Temporal Transcriptomic Profiling of the Developing Xenopus laevis Eye.

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA-sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and included regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth.

Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation.

Background: Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. Results: We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. Conclusion: These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.

Early-life exposure to five biodegradable plastics impairs eye development and visually-mediated behavior through disturbing hypothalamus-pituitary-thyroid (HPT) axis in zebrafish larvae.

Biodegradable plastics have been commonly developed and applied as an alternative to traditional plastics, which cause environmental plastic pollution. However, biodegradable plastics still present limitations such as stringent degradation conditions and slow degradation rate, and may cause harm to the environment and organisms. Consequently, in this study, zebrafish was used to evaluate the effects of five biodegradable microplastics (MPs), polyglycolic acid (PGA), polylactic acid (PLA), polybutylene succinate (PBS), polyhydroxyalkanoate (PHA) and polybutylene adipate terephthalate (PBAT) exposure on the early development, retina morphology, visually-mediated behavior, and thyroid signaling at concentrations of 1 mg/L and 100 mg/L. The results indicated that all MPs induced decreased survival rate, reduced body length, smaller eyes, and smaller heads, affecting the early development of zebrafish larvae. Moreover, the thickness of retinal layers, including inner plexiform layer (IPL), outer nuclear layer (ONL), and retinal ganglion layer (RGL) was decreased, and the expression of key genes related to eye and retinal development was abnormally altered after all MPs exposure. Exposure to PBS and PBAT led to abnormal visually-mediated behavior, indicating likely affected the visual function. All MPs could also cause thyroid system disorders, among which alterations in the thyroid hormone receptors (TRs) genes could affect the retinal development of zebrafish larvae. In summary, biodegradable MPs exhibited eye developmental toxicity and likely impaired the visual function in zebrafish larvae. This provided new evidence for revealing the effects of biodegradable plastics on aquatic organism development and environmental risks to aquatic ecosystems.

The intracellular domain of Sema6A is essential for development of the zebrafish retina.

Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development.

Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants.

Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position 71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1306 are likely benign. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.

Caspase-8 promotes NLRP3 inflammasome activation mediates eye development defects in zebrafish larvae exposed to perfulorooctane sulfonate (PFOS).

Epidemiological evidence showed that serum high perfluorooctane sulfonate (PFOS) levels are associated with multiple eye related diseases, but the potential underlying molecular mechanisms remain poorly understood. Zebrafish and photoreceptor cell (661w) models were used to investigate the molecular mechanism of PFOS induced eye development defects. Our results showed a novel molecular mechanism of PFOS-induced inflammation response-mediated photoreceptor cell death associated with eye development defects. Inhibition of Caspase-8 activation significantly decreased photoreceptor cell death in PFOS exposure. Mechanistically, Toll-like receptor 4 (TLR4) mediates activation of Caspase-8 promote activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome to elicit maturation of interleukin-1 beta (IL-1ß) via Caspase-1 activation, facilitating photoreceptor cell inflammation damage in PFOS exposure. In addition, we also made a novel finding that Caspase-3 activation was increased via Caspase-8 activation and directly intensified cell death. Our results show the important role of Caspase-8 activation in PFOS induced eye development defects and highlight Caspase-8 mediated activation of the NLRP3 inflammation triggers activation of Caspase-1 and promote the maturation of IL-1ß in retinal inflammatory injury.

Shedding a Light on Dark Genes: A Comparative Expression Study of PRR12 Orthologues during Zebrafish Development.

Haploinsufficiency of the PRR12 gene is implicated in a human neuro-ocular syndrome. Although identified as a nuclear protein highly expressed in the embryonic mouse brain, PRR12 molecular function remains elusive. This study explores the spatio-temporal expression of zebrafish PRR12 co-orthologs, prr12a and prr12b, as a first step to elucidate their function. In silico analysis reveals high evolutionary conservation in the DNA-interacting domains for both orthologs, with significant syntenic conservation observed for the prr12b locus. In situ hybridization and RT-qPCR analyses on zebrafish embryos and larvae reveal distinct expression patterns: prr12a is expressed early in zygotic development, mainly in the central nervous system, while prr12b expression initiates during gastrulation, localizing later to dopaminergic telencephalic and diencephalic cell clusters. Both transcripts are enriched in the ganglion cell and inner neural layers of the 72 hpf retina, with prr12b widely distributed in the ciliary marginal zone. In the adult brain, prr12a and prr12b are found in the cerebellum, amygdala and ventral telencephalon, which represent the main areas affected in autistic patients. Overall, this study suggests PRR12's potential involvement in eye and brain development, laying the groundwork for further investigations into PRR12-related neurobehavioral disorders.

Pax6 isoforms shape eye development: Insights from developmental stages and organoid models.

Pax6 is a critical transcription factor involved in the development of the central nervous system. However, in humans, mutations in Pax6 predominantly result in iris deficiency rather than neurological phenotypes. This may be attributed to the distinct functions of Pax6 isoforms, Pax6a and Pax6b. In this study, we investigated the spatial and temporal expression patterns of Pax6 isoforms during different stages of mouse eye development. We observed a strong correlation between Pax6a expression and the neuroretina gene Sox2, while Pax6b showed a high correlation with iris-component genes, including the mesenchymal gene Foxc1. During early patterning from E10.5, Pax6b was expressed in the hinge of the optic cup and neighboring mesenchymal cells, whereas Pax6a was absent in these regions. At E14.5, both Pax6a and Pax6b were expressed in the future iris and ciliary body, coinciding with the integration of mesenchymal cells and Mitf-positive cells in the outer region. From E18.5, Pax6 isoforms exhibited distinct expression patterns as lineage genes became more restricted. To further validate these findings, we utilized ESC-derived eye organoids, which recapitulated the temporal and spatial expression patterns of lineage genes and Pax6 isoforms. Additionally, we found that the spatial expression patterns of Foxc1 and Mitf were impaired in Pax6b-mutant ESC-derived eye organoids. This in vitro eye organoids model suggested the involvement of Pax6b-positive local mesodermal cells in iris development. These results provide valuable insights into the regulatory roles of Pax6 isoforms during iris and neuroretina development and highlight the potential of ESC-derived eye organoids as a tool for studying normal and pathological eye development.

Human cytomegalovirus infection coopts chromatin organization to diminish TEAD1 transcription factor activity.

Human cytomegalovirus (HCMV) infects up to 80% of the world's population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hours after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon-6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV's established roles in these processes.

Inter-plane feedback coordinates cell morphogenesis and maintains 3D tissue organization in the Drosophila pupal retina.

How complex organs coordinate cellular morphogenetic events to achieve three-dimensional (3D) form is a central question in development. The question is uniquely tractable in the late Drosophila pupal retina, where cells maintain stereotyped contacts as they elaborate the specialized cytoskeletal structures that pattern the apical, basal and longitudinal planes of the epithelium. In this study, we combined cell type-specific genetic manipulation of the cytoskeletal regulator Abelson (Abl) with 3D imaging to explore how the distinct cellular morphogenetic programs of photoreceptors and interommatidial pigment cells (IOPCs) organize tissue pattern to support retinal integrity. Our experiments show that photoreceptor and IOPC terminal differentiation is unexpectedly interdependent, connected by an intercellular feedback mechanism that coordinates and promotes morphogenetic change across orthogonal tissue planes to ensure correct 3D retinal pattern. We propose that genetic regulation of specialized cellular differentiation programs combined with inter-plane mechanical feedback confers spatial coordination to achieve robust 3D tissue morphogenesis.

Osmosis as nature's method for establishing optical alignment.

For eyes to maintain optimal focus, precise coordination is required between lens optics and retina position, a mechanism that in vertebrates is governed by genetics, visual feedback, and possibly intraocular pressure (IOP).1 While the underlying processes have been intensely studied in vertebrates, they remain elusive in arthropods, though visual feedback may be unimportant.2 How do arthropod eyes remain functional while undergoing substantial growth? Here, we test whether a common physiological process, osmoregulation,3 could regulate growth in the sophisticated camera-type eyes of the predatory larvae of Thermonectus marmoratus diving beetles. Upon molting, their eye tubes elongate in less than an hour, and osmotic pressure measurements reveal that this growth is preceded by a transient increase in hemolymph osmotic pressure. Histological evaluation of support cells that determine the lens-to-retina spacing reveals swelling rather than the addition of new cells. In addition, as expected, treating larvae with hyperosmotic media post-molt leads to far-sighted (hyperopic) eyes due to a failure of proper lengthening of the eye tube and results in impaired hunting success. This study suggests that osmoregulation could be of ubiquitous importance for properly focused eyes.

CRK and NCK adaptors may functionally overlap in zebrafish neurodevelopment, as indicated by common binding partners and overlapping expression patterns.

CRK adaptor proteins are important for signal transduction mechanisms driving cell proliferation and positioning during vertebrate central nervous system development. Zebrafish lacking both CRK family members exhibit small, disorganized retinas with 50% penetrance. The goal of this study was to determine whether another adaptor protein might functionally compensate for the loss of CRK adaptors. Expression patterns in developing zebrafish, and bioinformatic analyses of the motifs recognized by their SH2 and SH3 domains, suggest NCK adaptors are well-positioned to compensate for loss of CRK adaptors. In support of this hypothesis, proteomic analyses found CRK and NCK adaptors share overlapping interacting partners including known regulators of cell adhesion and migration, suggesting their functional intersection in neurodevelopment.

Exposure of male adult zebrafish (Danio rerio) to triphenyl phosphate (TPhP) induces eye development disorders and disrupts neurotransmitter system-mediated abnormal locomotor behavior in larval offspring.

Triphenyl phosphate (TPhP) is a widely used organophosphorus flame retardant, which has become ubiquitous in the environment. However, little information is available regarding its transgenerational effects. This study aimed to investigate the developmental toxicity of TPhP on F1 larvae offspring of adult male zebrafish exposed to various concentrations of TPhP for 28 or 60 days. The findings revealed significant morphological changes, alterations in locomotor behavior, variations in neurotransmitter, histopathological changes, oxidative stress levels, and disruption of Retinoic Acid (RA) signaling in the F1 larvae. After 28 and 60 days of TPhP exposure, the F1 larvae exhibited a myopia-like phenotype with pathological alterations in the lens and retina. The genes involved in the RA signaling pathway were down-regulated following parental TPhP exposure. Swimming speed and total distance of F1 larvae were significantly reduced by TPhP exposure, and long-term exposure to environmental levels of TPhP had more pronounced effects on locomotor behavior and neurotransmitter levels. In conclusion, TPhP induced histological and morphological alterations in the eyes of F1 larvae, leading to visual dysfunction, disruption of RA signaling and neurotransmitter systems, and ultimately resulting in neurobehavioral abnormalities. These findings highlight the importance of considering the impact of TPhP on the survival and population reproduction of wild larvae.

Combined inactivation of RB and Hippo converts differentiating Drosophila photoreceptors into eye progenitor cells through derepression of homothorax.

The retinoblastoma (RB) and Hippo pathways interact to regulate cell proliferation and differentiation. However, the mechanism of interaction is not fully understood. Drosophila photoreceptors with inactivated RB and Hippo pathways specify normally but fail to maintain their neuronal identity and dedifferentiate. We performed single-cell RNA sequencing to elucidate the cause of dedifferentiation and to determine the fate of these cells. We find that dedifferentiated cells adopt a progenitor-like fate due to inappropriate activation of the retinal differentiation suppressor homothorax (hth) by Yki/Sd. This results in the activation of a distinct Yki/Hth transcriptional program, driving photoreceptor dedifferentiation. We show that Rbf physically interacts with Yki and, together with the GAGA factor, inhibits the hth expression. Thus, RB and Hippo pathways cooperate to maintain photoreceptor differentiation by preventing inappropriate expression of hth in differentiating photoreceptors. Our work highlights the importance of both RB and Hippo pathway activities for maintaining the state of terminal differentiation.

SWI/SNF complexes are required for retinal pigmented epithelium differentiation and for the inhibition of cell proliferation and neural differentiation programs.

During embryonic development, tissue-specific transcription factors and chromatin remodelers function together to ensure gradual, coordinated differentiation of multiple lineages. Here, we define this regulatory interplay in the developing retinal pigmented epithelium (RPE), a neuroectodermal lineage essential for the development, function and maintenance of the adjacent retina. We present a high-resolution spatial transcriptomic atlas of the developing mouse RPE and the adjacent ocular mesenchyme obtained by geographical position sequencing (Geo-seq) of a single developmental stage of the eye that encompasses young and more mature ocular progenitors. These transcriptomic data, available online, reveal the key transcription factors and their gene regulatory networks during RPE and ocular mesenchyme differentiation. Moreover, conditional inactivation followed by Geo-seq revealed that this differentiation program is dependent on the activity of SWI/SNF complexes, shown here to control the expression and activity of RPE transcription factors and, at the same time, inhibit neural progenitor and cell proliferation genes. The findings reveal the roles of the SWI/SNF complexes in controlling the intersection between RPE and neural cell fates and the coupling of cell-cycle exit and differentiation.

Restoration of functional PAX6 in aniridia patient iPSC-derived ocular tissue models using repurposed nonsense suppression drugs.

Congenital aniridia is a rare, pan-ocular disease causing severe sight loss, with only symptomatic intervention offered to patients. Approximately 40% of aniridia patients present with heterozygous nonsense variants in PAX6, resulting in haploinsufficiency. Translational readthrough-inducing drugs (TRIDs) have the ability to weaken the recognition of in-frame premature termination codons (PTCs), permitting full-length protein to be translated. We established induced pluripotent stem cell (iPSC)-derived 3D optic cups and 2D limbal epithelial stem cell (LESC) models from two aniridia patients with prevalent PAX6 nonsense mutations. Both in vitro models show reduced PAX6 protein levels, mimicking the disease. The repurposed TRIDs amlexanox and 2,6-diaminopurine (DAP) and the positive control compounds ataluren and G418 were tested for their efficiency. Amlexanox was identified as the most promising TRID, increasing full-length PAX6 levels in both models and rescuing the disease phenotype through normalization of VSX2 and cell proliferation in the optic cups and reduction of ABCG2 protein and SOX10 expression in LESCs. This study highlights the significance of patient iPSC-derived cells as a new model system for aniridia and proposes amlexanox as a new putative treatment for nonsense-mediated aniridia.

Retinoic Acid Receptor-Related Orphan Receptors (RORs) in Eye Development and Disease.

The retinoic acid receptor-related orphan receptors (RORs) are ligand-mediated transcription factors with important biological roles in regulating circadian rhythms, metabolism, immunity, angiogenesis, inflammation, and development. They belong to the superfamily of nuclear receptors and include three family members: RORα, RORß, and RORγ. Currently identified ROR ligands include cholesterol and cholesterol derivatives for RORα and RORγ, and stearic acid and all-trans retinoic acid for RORß. Aberrant signaling of the RORs is involved in the pathogenesis of several human diseases including autoimmune diseases, metabolic disorders, and certain cancers. In the eye, RORs regulate normal development of the lens and the retina, and also contribute to potentially blinding eye diseases, especially retinal vascular diseases. Here, we review the role of RORs in eye development and disease to highlight their potential as druggable targets for therapeutic development in retinal vascular and degenerative diseases.

Ultrasonographic monitoring of fetal eye growth parameters throughout gestation in the common marmoset (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is considered an ideal species for developing genetically modified nonhuman primates (NHP) models of human disease, particularly eye disease. They have been proposed as a suitable bridge between rodents and other NHP models due to their similar ophthalmological features to humans. Prenatal ultrasonography is an accurate and reliable diagnostic tool for monitoring fetal development and congenital malformation. We monitored fetal eye growth and development using noninvasive ultrasonography in 40 heads of clinically normal fetuses during pregnancy to establish the criteria for studying congenital eye anomalies in marmosets. The coronal, sagittal, and transverse planes were useful to identify the facial structures for any associated abnormalities. For orbital measurements, biorbital distance (BOD), ocular diameter (OD), interorbital distance (IOD), and total axial length (TAL) were measured in the transverse plane and carefully identified for intraorbital structures. As a result, high correlations were observed between delivery-based gestational age (GA) and biparietal diameter (BPD), BOD, OD, and TAL. The correlation assessments based on BOD provide more reliable results for monitoring eye growth and development in normal marmosets than any other parameters since BOD has the highest correlation coefficient according to both delivery-based GA and BPD among ocular measurements. In conclusion, orbital measurements by prenatal ultrasonography provide reliable indicators of marmoset eye growth, and it could offer early diagnostic criteria to facilitate the development of eye disease models and novel therapies such as genome editing technologies in marmosets.

Knockout of sws2a and sws2b in Medaka (Oryzias latipes) Reveals Their Roles in Regulating Vision-Guided Behavior and Eye Development.

The medaka (Oryzias latipes) is an excellent vertebrate model for studying the development of the retina. Its genome database is complete, and the number of opsin genes is relatively small compared to zebrafish. Short wavelength sensitive 2 (sws2), a G-protein-coupled receptor expressed in the retina, has been lost in mammals, but its role in eye development in fish is still poorly understood. In this study, we established a sws2a and sws2b knockout medaka model by CRISPR/Cas9 technology. We discovered that medaka sws2a and sws2b are mainly expressed in the eyes and may be regulated by growth differentiation factor 6a (gdf6a). Compared with the WT, sws2a-/- and sws2b-/- mutant larvae displayed an increase in swimming speed during the changes from light to dark. We also observed that sws2a-/- and sws2b-/- larvae both swam faster than WT in the first 10 s of the 2 min light period. The enhanced vision-guided behavior in sws2a-/- and sws2b-/- medaka larvae may be related to the upregulation of phototransduction-related genes. Additionally, we also found that sws2b affects the expression of eye development genes, while sws2a is unaffected. Together, these findings indicate that sws2a and sws2b knockouts increase vision-guided behavior and phototransduction, but on the other hand, sws2b plays an important role in regulating eye development genes. This study provides data for further understanding of the role of sws2a and sws2b in medaka retina development.