Regulator Of Eye Development Research Articles

Novel and recurrent variants in PAX6 in four patients with ocular phenotypes from Southeast Asia.

Aniridia is an autosomal dominant condition characterized by the complete or partial absence of the iris, often with additional presentations such as foveal hypoplasia, nystagmus, cataract, glaucoma and other ocular abnormalities. Most cases are caused by heterozygous mutations in the paired box 6 gene (PAX6), which codes for a transcription factor that regulates eye development. Four patients from our hospital who presented with ocular phenotypes were recruited for research sequencing with informed consent. Sanger sequencing of PAX6 coding exons or exome sequencing was performed on genomic DNA from venous blood samples. Variants in PAX6 were identified in the four patients. Two variants are recurrent single-nucleotide substitutions - one is a substitution found in a patient with bilateral aniridia, whereas the other is a splice variant in a patient with nystagmus and neuroblastoma. The other two variants are novel and found in two patients with isolated aniridia. Both are small duplications that are predicted to lead to premature termination. For the recurrent variants, the comparison of phenotypes for patients with identical variants would shed light on the mechanisms of pathogenesis, and the discovery of two novel variants expands the spectrum of PAX6 mutations.

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.

Functional Differentiation of BMP7 Genes in Zebrafish: bmp7a for Dorsal-Ventral Pattern and bmp7b for Melanin Synthesis and Eye Development.

Bone morphogenetic protein 7 (BMP7) belongs to the transforming growth factor β (TGF-β) family, which not only induces cartilage and bone formation, but also regulates eye development and melanoma tumorigenesis in mammals. In teleosts, BMP7 differentiates into two subtypes, bmp7a and bmp7b, which have clearly differentiated structures. To fully understand the functional differentiation of bmp7a and bmp7b in fish species, we successfully constructed bmp7a and bmp7b gene deletion mutants in zebrafish using CRISPR/Cas9-mediated gene editing technology. Our results showed that bmp7a mutation caused abnormal development of the embryo’s dorsal-ventral pattern that led to death; bmp7b mutation induced growth inhibition and increased melanin production in the skin and eye of mutants. Histological analysis revealed that melanin in the retina of the eyes in bmp7b mutants increased, and behavioral observation showed that the vision and sensitivity to food of the mutants were reduced. Transcriptome analysis of the skin and eye tissues showed that the expression changes of wnt7ba and gna14 in bmp7b mutants might promote the increase of melanin. Additionally, the eye transcriptome analysis indicated that changes in the structure of the eyes in bmp7b mutants led to defects in phototransduction, and seven DEGs (rgs9a, rgs9b, rcvrn2, guca1d, grk1b, opn1mw4, and gc2) were identified as key candidate genes that affected the photonic response of the eyes. The study revealed the functional differentiation of bmp7a and bmp7b in teleosts and the first report about the inhibitory effect of bmp7b on melanogenesis may provide useful information for the future research on human melanoma-related diseases.

Shutdown corner, a large deletion mutant isolated from a haploid mutagenesis screen in zebrafish.

Morphogenesis, the formation of three-dimensional organ structures, requires precise coupling of genetic regulation and complex cell behaviors. The genetic networks governing many morphogenetic systems, including that of the embryonic eye, are poorly understood. In zebrafish, several forward genetic screens have sought to identify factors regulating eye development. These screens often look for eye defects at stages after the optic cup is formed and when retinal neurogenesis is under way. This approach can make it difficult to identify mutants specific for morphogenesis, as opposed to neurogenesis. To this end, we carried out a forward genetic, small-scale haploid mutagenesis screen in zebrafish (Danio rerio) to identify factors that govern optic cup morphogenesis. We screened ∼100 genomes and isolated shutdown corner (sco), a mutant that exhibits multiple tissue defects and harbors a ∼10-Mb deletion that encompasses 89 annotated genes. Using a combination of live imaging and antibody staining, we found cell proliferation, cell death, and tissue patterning defects in the sco optic cup. We also observed other phenotypes, including paralysis, neuromuscular defects, and ocular vasculature defects. To date, the largest deletion mutants reported in zebrafish are engineered using CRISPR-Cas9 and are less than 300 kb. Because of the number of genes within the deletion interval, shutdown corner [Df(Chr05:sco)z207] could be a useful resource to the zebrafish community, as it may be helpful for gene mapping, understanding genetic interactions, or studying many genes lost in the mutant.

PAX6 missense variants in two families with isolated foveal hypoplasia and nystagmus: evidence of paternal postzygotic mosaicism

PAX6 is considered the master regulator of eye development, the majority of variants affecting this gene cause the pan-ocular developmental eye disorder aniridia. Although no genotype-phenotype correlations are clearly established, missense variants affecting the DNA-binding paired domain of PAX6 are usually associated with non-aniridia phenotypes like microphthalmia, coloboma or isolated foveal hypoplasia. In this study, we report two missense heterozygous variants in the paired domain of PAX6 resulting in isolated foveal hypoplasia with nystagmus in two independent families: c.112 C > G; p.(Arg38Gly) and c.214 G > C; p.(Gly72Arg) in exons 5 and 6, respectively. Furthermore, we provide evidence that paternal postzygotic mosaicism is the cause of inheritance, with clinically unaffected fathers and reduced affected allele fraction. This work contributes to increase the phenotypic spectrum caused by PAX6 variants, and to our knowledge, is the first report to describe the presence of postzygotic parental mosaicism causing isolated foveal hypoplasia with nystagmus. These results support the growing evidence that suggest an overestimation of sporadic cases with PAX6 variants, which has strong implications for both genetic counselling and family planning.

Eye Degeneration and Loss of otx5b Expression in the Cavefish Sinocyclocheilus tileihornes

Cave animals possess remarkable phenotypes associated with existence in their dark environments. The Chinese cavefish Sinocyclocheilus tileihornes shows substantial eye degeneration, a trait shared by most cave species. The extent to which independent evolution of troglomorphic traits uses convergent molecular genetic mechanisms is as yet unknown. We performed transcriptome-wide gene expression profiling in S. tileihornes eyes and compared results with those from the closely related surface species S. angustiporus and an independently derived congeneric cavefish, S. anophthalmus. In total, 52.85 million 100 bp long paired-end clean reads were generated for S. tileihornes, and we identified differentially expressed genes between the three possible pairs of species. Functional analysis of genes differentially expressed between S. tileihornes and S. angustiporus revealed that phototransduction (KEGG id: dre04744) was the most significantly enriched pathway, indicating the obvious differences in response to captured photons between the cavefish S. tileihornes and the surface species S. angustiporus. Analysis of key genes regulating eye development showed complete absence of otx5b (orthodenticle homolog 5) expression in S. tileihornes eyes, probably related to degradation of rods, but normal expression of crx (cone-rod homeobox). The enriched pathways and Otx5 are involved in phototransduction, photoreceptor formation, and regulation of photoreceptor-related gene expression. Unlike the S. tileihornes reported here, S. anophthalmus has reduced crx and otx5 expression. These results show that different species of cavefish within the same genus that independently evolved troglodyte characteristics can have different genetic mechanisms of eye degeneration.

Determination of Expression Patterns of Seven De-sumoylation Enzymes in Major Ocular Cell Lines.

Accumulated evidence have well established that protein sumoylation plays multiple roles in various cellular processes. In the vertebrate eye, we and others have demonstrated that sumoylation displays indispensable roles in regulating eye development. Various ocular cell lines including human embryonic cell line (FHL124), the SV40-large T-transformed human lens epithelial cell line (HLE), the SV40-large T-transformed mouse lens epithelial cell line (αTN4-1), the rabbit lens epithelial cell line (N/N1003A) and the human retina pigment epithelial cell line (ARPE-19) have been extensively used for studying various cellular functions and disease processes including sumoylation functions, and mechanisms for cataract and age-related macular degeneration (AMD). However, the sumoylation enzyme systems have not been well established. FHL124, HLE, αTN4-1, N/N1003A and ARPE-19 were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% FBS and 1% penicillin & streptomycin. The expression levels of seven SENP mRNAs were analyzed with qRT-PCR, and the expression levels of seven SENP proteins were detected with Western blot analysis. Using both qRT-PCR and Western blot analysis, we have obtained the followings: 1). The 3 human ocular cell lines, FHL124, HLE and ARPE-19 express all types of SENP mRNA and proteins. 2). In mouse lens epithelial cell line αTN4-1, and rabbit lens epithelial cells line N/N1003A, however, only the mRNAs for SENP1, 2, 3, 6 and 7 are expressed. At the protein level, SENP8 was absent in both αTN4-1 and N/N1003A cells; 3). Each cell line has different dominant SENP enzymes. For FHL124, SENP3, 5, 7 and 8 proteins are relatively dominant. SENP3, 5 and 6 are the major de-sumoylation enzymes in HLE cells. Different from human lens epithelial cells, FHL124 and HLE, human retina pigment epithelial cells (ARPE-19) have SENP3, 7, and 8 as the dominant forms of de-sumoylation enzymes. For mouse lens epithelial cells, SENP1, 3 and 7 are the major de-sumoylation enzymes. On the other hand, the rabbit lens epithelial cells have SENP1, 2 and 7 as the major isoforms. Our results for the first time defined the differential expression patterns of the seven types of de-sumoylation enzymes (SENPs) in 5 major ocular cell lines. These results help to establish the basis for the future study of sumoylation functions and the related mechanisms in vertebrate eye.

Altered Expression Patterns of the Sumoylation Enzymes E1, E2 and E3 Are Associated with Glucose Oxidase- and UVA-Induced Cataractogenesis.

Protein sumoylation is a well established regulatory mechanism that regulates chromatin structure and dynamics, cell proliferation and differentiation, stress response and cell apoptosis. In the vertebrate eye, we and others have shown that sumoylation plays an indispensable role in regulating eye development. During stress induction and aging process, the ocular tissues gradually loss their normality and develop major ocular diseases such as cataract and aging-related macular degeneration. We have recently demonstrated that sumoylation actively regulates differentiation of lens cells, whether this process is implicated in lens pathogenesis remains to be investigated. In this study, we have demonstrated that transparent mouse lenses treated with glucose oxidase and UVA irradiation undergo in vitro cataract formation, and associated with this process, the expression patterns of the 3 sumoylation enzymes have been found significantly altered. Four-week-old C57BL/6J mice were used in our experiment. Lenses were carefully excised from eyes and cultured in M199 medium (Sigma 3769) for at least 12 hours. Transparent lenses (without surgical damage) were selected for experimentation. The lenses were exposed to UVA for 60 min or treated with 30 mU/mL glucose oxidase (GO, MP Biomedicals, 1673) to induce cataract formation. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. we have obtained the following results: 1) Both GO treatment and UVA irradiation can induce cataract formation in the in vitro cultured mouse lenses; 2) With GO treatment, the mRNAs and proteins for the 5 sumoylation enzymes were all significantly downregulated; 3) With UVA irradiation, the changes in the expression patterns of the mRNAs and proteins for the SAE1, UBA2 , UBC9 and PIAS1 were opposite, while the mRNAs were upregulated either significantly (for SAE1, UBA2 and UBC9) or slightly (PIAS1), the proteins for all 4 sumoylation enzymes were downregulated; For RanBP2, the UVA induced changes in both mRNA and protein are consist with the GO treatment. Under GO and UVA irradiation conditions, the expression levels of both mRNA and protein for the three major sumoylation enzymes were significantly changed. Our results suggest that altered expression patterns of the sumoylation enzymes are associated with oxidative stressinduced cataractogenesis.

Genes and regulation of eye development

SummaryDevelopmental ocular disorders have a strong genetic component. Our laboratory has been applying whole exome sequencing (WES) to identify novel human genes associated with abnormal eye development for many years. WES provides the opportunity to simultaneously screen coding regions of all genes in order to identify causative mutations. While this comprehensive examination greatly improves the chances of mutation detection, interpretation of the large volume of data is complicated. Since many developmental processes are conserved in vertebrates, studies of zebrafish orthologs of human disease genes can provide critical information about their embryonic function and determine other genes involved in the same pathway. With this, studies of zebrafish mutants help to identify an additional pool of relevant gene candidates for corresponding human phenotypes. Moreover, zebrafish model allows for rapid in vivo testing of promising human variants by overexpression and/or rescue experiments. Our utilization of this approach in studies of zebrafish orthologs of several transcription factors, such as PITX2, PITX3 and FOXE3 associated with Axenfeld‐Rieger syndrome, anterior segment dysgenesis, cataracts and microphthalmia, will be presented.

The exocyst is required for photoreceptor ciliogenesis and retinal development

We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on exoc5, a central component of the exocyst complex, by analyzing both exoc5 zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate exoc5 mutant zebrafish lines phenocopied exoc5 morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). exoc5 knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. exoc5 mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our Exoc5 fl/fl mouse line crossed with a rhodopsin-Cre driver line. In Exoc5 photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins.

The Male Abnormal Gene Family 21 (Mab21) Members Regulate Eye Development.

The male abnormal gene family contains 3 members, named mab21l1, mab21l2 and mab21l3. Since their first discovery in C. elegans, homologues of mab21l1 and mab21l2 have been found in Drosophila, Zebrafish, Xenopus, chicken, mouse and human. A number of studies have revealed that mab21 gene family members, mab21l1 and mab21l2, play important roles in regulating eye development. Here, we review the functions of the mab genes in regulating ocular development.

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation.

The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/β-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/β-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts.

A New Gestational Diabetes Mellitus Model, Hyperglycemia-Induced Eye Malformation via Inhibiting Pax6 in Chick Embryo

ABSTRACTGestational diabetes mellitus (GDM) is one of the leading causes of fetal malformations. However, few models have been developed to study the underlying mechanisms of GDM-induced fetal eye malformation. In this study, a high concentration of glucose (0.2 mmol per egg) was injected into the air sac of chick embryos on embryo development day (EDD) 1 to develop a hyperglycemia model. Results showed that 47.3% of embryonic eye malformation happened on EDD 5. In this model, the key genes regulating eye development, Pax6, Six3 and Otx2, were downregulated by hyperglycemia. Among these genes, the expression of Pax6 was the most vulnerable to hyperglycemia, being suppressed by 70%. A reduction in Pax6 gene expression induced eye malformation in chick embryos. However, increased expression of Pax6 in chick embryos could rescue hyperglycemia-induced eye malformation. Hyperglycemia stimulated O-linked N-acetylglucosaminylation, which caused oxidative stress in chick embryos. Pax6 was found to be vulnerable to free radicals, but the antioxidant edaravone could restore Pax6 expression and reverse eye malformation. These results illustrated a successful establishment of a new chick embryo model to study the molecular mechanism of hyperglycemia-induced eye malformation. The suppression of the Pax6 gene is probably mediated by oxidative stress and could be a crucial target for the therapy of GDM-induced embryonic eye malformation.

Regulation of Eye Development by Protein Serine/Threonine Phosphatases-1 and -2A.

The protein serine/threonine phosphatases-1 and -2A are major cellular phosphatases, playing a fundamental role in organisms from prokaryotes to eukaryotes. They contribute to 90% dephosphorylation in eukaryote proteins. In the eye, both phosphatases are highly expressed and display important functions in regulating normal eye development. Moreover, they are implicated in pathogenesis through modulation of stress-induced apoptosis. Here we review the recent progresses on these aspects.

Proanthocyanidins Prevent High Glucose-Induced Eye Malformation by Restoring Pax6 Expression in Chick Embryo.

Gestational diabetes mellitus (GDM) is one of the leading causes of offspring malformations, in which eye malformation is an important disease. It has raised demand for therapy to improve fetal outcomes. In this study, we used chick embryo to establish a GDM model to study the protective effects of proanthocyanidins on eye development. Chick embryos were exposed to high glucose (0.2 mmol/egg) on embryo development day (EDD) 1. Proanthocyanidins (1 and 10 nmol/egg) were injected into the air sac on EDD 0. Results showed that both dosages of proanthocyanidins could prevent the eye malformation and rescue the high glucose-induced oxidative stress significantly, which the similar effects were showed in edaravone. However, proanthocyanidins could not decrease the glucose concentration of embryo eye. Moreover, the key genes regulating eye development, Pax6, was down-regulated by high glucose. Proanthocyanidins could restore the suppressed expression of Pax6. These results indicated proanthocyanidins might be a promising natural agent to prevent high glucose-induced eye malformation by restoring Pax6 expression.

The Hippo pathway effector Yki downregulates Wg signaling to promote retinal differentiation in the Drosophila eye

The evolutionarily conserved Hippo signaling pathway is known to regulate cell proliferation and maintain tissue homeostasis during development. We found that activation of Yorkie (Yki), the effector of the Hippo signaling pathway, causes separable effects on growth and differentiation of the Drosophila eye. We present evidence supporting a role for Yki in suppressing eye fate by downregulation of the core retinal determination genes. Other upstream regulators of the Hippo pathway mediate this effect of Yki on retinal differentiation. Here, we show that, in the developing eye, Yki can prevent retinal differentiation by blocking morphogenetic furrow (MF) progression and R8 specification. The inhibition of MF progression is due to ectopic induction of Wingless (Wg) signaling and Homothorax (Hth), the negative regulators of eye development. Modulating Wg signaling can modify Yki-mediated suppression of eye fate. Furthermore, ectopic Hth induction due to Yki activation in the eye is dependent on Wg. Last, using Cut (Ct), a marker for the antennal fate, we show that suppression of eye fate by hyperactivation of yki does not change the cell fate (from eye to antenna-specific fate). In summary, we provide the genetic mechanism by which yki plays a role in cell fate specification and differentiation - a novel aspect of Yki function that is emerging from multiple model organisms.

MicroRNAs that target RGS5.

An earlier meta-analysis on gene expression data derived from four microarray, two cDNA library, and one SAGE experiment had identified RGS5 as one of only ten non-housekeeping genes that were reported to be expressed in human trabecular meshwork (TM) cells by all studies. RGS5 encodes regulator of G-protein signaling-5. The TM tissue is the route of aqueous fluid outflow, and is relevant to the pathology of glaucoma. MicroRNAs constitute the most recently identified components of the cellular machinery for gene regulation in eukaryotic cells. Given our long standing interest in glaucoma, we aimed to identify miRNAs that may target RGS5. Eight miRNAs were selected for study using bioinformatics tools and available data on miRNAs expressed in the eye. Their effects were assessed using the dual luciferase assay. 3'-UTR segments of RGS5 mRNA were cloned downstream of a luciferase coding gene in psiCHECK2 vectors, and these were co-transfected with each of the miRNAs into HEK293 cells. The outcomes evidenced that one or more of the segments are in fact targeted by miR-7, miR-9, miR-96, miR-23a, miR-23b, miR-204, and miR-211. Down regulations by the miRNAs were statistically significant. The effect of miR-204 is considered particularly important as this miRNA is well known to regulate eye development and to affect multiple ocular functions. Our results justify further studies on regulation of RGS5 expression and RGS5 downstream functions by these miRNAs.

Mapping Gene Regulatory Networks in Drosophila Eye Development by Large-Scale Transcriptome Perturbations and Motif Inference

Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, we performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, we derived direct TF-DNA interactions using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, we found network motifs, cis-regulatory codes, and regulators of eye development. We validate the predicted target regions of Grainyhead by ChIP-seq and identify this factor as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions.

English

Aniridia are rare developmental anomalies present in 1.8/100, 000 live births. Aniridia occurs due to abnormal neuroectodermal development secondary to mutations in paired box gene 6 (PAX6) on band p13 of chromosome 11 which regulates eye development. Aniridia may be congenital or traumatic. It may occur in isolation or be associated with a number of syndromes such as WAGR. Two third of cases are familial and one third is sporadic. We report a family of aniridia with different clinical spectrum of features.

Excess caffeine exposure impairs eye development during chick embryogenesis

Caffeine has been an integral component of our diet and medicines for centuries. It is now known that over consumption of caffeine has detrimental effects on our health, and also disrupts normal foetal development in pregnant mothers. In this study, we investigated the potential teratogenic effect of caffeine over-exposure on eye development in the early chick embryo. Firstly, we demonstrated that caffeine exposure caused chick embryos to develop asymmetrical microphthalmia and induced the orbital bone to develop abnormally. Secondly, caffeine exposure perturbed Pax6 expression in the retina of the developing eye. In addition, it perturbed the migration of HNK-1+ cranial neural crest cells. Pax6 is an important gene that regulates eye development, so altering the expression of this gene might be the cause for the abnormal eye development. Thirdly, we found that reactive oxygen species (ROS) production was significantly increased in eye tissues following caffeine treatment, and that the addition of anti-oxidant vitamin C could rescue the eyes from developing abnormally in the presence of caffeine. This suggests that excess ROS induced by caffeine is one of the mechanisms involved in the teratogenic alterations observed in the eye during embryogenesis. In sum, our experiments in the chick embryo demonstrated that caffeine is a potential teratogen. It causes asymmetrical microphthalmia to develop by increasing ROS production and perturbs Pax6 expression.