Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.     

Lmx1b is essential for survival of periocular mesenchymal cells and influences Fgf-mediated retinal patterning in zebrafish

To gain insight into the mechanisms of Lmx1b function during ocular morphogenesis, we have studied the roles of lmx1b.1 and lmx1b.2 during zebrafish eye development. In situ hybridization and characterization of transgenic lines in which GFP is expressed under lmx1b.1 regulatory sequence show that these genes are expressed in periocular tissues and in a pattern conserved with other vertebrates. Anti-sense morpholinos against lmx1b.1 and lmx1b.2 result in defective migration of periocular mesenchymal cells around the eye and lead to apoptosis of these cells. These defects in the periocular mesenchyme are correlated with a failure in fusion of the choroid fissure or in some instances, more severe ventral optic cup morphogenesis phenotypes. Indeed, by blocking the death of the periocular mesenchyme in Lmx1b morphants, optic vesicle morphogenesis is largely restored. Within the retina of lmx1b morphants, Fgf activity is transiently up-regulated and these morphants show defective naso-temporal patterning. Epistasis experiments indicate that the increase in Fgf activity is partially responsible for the ocular anomalies caused by loss of Lmx1b function. Overall, we propose zebrafish lmx1b.1 and lmx1b.2 promote the survival of periocular mesenchymal cells that influence multiple signaling events required for proper ocular development.     

Retinoic acid-dependent eye morphogenesis is orchestrated by neural crest cells

Using genetic approaches in the mouse, we show that the primary target tissue of retinoic acid (RA) action during eye morphogenesis is not the retina nor the corneal ectoderm, which both express RA-synthesizing retinaldehyde dehydrogenases (RALDH1 and RALDH3), but the neural crest cell-derived periocular mesenchyme (POM), which is devoid of RALDH. In POM, the effects of the paracrine RA signal are mediated by the nuclear RA receptors heterodimers RXRalpha/RARbeta and RXRalpha/RARgamma. These heterodimers appear to control: (1) the remodeling of the POM through activation of Eya2-related apoptosis; (2) the expression of Foxc1 and Pitx2, which play crucial roles in anterior eye segment development; and (3) the growth of the ventral retina. We additionally show that RALDH1 and RALDH3 are the only enzymes that are required for RA synthesis in the eye region from E10.5 to E13.5, and that patterning of the dorsoventral axis of the retina does not require RA.     

Neural crest derivatives in ocular development: Discerning the eye of the storm

Neural crest cells (NCCs) are vertebrate‐specific transient, multipotent, migratory stem cells that play a crucial role in many aspects of embryonic development. These cells emerge from the dorsal neural tube and subsequently migrate to different regions of the body, contributing to the formation of diverse cell lineages and structures, including much of the peripheral nervous system, craniofacial skeleton, smooth muscle, skin pigmentation, and multiple ocular and periocular structures. Indeed, abnormalities in neural crest development cause craniofacial defects and ocular anomalies, such as Axenfeld‐Rieger syndrome and primary congenital glaucoma. Thus, understanding the molecular regulation of neural crest development is important to enhance our knowledge of the basis for congenital eye diseases, reflecting the contributions of these progenitors to multiple cell lineages. Particularly, understanding the underpinnings of neural crest formation will help to discern the complexities of eye development, as these NCCs are involved in every aspect of this process. In this review, we summarize the role of ocular NCCs in eye development, particularly focusing on congenital eye diseases associated with anterior segment defects and the interplay between three prominent molecules, PITX2, CYP1B1, and retinoic acid, which act in concert to specify a population of neural crest‐derived mesenchymal progenitors for migration and differentiation, to give rise to distinct anterior segment tissues. We also describe recent findings implicating this stem cell population in ocular coloboma formation, and introduce recent evidence suggesting the involvement of NCCs in optic fissure closure and vascular development. Birth Defects Research (Part C) 105:87–95, 2015. © 2015 Wiley Periodicals, Inc.     

Morphogenesis of the anterior segment in the zebrafish eye

Background  

The ocular anterior segment is critical for focusing incoming light onto the neural retina and for regulating intraocular pressure. It is comprised of the cornea, lens, iris, ciliary body, and highly specialized tissue at the iridocorneal angle. During development, cells from diverse embryonic lineages interact to form the anterior segment. Abnormal migration, proliferation, differentiation, or survival of these cells contribute to diseases of the anterior segment such as corneal dystrophy, lens cataract, and glaucoma. Zebrafish represent a powerful model organism for investigating the genetics and cell biology of development and disease. To lay the foundation for genetic studies of anterior segment development, we have described the morphogenesis of this structure in zebrafish.     

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.     

Keratocan-deficient mice display alterations in corneal structure

Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera-/- cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera-/- cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera-/- mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.     

Neural crest derivatives in ocular and periocular structures

In vertebrates, the eye is an ectodermal compound structure associating neurectodermal and placodal anlagen. In addition, it benefits early on from a mesenchymal ectoderm-derived component, the neural crest. In this respect, the construction of chimeras between quail and chick has been a turning point, instrumental in appraising the contribution of the cephalic neural crest to the development of ocular and periocular structures. Given the variety of crest derivatives underscored in the developing eye, this study illustrates the fascinating ability of this unique structure to finely adapt its differentiation to microenvironmental cues. This analysis of neural crest cell contribution to ocular development emphasizes their paramount role to design the anterior segment of the eye, supply refracting media and contribute to the homeostasy of the anterior optic chamber.     

What's retinoic acid got to do with it? Retinoic acid regulation of the neural crest in craniofacial and ocular development

Retinoic acid (RA), the active derivative of vitamin A (retinol), is an essential morphogen signaling molecule and major regulator of embryonic development. The dysregulation of RA levels during embryogenesis has been associated with numerous congenital anomalies, including craniofacial, auditory, and ocular defects. These anomalies result from disruptions in the cranial neural crest, a vertebrate‐specific transient population of stem cells that contribute to the formation of diverse cell lineages and embryonic structures during development. In this review, we summarize our current knowledge of the RA‐mediated regulation of cranial neural crest induction at the edge of the neural tube and the migration of these cells into the craniofacial region. Further, we discuss the role of RA in the regulation of cranial neural crest cells found within the frontonasal process, periocular mesenchyme, and pharyngeal arches, which eventually form the bones and connective tissues of the head and neck and contribute to structures in the anterior segment of the eye. We then review our understanding of the mechanisms underlying congenital craniofacial and ocular diseases caused by either the genetic or toxic disruption of RA signaling. Finally, we discuss the role of RA in maintaining neural crest‐derived structures in postembryonic tissues and the implications of these studies in creating new treatments for degenerative craniofacial and ocular diseases.     

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.     

Six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye

The establishment of retinal identity and the subsequent patterning of the optic vesicle are the key steps in early vertebrate eye development. To date little is known about the nature and interaction of the genes controlling these steps. So far few genes have been identified that, when over-expressed, can initiate ectopic eye formation. Of note is Six3, which is expressed exclusively in the anterior neural plate. However, 'loss of function' analysis has not been reported. Using medaka fish, we show that vertebrate Six3 is necessary for patterning of the anterior neuroectoderm including the retina anlage. Inactivation of Six3 function by morpholino knock-down results in the lack of forebrain and eyes. Corroborated by gain-of-function experiments, graded interference reveals an additional role of Six3 in the proximodistal patterning of the optic vesicle. During both processes of vertebrate eye formation, Six3 cooperates with Pax6.     

A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens

The anterior segment of the vertebrate eye includes the cornea, iris, ciliary body, trabecular meshwork, and lens. Although malformations of these structures have been implicated in many human eye diseases, little is known about the molecular mechanisms that control their development. To identify genes involved in anterior segment formation, we developed a large-scale in situ hybridization screen and examined the spatial and temporal expression of over 1000 genes during eye development. This screen identified 62 genes with distinct expression patterns in specific eye structures, including several expressed in novel patterns in the anterior segment. Using these genes as developmental markers, we tested for the presence of inductive signals that control the differentiation of anterior segment tissues. Organ culture recombination experiments showed that a chick lens is capable of inducing the expression of markers of the presumptive iris and ciliary body in the developing mouse neural retina. The inducing activity from the lens acts only over short ranges and is present at multiple stages of eye development. These studies provide molecular evidence that an evolutionarily conserved signal from the lens controls tissue specification in the developing optic cup.