Early Eye Development Research Articles

A Novel Mutation in Cse1l Disrupts Brain and Eye Development with Specific Effects on Pax6 Expression.

Forward genetics in the mouse continues to be a useful and unbiased approach to identifying new genes and alleles with previously unappreciated roles in mammalian development and disease. Here, we report a new mouse allele of Cse1l that was recovered from an ENU mutagenesis screen. Embryos homozygous for the anteater allele of Cse1l display a number of variable phenotypes, with craniofacial and ocular malformations being the most obvious. We provide evidence that Cse1l is the causal gene through complementation with a novel null allele of Cse1l generated by CRISPR-Cas9 editing. While the variability in the anteater phenotype was high enough to preclude a detailed molecular analysis, we demonstrate a very penetrant reduction in Pax6 levels in the developing eye along with significant ocular developmental phenotypes. The eye gene discovery tool iSyTE shows Cse1l to be significantly expressed in the lens from early eye development stages in embryos through adulthood. Cse1l has not previously been shown to be required for organogenesis as homozygosity for a null allele results in very early lethality. Future detailed studies of Cse1l function in craniofacial and neural development will be best served with a conditional allele to circumvent the variable phenotypes we report here. We suggest that human next-generation (whole genome or exome) sequencing studies yielding variants of unknown significance in CSE1L could consider these findings as part of variant analysis.

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis.

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

Chemical Properties Determine Solubility and Stability in βγ-Crystallins of the Eye Lens.

βγ-Crystallins are the primary structural and refractive proteins found in the vertebrate eye lens. Because crystallins are not replaced after early eye development, their solubility and stability must be maintained for a lifetime, which is even more remarkable given the high protein concentration in the lens. Aggregation of crystallins caused by mutations or post-translational modifications can reduce crystallin protein stability and alter intermolecular interactions. Common post-translational modifications that can cause age-related cataracts include deamidation, oxidation, and tryptophan derivatization. Metal ion binding can also trigger reduced crystallin solubility through a variety of mechanisms. Interprotein interactions are critical to maintaining lens transparency: crystallins can undergo domain swapping, disulfide bonding, and liquid-liquid phase separation, all of which can cause opacity depending on the context. Important experimental techniques for assessing crystallin conformation in the absence of a high-resolution structure include dye-binding assays, circular dichroism, fluorescence, light scattering, and transition metal FRET.

An E3 ubiquitin ligase, cullin-4 regulates retinal differentiation in Drosophila eye.

During organogenesis, cell proliferation is followed by the differentiation of specific cell types to form an organ. Any aberration in differentiation can result in developmental defects, which can result in a partial to a near-complete loss of an organ. We employ the Drosophila eye model to understand the genetic and molecular mechanisms involved in the process of differentiation. In a forward genetic screen, we identified, cullin-4 (cul-4), which encodes an E3 ubiquitin ligase, to play an important role in retinal differentiation. During development, cul-4 is known to be involved in protein degradation, regulation of genomic stability, and regulation of cell cycle. Previously, we have reported that cul-4 regulates cell death during eye development by downregulating Wingless (Wg)/Wnt signaling pathway. We found that loss-of-function of cul-4 results in a reduced eye phenotype, which can be due to onset of cell death. However, we found that loss-of-function of cul-4 also affects retinal development by downregulating retinal determination (RD) gene expression. Early markers of retinal differentiation are dysregulated in cul-4 loss of function conditions, indicating that cul-4 is necessary for differentiation. Furthermore, loss-of-function of cul-4 ectopically induces expression of negative regulators of eye development like Wg and Homothorax (Hth). During eye development, Wg is known to block the progression of a synchronous wave of differentiation referred to as Morphogenetic furrow (MF). In cul-4 loss-of-function background, expression of dpp-lacZ, a MF marker, is significantly downregulated. Our data suggest a new role of cul-4 in retinal differentiation. These studies may have significant bearings on our understanding of early eye development.

The Use of Induced Pluripotent Stem Cells as a Model for Developmental Eye Disorders.

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.

Effects of topical pilocarpine on ocular growth and refractive development in rabbits.

This study aimed to investigate whether topical pilocarpine affects ocular growth and refractive development as well as the underlying biochemical processes in early eye development in rabbits. Twenty three-week-old New Zealand white rabbits were treated with 0.5% pilocarpine in the right eye for 6 weeks. The left eyes served as contralateral controls. The effects of pilocarpine on refractive error, corneal curvature and ocular biometrics were assessed using streak retinoscopy, keratometry, and A-scan ultrasonography, respectively. Eyeballs were enucleated for histological analysis. The ciliary body and sclera were homogenized to determine the mRNA and protein expression levels of five subtypes of muscarinic receptors. Compared to control eyes, pilocarpine-treated eyes exhibited approximately -1.63 ± 0.54 D myopia accompanied by a 0.11 ± 0.04 mm increase in axial length (AL) (p < 0.001, respectively). The anterior chamber depth (ACD) was reduced, whereas the lens thickness (LT) and vitreous chamber depth (VCD) increased (p < 0.001, respectively). Corneal curvature decreased over time but was not significantly different between treated and control eyes. The mRNA and protein expression levels of five subtypes of muscarinic receptors were upregulated in the ciliary body and downregulated in the sclera. Based on these results, pilocarpine can induce myopic shift, increase LT, elongate VCD and AL, and reduce muscarinic receptor expression in the sclera early in development. These changes raise the possibility that pilocarpine may promote axial elongation in ocular development and facilitate the emmetropization of hyperopic eyes.

MiR-199 plays both positive and negative regulatory roles in Xenopus eye development.

To investigate microRNA (miR) functions in early eye development, we asked whether eye field transcription factors (EFTFs) are targets of miR-dependent regulation in Xenopus embryos. Argonaute (AGO) ribonucleoprotein complexes, including miRs and targeted mRNAs, were coimmunoprecipitated from transgenic embryos expressing myc-tagged AGO under the control of the rax1 promoter; mRNAs for all EFTFs coimmunoprecipitated with Ago in late neurulae. Computational predictions of miR binding sites within EFTF 3'UTRs identified miR-199a-3p ("miR-199") as a candidate regulator of EFTFs, and miR-199 was shown to regulate rax1 in vivo. Targeted overexpression of miR-199 led to small eyes, a reduction in EFTF expression, and reduced cell proliferation. Inhibition of interactions between mir-199 and the rax1 3'UTR reversed the small eye phenotype. Although targeted knockdown of miR-199 left the eye field intact, it reduced optic cup outgrowth and disrupted eye formation. Computational identification of candidate miR-199 targets within the Xenopus transcriptome led to the identification of ptk7 as a candidate regulator. Targeted overexpression of ptk7 resulted in abnormal optic cup formation and a reduction or loss of eye development, recapitulating the range of eye phenotypes seen following miR-199 knockdown. Our results indicate that miR-199 plays both positive and negative regulatory roles in eye development.

Optic cup morphogenesis requires neural crest-mediated basement membrane assembly.

ABSTRACTOrganogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated ‘The people behind the papers’ interview.

Embryonic development and microsatellite-based parentage assignment of seaweed pipefish Syngnathus schlegeli

In this study, the embryonic development of Syngnathus schlegeli is first described based on external morphological characteristics and then the genetic relationship between embryos is investigated using a microsatellite-based approach. Embryonic development proceeds from fertilized egg to prerelease of larva and can be categorized into four periods: early embryogenesis, eye development, snout formation and larva, which can be further divided into 15 stages. Embryos in the brood pouch are all in the same developmental stage. However, multi-maternity is observed in all sampled broods. Microsatellite-based parentage analysis suggests that pregnant males had, per male, mated with and received eggs from 3 to 7 females, with average being 4.3. The embryos of full sibs within a brood pouch were clustered, and seven patterns of parentage were observed. Combining previous studies with the evidence obtained from genotyped eggs inside the brood pouch in this study, we can confirm that the mating pattern of S. schlegeli is polygamous.

The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development.

Mutations in the key transcription factor, SOX2, alone account for 20% of anophthalmia (no eye) and microphthalmia (small eye) birth defects in humans-yet its regulation is not well understood, especially on the post-transcription level. We report the unprecedented finding that the conserved RNA-binding motif protein, RBM24, positively controls Sox2 mRNA stability and is necessary for optimal SOX2 mRNA and protein levels in development, perturbation of which causes ocular defects, including microphthalmia and anophthalmia. RNA immunoprecipitation assay indicates that RBM24 protein interacts with Sox2 mRNA in mouse embryonic eye tissue. and electrophoretic mobility shift assay shows that RBM24 directly binds to the Sox2 mRNA 3'UTR, which is dependent on AU-rich elements (ARE) present in the Sox2 mRNA 3'UTR. Further, we demonstrate that Sox2 3'UTR AREs are necessary for RBM24-based elevation of Sox2 mRNA half-life. We find that this novel RBM24-Sox2 regulatory module is essential for early eye development in vertebrates. We show that Rbm24-targeted deletion using a constitutive CMV-driven Cre in mouse, and rbm24a-CRISPR/Cas9-targeted mutation or morpholino knockdown in zebrafish, results in Sox2 downregulation and causes the developmental defects anophthalmia or microphthalmia, similar to human SOX2-deficiency defects. We further show that Rbm24 deficiency leads to apoptotic defects in mouse ocular tissue and downregulation of eye development markers Lhx2, Pax6, Jag1, E-cadherin and gamma-crystallins. These data highlight the exquisite specificity that conserved RNA-binding proteins like RBM24 mediate in the post-transcriptional control of key transcription factors, namely, SOX2, associated with organogenesis and human developmental defects.

Agr2-interacting Prod1-like protein Tfp4 from Xenopus laevis is necessary for early forebrain and eye development as well as for the tadpole appendage regeneration.

The Agr family genes, Ag1, Agr2, and Agr3, encode for the thioredoxin domain containing secreted proteins and are specific only for vertebrates. These proteins are attracting increasing attention due to their involvement in many physiological and pathological processes, including exocrine secretion, cancer, regeneration of the body appendages, and the early brain development. At the same time, the mode by which Agrs regulate intracellular processes are poorly understood. Despite that the receptor to Agr2, the membrane anchored protein Prod1, has been firstly discovered in Urodeles, and it has been shown to interact with Agr2 in the regenerating limb, no functional homologs of Prod1 were identified in other vertebrates. This raises the question of the mechanisms by which Agrs can regulate regeneration in other lower vertebrates. Recently, we have identified that Tfp4 (three-fingers Protein 4), the structural and functional homolog of Prod1 in Anurans, interacts with Agr2 in Xenopus laevis embryos. In the present work we show by several methods that the activity of Tfp4 is essential for the tadpole tail regeneration as well as for the early eye and forebrain development during embryogenesis. These data show for the first time the common molecular mechanism of regeneration regulation in amphibians by interaction of Prod1 and Agr2 proteins.

Larval development of mandi-pintado Pimelodus britskii (Siluriformes: Pimelodidae).

This study investigated the morphology, morphometric and meristic characters of 117 larval Pimelodus britskii showing early development of head, eye, barbel and snout. Body and mouth pigmentation increased throughout development; the mouth was ventrally situated in the yolk-sac stage, becoming subterminal afterwards, and an embryonic fin was visible in all four stages observed. Post-flexion larval P. bristskii are distinguished from larval P. ortmanni by having 47-50 myomeres (v. 36).

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.

Generation and characterization of pathogenic Mab21l2(R51C) mouse model.

MAB21L2(R51C) is one of the five documented MAB21L2 mutations in human patients with bilateral eye malformations identified via whole exome sequencing. In addition to the eye abnormality, patients with MAB21L2 R51C/+ mutation also have skeletal dysplasia and intellectual disability. To evaluate the pathology of this mutant allele systematically in understanding the functional role of MAB21L2 in human development, we introduce the R51C mutation into the mouse genome by CRISPR/Cas9 system to generate a mouse model for detailed characterization. The Mab21l2 R51C/+ mice have eyeless phenotype and skeletal abnormalities. Micro-computed tomography (micro-CT) analysis showed the Mab21l2 R51C/+ mice have no eye balls but with two abnormal tissues underneath the brain. Histological analysis revealed that the early eye development in the mutant embryos is interrupted. In addition, Mab21l2 R51C/+ mice also have joint fusion phenotype; the humerus is fused with radius, whereas femur is fused with tibia. Limbs in the mutant animals are distinctly shorter than the wild type; and deltoid tuberosities in humeri are absent in these Mab21l2 R51C/+ mice. In summary, we showed that our Mab21l2 R51C/+ mutant mice have recapitulated the pathological features in eye and bone of human patients. Further analyses of the mutant phenotype with molecular markers will provide insight on how MAB21L2 guides the optic differentiation and skeletogenesis, revealing specific underlying pathogenic mechanism of the MAB21L2(R51C) mutation.

Loss of zebrafish Ataxin-7, a SAGA subunit responsible for SCA7 retinopathy, causes ocular coloboma and malformation of photoreceptors.

Polyglutamine (polyQ) expansion in Ataxin-7 (ATXN7) results in spinocerebellar ataxia type 7 (SCA7) and causes visual impairment. SCA7 photoreceptors progressively lose their outer segments (OSs), a structure essential for their visual function. ATXN7 is a subunit of the transcriptional coactivator Spt-Ada-Gcn5 Acetyltransferase complex, implicated in the development of the visual system in flies. To determine the function of ATXN7 in the vertebrate eye, we have inactivated ATXN7 in zebrafish. While ATXN7 depletion in flies led to gross retinal degeneration, in zebrafish, it primarily results in ocular coloboma, a structural malformation responsible for pediatric visual impairment in humans. ATXN7 inactivation leads to elevated Hedgehog signaling in the forebrain, causing an alteration of proximo-distal patterning of the optic vesicle during early eye development and coloboma. At later developmental stages, malformations of photoreceptors due to incomplete formation of their OSs are observed and correlate with altered expression of crx, a key transcription factor involved in the formation of photoreceptor OS. Therefore, we propose that a primary toxic effect of polyQ expansion is the alteration of ATXN7 function in the daily renewal of OS in SCA7. Together, our data indicate that ATXN7 plays an essential role in vertebrate eye morphogenesis and photoreceptor differentiation, and its loss of function may contribute to the development of human coloboma.

All eyes on rapid adaptive evolution.

A new study reveals that DNA methylation-mediated repression of gene expression regulates early eye development in blind cave morphs of Astyanax mexicanus.

An epigenetic mechanism for cavefish eye degeneration.

Coding and non-coding mutations in DNA contribute significantly to phenotypic variability during evolution. However, less is known about the role of epigenetics in this process. Although previous studies have identified eye development genes associated with the loss of eyes phenotype in the Pachón blind cave morph of the Mexican tetra Astyanax mexicanus, no inactivating mutations have been found in any of these genes. Here we show that excess DNA methylation-based epigenetic silencing promotes eye degeneration in blind cave Astyanax mexicanus. By performing parallel analyses in Astyanax mexicanus cave and surface morphs and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development. The most significantly hypermethylated and down-regulated genes in the cave morph are also linked to human eye disorders, suggesting the function of these genes is conserved across the vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution.

The role of the Rx homeobox gene in retinal progenitor proliferation and cell fate specification

The Retinal homeobox gene (Rx; also Rax) plays a crucial role in the early development of the vertebrate eye. Germline deletion of Rx in mice results in the failure of optic vesicle formation, leading to anophthalmia. Recent research using conditional mouse knockout models provides some clues to the role of Rx in eye development following optic vesicle formation. However, the functions of Rx in embryonic retinogenesis are still not fully understood. We investigated the function of Rx in the mouse neural retina using a conditional knockout where the Pax6α-Cre driver deletes Rx activity in early retinal progenitors. The deletion of Rx activity causes a loss of retinal lamination, a depletion of retinal progenitors, and a change in retinal cell fate in our conditional knockout model. The deletion of Rx leads to an absence of late-born retinal neurons (rods and bipolar cells) and Müller glia at postnatal ages, as well as a loss of the early-born cone photoreceptors. Decreased BrdU labeling in the Rx-deleted portion of the retina suggests a loss of retinal progenitors via early cell cycle exit, which likely prevents the formation of late-born cells. As early-born cells, cone photoreceptors should not be as affected by early cell cycle exit of retinal progenitors. However, embryonic cone photoreceptor labeling is also markedly reduced in Rx-deleted retinas. Together these data demonstrate the importance of Rx for retinal progenitor proliferation and a specific requirement of Rx for cone formation in mice.

New GJA8 variants and phenotypes highlight its critical role in a broad spectrum of eye anomalies.

GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype-phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development.