Eye Field Transcription Factors Research Articles

Stage-specific dynamic reorganization of genome topology shapes transcriptional neighborhoods in developing human retinal organoids

We have generated a high-resolution Hi-C map of developing human retinal organoids to elucidate spatiotemporal dynamics of genomic architecture and its relationship with gene expression patterns. We demonstrate progressive stage-specific alterations in DNA topology and correlate these changes with transcription of cell-type-restricted gene markers during retinal differentiation. Temporal Hi-C reveals a shift toward A compartment for protein-coding genes and B compartment for non-coding RNAs, displaying high and low expression, respectively. Notably, retina-enriched genes are clustered near lost boundaries of topologically associated domains (TADs), and higher-order assemblages (i.e., TAD cliques) localize in active chromatin regions with binding sites for eye-field transcription factors. These genes gain chromatin contacts at their transcription start site as organoid differentiation proceeds. Our study provides a global view of chromatin architecture dynamics associated with diversification of cell types during retinal development and serves as a foundational resource for in-depth functional investigations of retinal developmental traits.

Lactate-dependent transcriptional regulation controls mammalian eye morphogenesis

Mammalian retinal metabolism favors aerobic glycolysis. However, the role of glycolytic metabolism in retinal morphogenesis remains unknown. We report that aerobic glycolysis is necessary for the early stages of retinal development. Taking advantage of an unbiased approach that combines the use of eye organoids and single-cell RNA sequencing, we identify specific glucose transporters and glycolytic genes in retinal progenitors. Next, we determine that the optic vesicle territory of mouse embryos displays elevated levels of glycolytic activity. At the functional level, we show that removal of Glucose transporter 1 and Lactate dehydrogenase A gene activity from developing retinal progenitors arrests eye morphogenesis. Surprisingly, we uncover that lactate-mediated upregulation of key eye-field transcription factors is controlled by the epigenetic modification of histone H3 acetylation through histone deacetylase activity. Our results identify an unexpected bioenergetic independent role of lactate as a signaling molecule necessary for mammalian eye morphogenesis.

Efficient embryoid-based method to improve generation of optic vesicles from human induced pluripotent stem cells.

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

Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) is valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in Best Disease patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

Transcription Factor Overexpression Drives Reliable Differentiation of Retinal Pigment Epithelium from Human Induced Pluripotent Stem Cells

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) are valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

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.

Teratogenic Rubella Virus Alters the Endodermal Differentiation Capacity of Human Induced Pluripotent Stem Cells

The study of congenital virus infections in humans requires suitable ex vivo platforms for the species-specific events during embryonal development. A prominent example for these infections is rubella virus (RV) which most commonly leads to defects in ear, heart, and eye development. We applied teratogenic RV to human induced pluripotent stem cells (iPSCs) followed by differentiation into cells of the three embryonic lineages (ecto-, meso-, and endoderm) as a cell culture model for blastocyst- and gastrulation-like stages. In the presence of RV, lineage-specific differentiation markers were expressed, indicating that lineage identity was maintained. However, portrait analysis of the transcriptomic expression signatures of all samples revealed that mock- and RV-infected endodermal cells were less related to each other than their ecto- and mesodermal counterparts. Markers for definitive endoderm were increased during RV infection. Profound alterations of the epigenetic landscape including the expression level of components of the chromatin remodeling complexes and an induction of type III interferons were found, especially after endodermal differentiation of RV-infected iPSCs. Moreover, the eye field transcription factors RAX and SIX3 and components of the gene set vasculogenesis were identified as dysregulated transcripts. Although iPSC morphology was maintained, the formation of embryoid bodies as three-dimensional cell aggregates and as such cellular adhesion capacity was impaired during RV infection. The correlation of the molecular alterations induced by RV during differentiation of iPSCs with the clinical signs of congenital rubella syndrome suggests mechanisms of viral impairment of human development.

Defined Conditions for Differentiation of Functional Retinal Ganglion Cells From Human Pluripotent Stem Cells.

We aimed to establish an efficient method for retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs) using defined factors. To define the contribution of specific signal pathways to RGC development and optimize the differentiation of hPSCs toward RGCs, we examined RGC differentiation in three stages: (1) eye field progenitors expressing the eye field transcription factors (EFTFs), (2) RGC progenitors expressing MATH5, and (3) RGCs expressing BRN3B and ISLET1. By monitoring the condition that elicited the highest yield of cells expressing stage-specific markers, we determined the optimal concentrations and combinations of signaling pathways required for efficient generation of RGCs from hPSCs. Precise modulation of signaling pathways, including Wnt, insulin growth factor-1, and fibroblast growth factor, in combination with mechanical isolation of neural rosette cell clusters significantly enriched RX and PAX6 double-positive eye field progenitors from hPSCs by day 12. Furthermore, Notch signal inhibition facilitated differentiation into MATH5-positive progenitors at 90% efficiency by day 20, and these cells further differentiated to BRN3B and ISLET1 double-positive RGCs at 45% efficiency by day 40. RGCs differentiated via this method were functional as exemplified by their ability to generate action potentials, express microfilament components on neuronal processes, and exhibit axonal transportation of mitochondria. This protocol offers highly defined culture conditions for RGC differentiation from hPSCs and in vitro disease model and cell source for transplantation for diseases related to RGCs.

Generation of Photoreceptor Precursors from Mouse Embryonic Stem Cells.

Embryonic stem cell (ESC) differentiation can be used to model development and to produce transplantable cells of the desired phenotype. ESCs can reproducibly generate retinal cells but the derivation of photoreceptor precursors is variable and depends on an array of exogenous factors and intrinsic cell-cell interactions. In this work, we have defined the use of exogenous signaling factors, dissociation, and adherent versus 3-dimensional (3D) conditions on the derivation of retinal cells from pluripotent mouse ESCs. Differentiation protocols were chosen based on the developmental stage and cell population of interest and evaluated by expression of developmental stage- and lineage-specific marker genes. We present a relatively simple protocol that guides differentiating ESCs through stages that correspond to the sequence of in vivo developmental events and is optimized for studying the time frame between eye field formation and photoreceptor precursor development in the equivalent of embryonic retina. Step-wise exposure of adherent cultures to exogenous factors facilitated expression of eye field transcription factors and limited non-specific differentiation. Dissociation after the establishment of eye field and retinal progenitor cell gene expression did not cause substantial loss in expression of markers of mature photoreceptors. Finally, 3D organoids improved expression of photoreceptor genes and region-specific architecture but required more technical manipulation. We demonstrate the usefulness of this ESC-retinal induction protocol in screening for factors that improve photoreceptor precursor yield by evaluating response to alterations in Activin signaling.

Analysis of expression of transcription factors in early human retina

The retina originates in the central nervous system. Due to its accessibility and simplicity, the retina has become an invaluable model for studying the basic mechanisms involved in development. To date, considerable knowledge regarding the interactions among genes that coordinate retinal development has been gained from extensive research in model animals. However, our understanding of retinal development in humans remains undeveloped. Here, we analyze the expression of transcription factors that are involved in the early development of the retina in human embryos at 6–12 weeks post-conception. Our work demonstrates that early developing neural retinas can be divided into two layers, the outer and inner neuroblast layers. Eye-field transcription factors and those related to the early development of the retina have distinct expression patterns in the two layers. Cell-type-specific transcription factors emerge at 8 weeks. These data provide clear and systemic structures for early retinal development in human.

Tbx3 represses bmp4 expression and, with Pax6, is required and sufficient for retina formation.

Vertebrate eye formation begins in the anterior neural plate in the eye field. Seven eye field transcription factors (EFTFs) are expressed in eye field cells and when expressed together are sufficient to generate retina from pluripotent cells. The EFTF Tbx3 can regulate the expression of some EFTFs; however, its role in retina formation is unknown. Here, we show that Tbx3 represses bmp4 transcription and is required in the eye field for both neural induction and normal eye formation in Xenopus laevis Although sufficient for neural induction, Tbx3-expressing pluripotent cells only form retina in the context of the eye field. Unlike Tbx3, the neural inducer Noggin can generate retina both within and outside the eye field. We found that the neural and retina-inducing activity of Noggin requires Tbx3. Noggin, but not Tbx3, induces Pax6 and coexpression of Tbx3 and Pax6 is sufficient to determine pluripotent cells to a retinal lineage. Our results suggest that Tbx3 represses bmp4 expression and maintains eye field neural progenitors in a multipotent state; then, in combination with Pax6, Tbx3 causes eye field cells to form retina.

Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

SummaryRetinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs) toward eye field fates. Inhibition of Wnt/β-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine.

The GIPC1-Akt1 Pathway Is Required for the Specification of the Eye Field in Mouse Embryonic Stem Cells.

During early patterning of the neural plate, a single region of the embryonic forebrain, the eye field, becomes competent for eye development. The hallmark of eye field specification is the expression of the eye field transcription factors (EFTFs). Experiments in fish, amphibians, birds, and mammals have demonstrated largely conserved roles for the EFTFs. Although some of the key signaling events that direct the synchronized expression of these factors to the eye field have been elucidated in fish and frogs, it has been more difficult to study these mechanisms in mammalian embryos. In this study, we have used two different methods for directed differentiation of mouse embryonic stem cells (mESCs) to generate eye field cells and retina in vitro to test for a role of the PDZ domain-containing protein GIPC1 in the specification of the mammalian eye primordia. We find that the overexpression of a dominant-negative form of GIPC1 (dnGIPC1), as well as the downregulation of endogenous GIPC1, is sufficient to inhibit the development of eye field cells from mESCs. GIPC1 interacts directly with IGFR and participates in Akt1 activation, and pharmacological inhibition of Akt1 phosphorylation mimics the dnGIPC1 phenotype. Our data, together with previous studies in Xenopus, support the hypothesis that the GIPC1-PI3K-Akt1 pathway plays a key role in eye field specification in vertebrates.

Dental stem cells: a future asset of ocular cell therapy.

Regenerative medicine using patient's own stem cells (SCs) to repair dysfunctional tissues is an attractive approach to complement surgical and pharmacological treatments for aging and degenerative disorders. Recently, dental SCs have drawn much attention owing to their accessibility, plasticity and applicability for regenerative use not only for dental, but also other body tissues. In ophthalmology, there has been increasing interest to differentiate dental pulp SC and periodontal ligament SC (PDLSC) towards ocular lineage. Both can commit to retinal fate expressing eye field transcription factors and generate rhodopsin-positive photoreceptor-like cells. This proposes a novel therapeutic alternative for retinal degeneration diseases. Moreover, as PDLSC shares similar cranial neural crest origin and proteoglycan secretion with corneal stromal keratoctyes and corneal endothelial cells, this offers the possibility of differentiating PDLSC to these corneal cell types. The advance could lead to a shift in the medical management of corneal opacities and endothelial disorders from highly invasive corneal transplantation using limited donor tissue to cell therapy utilizing autologous cells. This article provides an overview of dental SC research and the perspective of utilizing dental SCs for ocular regenerative medicine.

Distinct evolutionary rate in the eye field transcription factors found by estimation of ancestral protein structure

Eye-field transcription factors (EFTFs) are a set of genes that compose a regulatory network for eye development in animals, which are highly conserved among various animal phyla. To investigate the processes of conservation and diversification of the transcription factors for eye development, we examined the structural changes in the EFTF proteins by estimating the ancestral sequences with the available genome information. Among the different types of EFTFs, we selected otx2, tbx3, rx1, pax6, six3/6, lhx2 and nr2e1 because they are highly conserved in bilaterian animals. We searched the genome sequences of representative animal phyla for EFTF protein sequences. With deduced ancestral sequences and three-dimensional structures of EFTFs, we traced the evolutionary changes in amino acid residues and found that the DNA-binding domains were always more conserved than other regions, and that the other regions showed distinct evolutionary rates. The EFTF rx1, which resides at the pivotal part of the EFTF network, had a faster evolutionary rate than the others. These results indicated that the evolutionary rates of each protein in the EFTF network, which were expected to be consistent with each other to maintain the interactions in the network, were not constant among or within the factors, but rather, varied to a significant extent.

Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character

The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.

Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation.

Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.

Directing Adult Human Periodontal Ligament–Derived Stem Cells to Retinal Fate

To investigate the retinal fate competence of human postnatal periodontal ligament (PDL)-derived stem cells (PDLSC) through a directed differentiation mimicking mammalian retinogenesis. Human teeth were collected from healthy subjects younger than 35 years old. Primary PDLSC were isolated by collagenase digestion and cultivated. PDLSC at passage 3 were cultured in the induction media containing Noggin (antagonist of bone morphogenic protein) and Dkk-1 (antagonist of Wnt/β-catenin signaling). Gene expression of neural crest cells, retinal progenitors, and retinal neurons, including photoreceptors, was revealed by RNA analyses, immunofluorescence, and flow cytometry. The neuronal-like property of differentiated cells in response to excitatory glutamate was examined by fluo-4-acetoxymethyl calcium imaging assay. Primary human PDLSC stably expressed marker genes for neural crest (Notch1, BMP2, Slug, Snail, nestin, and Tuj1), mesenchymal stem cell (CD44, CD90, and vimentin), and embryonic stem cell (c-Myc, Klf4, Nanog, and SSEA4). Under low attachment culture, PDLSC generated neurospheres expressing nestin, p75/NGFR, Pax6, and Tuj1 (markers of neural progenitors). When neurospheres were plated on Matrigel-coated surface, they exhibited rosette-like outgrowth. They expressed eye field transcription factors (Pax6, Rx, Lhx, Otx2). By flow cytometry, 94% of cells were Pax6(nuclear)Rx(+), indicative of retinal progenitors. At prolonged induction, they expressed photoreceptor markers (Nrl, rhodopsin and its kinase) and showed significant responsiveness to excitatory glutamate. Primary human PDLSC could be directed to retinal progenitors with competence for photoreceptor differentiation. Human neural crest-derived PDL is readily accessible and can be an ample autologous source of undifferentiated cells for retinal cell regeneration.

LHX2 Is Necessary for the Maintenance of Optic Identity and for the Progression of Optic Morphogenesis

Eye formation is regulated by a complex network of eye field transcription factors (EFTFs), including LIM-homeodomain gene LHX2. We disrupted LHX2 function at different stages during this process using a conditional knock-out strategy in mice. We find that LHX2 function is required in an ongoing fashion to maintain optic identity across multiple stages, from the formation of the optic vesicle to the differentiation of the neuroretina. At each stage, loss of Lhx2 led to upregulation of a set of molecular markers that are normally expressed in the thalamic eminence and in the anterodorsal hypothalamus in a portion of the optic vesicle or retina. Furthermore, the longer LHX2 function was maintained, the further optic morphogenesis progressed. Early loss of function caused profound mispatterning of the entire telencephalic-optic-hypothalamic field, such that the optic vesicle became mispositioned and appeared to arise from the diencephalic-telencephalic boundary. At subsequent stages, loss of Lhx2 did not affect optic vesicle position but caused arrest of optic cup formation. If Lhx2 was selectively disrupted in the neuroretina from E11.5, the neuroretina showed gross dysmorphology along with aberrant expression of markers specific to the thalamic eminence and anterodorsal hypothalamus. Our findings indicate a continual requirement for LHX2 throughout the early stages of optic development, not only to maintain optic identity by suppressing alternative fates but also to mediate multiple steps of optic morphogenesis. These findings provide new insight into the anophthalmic phenotype of the Lhx2 mutant and reveal novel roles for this transcription factor in eye development.

Eye Development and Retinogenesis

Three embryonic tissue sources-the neural ectoderm, the surface ectoderm, and the periocular mesenchyme-contribute to the formation of the mammalian eye. For this reason, the developing eye has presented an invaluable system for studying the interactions among cells and, more recently, genes, in specifying cell fate. This article describes how the eye primordium is specified in the anterior neural plate by four eye field transcription factors and how the optic vesicle becomes regionalized into three distinct tissue types. Specific attention is given to how cross talk between the optic vesicle and surface ectoderm contributes to lens and optic cup formation. This article also describes how signaling networks and cell movements set up axes in the optic cup and establish the multiple cell fates important for vision. How multipotent retinal progenitor cells give rise to the six neuronal and one glial cell type in the mature retina is also explained. Finally, the history and progress of cellular therapeutics for the treatment of degenerative eye disease is outlined. Throughout this article, special attention is given to how disruption of gene function causes ocular malformation in humans. Indeed, the accessibility of the eye has contributed much to our understanding of the basic processes involved in mammalian development.