FGF-mediated induction of ciliary body tissue in the chick eye

Upon morphogenesis, the simple neuroepithelium of the optic vesicle gives rise to four basic tissues in the vertebrate optic cup: pigmented epithelium, sensory neural retina, secretory ciliary body and muscular iris. Pigmented epithelium and neural retina are established through interactions with specific environments and signals: periocular mesenchyme/BMP specifies pigmented epithelium and surface ectoderm/FGF specifies neural retina. The anterior portions (iris and ciliary body) are specified through interactions with lens although the molecular mechanisms of induction have not been deciphered. As lens is a source of FGF, we examined whether this factor was involved in inducing ciliary body. We forced the pigmented epithelium of the embryonic chick eye to express FGF4. Infected cells and their immediate neighbors were transformed into neural retina. At a distance from the FGF signal, the tissue transitioned back into pigmented epithelium. Ciliary body tissue was found in the transitioning zone. The ectopic ciliary body was never in contact with the lens tissue. In order to assess the contribution of the lens on the specification of normal ciliary body, we created optic cups in which the lens had been removed while still pre-lens ectoderm. Ciliary body tissue was identified in the anterior portion of lens-less optic cups. We propose that the ciliary body may be specified at optic vesicle stages, at the same developmental stage when the neural retina and pigmented epithelium are specified and we present a model as to how this could be accomplished through overlapping BMP and FGF signals.     

Roles for Dicer1 in the patterning and differentiation of the optic cup neuroepithelium

The embryonic ocular neuroepithilium generates a myriad of cell types, including the neuroretina, the pigmented epithelium, the ciliary and iris epithelia, and the iris smooth muscles. As in other regions of the developing nervous system, the generation of these various cell types requires a coordinated sequence of patterning, specification and differentiation events. We investigated the roles of microRNAs (miRNAs) in the development of optic cup (OC)-derived structures. We inactivated Dicer1, a key mediator of miRNA biosynthesis, within the OC in overlapping yet distinct spatiotemporal patterns. Ablation of Dicer1 in the inner layer of the OC resulted in patterning alteration, particularly at the most distal margins. Following loss of Dicer1, this region generated a cryptic population of cells with a mixed phenotype of neuronal and ciliary body (CB) progenitors. Notably, inactivation of Dicer1 in the retinal progenitors further resulted in abrogated neurogenesis, with prolongation of ganglion cell birth and arrested differentiation of other neuronal subtypes, including amacrine and photoreceptor cells. These alterations were accompanied by changes in the expression of Notch and Hedgehog signaling components, indicating the sensitivity of the pathways to miRNA activity. Moreover, this study revealed the requirement of miRNAs for morphogenesis of the iris and for the regulation of CB cell type proliferation and differentiation. Together, analysis of the three genetic models revealed novel, stage-dependent roles for miRNAs in the development of the ocular sub-organs, which are all essential for normal vision.     

Dominant inhibition of lens placode formation in mice

The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and αA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.     

Neuronal stem/progenitor cells in the vertebrate eye

We acquire information from the outside world through our eyes which contain the retina, the photosensitive component of the central nervous system. Once the adult mammalian retina is damaged, the retinal neuronal death causes a severe loss of visual function. It has been believed that the adult mammalian retina had no regenerative capacity. However, the identification of neuronal progenitor cells in the retina sheds some light on cellular therapies for damaged retinal regeneration. In this review, we highlight three potential stem/progenitor cells in the eye, the ciliary body epithelium cells, the iris pigmented epithelium cells, and Müller glia. In order to make them prime candidates for the possible treatment of retinal diseases, it is important to understand their basic characters. In addition, we discuss the key signaling molecules that function extracellularly and determine whether neuronal progenitors remain quiescent, proliferate, or differentiate. Finally, we introduce a secreted protein, Tsukushi, which is a possible candidate as a niche molecule for retinal stem/progenitor cells.     

Pigment cell development in rhesus monkey eyes: an electron microscopic and histochemical study

The ontogeny of pigment cells in the eyes of rhesus monkeys was studied by electron microscopy and histochemistry.In 60- to 80-day-old fetuses, the pigment epithelium of the iris and retina has already differentiated whereas stromal melanocytes of the uveal tract differentiate much later. The morphological and histochemical difference between melanocytes of the iris stroma and the choroid suggests that during embryonic development melanocytes migrate from the iris toward the ciliary body and choroid.Similarly, melanosomes of pigmented epithelial cells may have their origin in the epithelium of the anterior layer of the iris, which is metabolically more active than both the posterior layer and the pigment epithelium of the ciliary body and retina.     

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.     

Ciliary margin transdifferentiation from neural retina is controlled by canonical Wnt signaling

The epithelial layers of the ciliary body (CB) and iris are non-neural structures that differentiate from the anterior region of the eyecup, the ciliary margin (CM). We show here that activation of the canonical Wnt signaling pathway is sufficient and necessary for the normal development of anterior eye structures. Pharmacological activation of beta-catenin signaling with lithium (Li(+)) treatment in retinal explants in vitro induced the ectopic expression of the CM markers Otx1 and Msx1. Cre-mediated stabilization of beta-catenin expression in the peripheral retina in vivo induced a cell autonomous upregulation of CM markers at the expense of neural retina (NR) markers and inhibited neurogenesis. Consistent with a cell autonomous conversion to peripheral eye fates, the proliferation index in the region of the retina that expressed stabilized beta-catenin was identical to the wild-type CM and there was an expansion of CB-like structures at later stages. Conversely, Cre-mediated inactivation of beta-catenin reduced CM marker expression as well as the size of the CM and CB/iris. Aberrant CB development in both mouse models was also associated with a reduction in the number of retinal stem cells in vitro. In summary, activation of canonical Wnt signaling is sufficient to promote the development of peripheral eyecup fates at the expense of the NR and is also required for the normal development of anterior eyecup structures.     

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.     

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.     

Defining the proteome of human iris,ciliary body,retinal pigment epithelium,and choroid

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS‐PAGE. After in‐gel digestion, peptides were analyzed using LC‐MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false‐positive rates of <0.1% and <1%, respectively. Forty‐three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.     

Retinal stem cells and regeneration

The optic vesicle gives rise to several very different epithelial tissues, including the neural retina, the pigmented epithelium, the iris, the ciliary epithelium of the ciliary body and the optic stalk. Retinal regeneration can arise from several different cellular sources; in some species, the process involves interconversion, or transdifferentiation, among cells of the different tissue types. Therefore, prior to a discussion of retinal regeneration, we will briefly discuss current knowledge about the influence of signaling molecules in cell fate determination in ocular tissues. Next, we will detail the evidence for neurogenesis in the mature retina. Lastly, we will describe various types of regenerative phenomena that occur in the retina, from complete regeneration of functional retina in fish and amphibians, to the more limited neuronal production that occurs in avian and mammalian retinas.     

Immunolocalisation of P2Y receptors in the rat eye

Nucleotides present an important role in ocular physiology which has been demonstrated by recent works that indicate their involvement in many ocular processes. P2Y are important among P2 receptors since they can control tear production, corneal wound healing, aqueous humour dynamics and retinal physiology. Commercial antibodies have allowed us to investigate the distribution of P2Y receptors in the cornea, anterior and posterior chamber of the eye and retina. The P2Y₁ receptor was present mainly in cornea, ciliary processes, and trabecular meshwork. The P2Y₂ receptors were present in cornea, ciliary processes and retinal pigmented epithelium. P2Y₄ was present in cornea, ciliary processes, photoreceptors, outer plexiform layer and ganglion cell layer. The P2Y₆ presented almost an identical distribution as the P2Y₄ receptor. The P2Y₁₁ was also detectable in the retinal pigmented epithelium. The detailed distribution of the receptors clearly supports the recent findings indicating the relevant role of nucleotides in the ocular function.     

Generation of a second eye by embryonic transplantation of the antero‐ventral hemicephalon

Vertebrate ocular morphogenesis requires proper dorso‐ventral polarity within the optic vesicle, and loss of dorso‐ventral polarity results in failure of optic cup formation and domain specification, as shown by a reverse transplantation of the optic vesicle. We have shown previously that the ocular development depends not only on the signal within the antero‐ventral optic vesicle but also on the extraocular signals. In the present study, using embryonic transplantation of a discrete portion of the embryonic chick brain, we demonstrate formation of a second eye from the antero‐ventral hemicephalon when it was transplanted in the antero‐dorsal hemicephalon of the host embryo. The transplant consists of an antero‐ventral quadrant of the optic vesicle and the surrounding part of the anterior cephalon. The original dorso‐ventral polarity of the transplant was once cancelled and re‐established in accordance with that of the host embryo. Neither dorsal nor ventral cephalic halves in isolation did not develop into entire eye structures under the culture condition; the dorsal halves developed merely into the retinal pigmented epithelium and the ventral halves into the neural retina alone. The present study clearly suggests that extraocular dorsal and ventral signals counterbalance each other to specify the polarity of the optic vesicle.     

Lens-Specific Expression of PDGF-A Alters Lens Growth and Development

The vertebrate lens provides anin vivomodel to study the molecular mechanisms by which growth factors influence development decisions. In this study, we have investigated the expression patterns of platelet-derived growth factor (PDGF) and PDGF receptors during murine eye development byin situhybridization. Postnatally, PDGF-A is highly expressed in the iris and ciliary body, the ocular tissues closest to the germinative zone of the lens, a region where most proliferation of lens epithelial cells occurs. PDGF-A is also present in the corneal endothelium anterior to the lens epithelium in embryonic and early postnatal eyes. PDGF-B is expressed in the iris and ciliary body as well as in the vascular cells which surround the lens during early eye development. In the lens, expression of PDGF-α receptor (PDGF-αR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-αR in the lens epithelial cells and PDGF (A- and B-chains) in the ocular tissues adjacent to the lens suggests that PDGF signaling may play a key role in regulating lens development. To further examine how PDGF affects lens developmentin vivo,we generated transgenic mice that express human PDGF-A in the lens under the control of the αA-crystallin promoter. The transgenic mice exhibit lenticular defects that result in cataracts. The percentage of surface epithelial cells in S-phase is increased in transgenic lenses compared to their nontransgenic littermates. Higher than normal levels of cyclin A and cyclin D2 expression were also detected in transgenic lens epithelium. These results together suggest that PDGF-A can induce a proliferative response in lens epithelial cells. The lens epithelial cells in the transgenic mice also exhibit characteristics of differentiating fiber cells. For example, the transgenic lens epithelial cells are slightly elongated, contain larger and less condensed nuclei, and express fiber-cell-specific β-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cellsin vivo.Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.     

Developmental analysis of ocular morphogenesis in alpha A-crystallin/diphtheria toxin transgenic mice undergoing ablation of the lens.

The role of the lens in early eye development was examined in transgenic mice carrying the cytotoxic diphtheria toxin A gene driven by hamster alpha A-crystallin promoter sequences. Mice hemizygous for this construct are microphthalmic and contain a vacuolated and highly disorganized lens, whereas adult homozygous mice are completely ablated of the lens and lack a pupil, aqueous and posterior chamber, vitreous humor, iris, and ciliary body and show extensive convolution of the sensory retina. Developmental analysis of animals homozygous for the transgene revealed that the optic cup and lens vesicle form normally and that ablation of the lens occurs as a gradual degenerative process beginning between Days 12 and 13 of gestation. Degeneration of the lens vesicle coincides with retarded growth and development of the neuroretina, sclera, and cornea. The anterior lip of the optic cup fails to differentiate into the normal epithelium of the iris and ciliary body and the vitreous body does not develop. Although the retinal layers apparently form normally, retinal folding becomes prominent following lens degeneration. These results suggest that development of a functional lens from Embryonic Day 12.5 onward is critical for formation of the ciliary epithelium, iris, and vitreous body, as well as for appropriate growth, development, and maintenance of morphology of the retina, cornea, sclera, and optic nerve. Our results also provide information on the time course of DT-A-mediated cell destruction in vivo and are discussed in context with previous lens ablation studies and the importance of developmental analysis for interpretation of the extent to which morphogenetic aberrations are concurrent with or secondary to genetic ablation of the target tissue.     

BMP signaling is required for development of the ciliary body

The ciliary body in the eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During embryonic development, the ciliary epithelium is derived from the optic vesicle, but the molecular signals that control morphogenesis of the ciliary body are unknown. We report that lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye.