and treating the genetic basis of human diseases.

Novel mutations in ALDH1A3 associated with autosomal recessive anophthalmia/microphthalmia, and review of the literature

BackgroundAutosomal recessive anophthalmia and microphthalmia are rare developmental eye defects occurring during early fetal development. Syndromic and non-syndromic forms of anophthalmia and microphthalmia demonstrate extensive genetic and allelic heterogeneity. To date, disease mutations have been identified in 29 causative genes associated with anophthalmia and microphthalmia, with autosomal dominant, autosomal recessive and X-linked inheritance patterns described. Biallelic ALDH1A3 gene variants are the leading genetic causes of autosomal recessive anophthalmia and microphthalmia in countries with frequent parental consanguinity.MethodsThis study describes genetic investigations in two consanguineous Pakistani families with a total of seven affected individuals with bilateral non-syndromic clinical anophthalmia.ResultsUsing whole exome and Sanger sequencing, we identified two novel homozygous ALDH1A3 sequence variants as likely responsible for the condition in each family; missense mutation [NM_000693.3:c.1240G > C, p.Gly414Arg; Chr15:101447332G > C (GRCh37)] in exon 11 (family 1), and, a frameshift mutation [NM_000693.3:c.172dup, p.Glu58Glyfs*5; Chr15:101425544dup (GRCh37)] in exon 2 predicted to result in protein truncation (family 2).ConclusionsThis study expands the molecular spectrum of pathogenic ALDH1A3 variants associated with anophthalmia and microphthalmia, and provides further insight of the key role of the ALDH1A3 in human eye development.