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The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intra-ocular pressure and regulate exposure of the internal eye structures to light, respectively. The cellular and molecular factors that mediate the development of the ciliary body and iris from the ocular pigmented epithelium remain to be fully elucidated. Here, we have investigated the role of Notch signaling during the development of the anterior pigmented epithelium by using genetic loss- and gain-of-function approaches. Loss of canonical Notch signaling results in normal iris development but absence of the ciliary body. This causes progressive hypotony and over time leads to phthisis bulbi, a condition characterized by shrinkage of the eye and loss of structure/function. Conversely, Notch gain-of-function results in aniridia and profound ciliary body hyperplasia, which causes ocular hypertension and glaucoma-like disease. Collectively, these data indicate that Notch signaling promotes ciliary body development at the expense of iris formation and reveals novel animal models of human ocular pathologies., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Systematic toxicological study is still required to fully understand the hazard potentials of gold nanoparticles (AuNPs). Because their biomedical applications are rapidly evolving, we investigated developmental toxicity of AuNPs in an in vivo embryonic zebrafish model at exposure concentration ranges from 0.08 to 50mg/l. Exposure of zebrafish embryos to 1.3 nm AuNPs functionalized with a cationic ligand, N,N,N-trimethylammoniumethanethiol (TMAT-AuNPs), resulted in smaller malpigmented eyes. We determined that TMAT-AuNPs caused a significant increase of cell death in the eye, which was correlated with an increase in gene expression of p53 and bax. Expression patterns of key transcription factors regulating eye development (pax6a, pax6b, otx2, and rx1) and pigmentation (sox10) were both repressed in a concentration-dependent manner in embryos exposed to TMAT-AuNPs. Reduced spatial localization of pax6a, rx1, sox10, and mitfa was observed in embryos by whole-mount in situ hybridization. The swimming behavior of embryos exposed to sublethal concentrations of TMAT-AuNPs showed hypoactivity, and embryos exhibited axonal growth inhibition. Overall, these results demonstrated that TMAT-AuNPs disrupt the progression of eye development and pigmentation that continues to behavioral and neuronal damage in the developing zebrafish.

Background: In the cuttlefish Sepia officinalis, iridescence is known to play a role in patterning and communication. In iridophores, iridosomes are composed of reflectins, a protein family, which show great diversity in all cephalopod species. Iridosomes are established before hatching, but very little is known about how these cells are established, their distribution in embryos, or the contribution of each reflectin gene to iridosome structures., Results: Six reflectin genes are expressed during the development of iridosomes in Sepia officinalis. We show that they are expressed in numerous parts of the body before hatching. Evidence of the colocalization of two different genes of reflectin was found. Curiously, reflectin mRNA expression was no longer detectable at the time of hatchling, while reflectin proteins were present and gave rise to visible iridescence., Conclusion: These data suggest that several different forms of reflectins are simultaneously used to produce iridescence in S. officinalis and that mRNA production and translation are decoupled in time during iridosome development., (Copyright © 2013 Wiley Periodicals, Inc.)

Retinal stem cells (RSCs) are present within the pigmented ciliary epithelium (CE) of the adult human eye and produce progeny that differentiate in vitro into all neural retinal subtypes and retinal pigmented epithelium (RPE). We hypothesized that a RSC population, similar to the adult CE-derived RSC, is contained within pigmented colonies that arise in long-term cultures of human embryonic stem cells (hESCs) suggested to recapitulate retinal development in vitro. Single pigmented hESC-derived cells were isolated and plated in serum-free media containing growth factors and, after 2 weeks, clonal sphere colonies containing both pigmented and non-pigmented cells were observed. These colonies expressed the early retinal transcription factors Rx, Chx10 and Pax6, and could be dissociated and replated as single cells to form secondary clonal colonies. When allowed to differentiate, expression of markers for both RPE and neurons was observed. Rhodopsin expression was detected after explant co-culture and transplantation into the developing mouse eye as well as following treatment with soluble factors in vitro. We show that RSCs emerge in an in vitro model of retinal development and are a potential source of human photoreceptors for use in transplantation., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART-1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell-specific gene MART-1 (mlana). When MART-1::Cre BAC transgenic mice were bred with the ROSA26-R reporter line, ß-galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART-1::Cre line provides Cre recombinase activity in pigment-producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP-Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART-1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403-409, 2011., (2011 Wiley-Liss, Inc.)

Purpose: The optic cup is created through invagination of the optic vesicle. The morphogenetic rearrangement creates a double-layered cup, with a hinge (the Optic Cup Lip) where the epithelium bends back upon itself. Shortly after the optic cup forms, it is thought to be sub-divided into separate lineages: i) pigmented epithelium in the outer layer; ii) presumptive iris and ciliary body at the most anterior aspect of the inner layer; and iii) presumptive neural retina in the remainder of the inner layer. We test the native developmental potential of the anterior cup to determine if it normally contributes to the retina., Methods: Vital dye and green fluorescent protein (GFP) expressing replication-incompetent retroviral vectors were used to label cells in the nascent optic cup and follow their direct progeny throughout development. Label was applied to either the optic cup lip (n=40), or to the domain just posterior to the lip (n=20). Retroviral labeling is a permanent lineage marker and enabled the analysis of advanced stages of development., Results: Labeling within the optic cup gave rise to labeled progeny in the posterior optic cup that differentiated as neural retina (20 of 20). In contrast, labeling cells in the optic cup lip gave rise to progeny of labeled cells arrayed in a linear progression, from the lip into the neural retina (36 of 40). Label was retained in cells at the optic cup lip, regardless of age at examination. In older embryos, labeled progeny delaminated from the optic cup lip to differentiate as muscle of the pupillary margin., Conclusions: The data show that the cells at the optic cup lip are a common progenitor population for pigmented epithelium, anterior eye tissues (ciliary body, iris, and pupillary muscle) and retinal neurons. The findings are supportive of an interpretation where the optic cup lip is a specialized niche containing a multipotent progenitor population.

There are numerous scenarios in which replacing the diseased RPE monolayer is an attractive but as yet unrealised goal. The proof of concept that vision can be improved by placing a healthy neuroretina onto a different, healthy, underlying RPE layer is demonstrated in patch graft transplantations. The surgical procedure to relocate the neuroretina is both complex and is hampered by postoperative complications and as such newer replacement procedures are also being investigated including stem cell replacement therapies. Past studies have largely focused on using cell suspensions and have had disappointing outcomes largely due to the lack of control over cellular differentiation, incomplete attachment onto Bruch's membrane and subsequent integration into the existing RPE monolayer. The choice of which cells to transplant is still under investigation and is complicated by factors such as the ease of collection of an adequate sample, rejection following implantation, the age of the cells and ethical issues. In all these situations, however, understanding the mechanisms of cellular differentiation are likely to be prerequisite to future successes.The current research into replacing the RPE monolayer is briefly discussed with reference to our experiences comparing IPE and RPE cells in an in vitro environment.

The teleost Astyanax mexicanus is a single species consisting of two radically different forms: a sighted pigmented surface-dwelling form (surface fish) and a blind depigmented cave-dwelling form (cavefish). The two forms of Astyanax have favorable attributes, including descent from a common ancestor, ease of laboratory culture, and the ability to perform genetic analysis, permitting their use as a model system to explore questions in evolution and development. Here, we review current research on the molecular, cellular, and developmental mechanisms underlying the loss of eyes and pigmentation in Astyanax cavefish. Although functional eyes are lacking in adults, cavefish embryos begin to develop eye primordia, which subsequently degenerate. The major cause of eye degeneration appears to be apoptotic cell death of the lens, which prevents the growth of other optic tissues, including the retina. Ultimately, the loss of the eye is the cause of craniofacial differences between cavefish and surface fish. Lens apoptosis is induced by enhanced activity of the Hedgehog signaling system along the cavefish embryonic midline. The absence of melanin pigmentation in cavefish is due to a block in the ability of undifferentiated melanoblasts to accumulate L-tyrosine, the precursor of L-DOPA and melanin, in melanosomes. Genetic analysis has shown that this defect is caused by a hypomorphic mutation in the p/oca2 gene encoding an integral melanosomal membrane protein. We discuss how current studies of eye and pigment regression have revealed some of the mechanisms in which cavefish development has been changed during evolution.

The development of extraocular orbital structures, in particular the choroid and sclera, is regulated by a complex series of interactions between neuroectoderm, neural crest and mesoderm derivatives, although in many instances the signals that mediate these interactions are not known. In this study we have investigated the function of Indian hedgehog (Ihh) in the developing mammalian eye. We show that Ihh is expressed in a population of non-pigmented cells located in the developing choroid adjacent to the RPE. The analysis of Hh mutant mice demonstrates that the RPE and developing scleral mesenchyme are direct targets of Ihh signaling and that Ihh is required for the normal pigmentation pattern of the RPE and the condensation of mesenchymal cells to form the sclera. Our findings also indicate that Ihh signals indirectly to promote proliferation and photoreceptor specification in the neural retina. This study identifies Ihh as a novel choroid-derived signal that regulates RPE, sclera and neural retina development.

During vertebrate eye development, the transcription factor MITF plays central roles in neuroepithelial domain specification and differentiation of the retinal pigment epithelium. MITF is not a single protein but represents a family of isoforms generated from a common gene by alternative promoter/exon use. To address the question of the role and regulation of these isoforms, we first determined their expression patterns in developing mouse eyes and analyzed the role of some of them in genetic models. We found that two isoforms, A- and J-Mitf, are present throughout development in both retina and pigment epithelium, whereas H-Mitf is detected preferentially and D-Mitf exclusively in the pigment epithelium. We further found that a genomic deletion encompassing the promoter/exon regions of H-, D- and B-Mitf leads to novel mRNA isoforms and proteins translated from internal start sites. These novel proteins lack the normal, isoform-specific N-terminal sequences and are unable to support the development of the pigment epithelium, but are capable of inducing pigmentation in the ciliary margin and the iris. Moreover, in mutants of the retinal Mitf regulator Chx10 (Vsx2), reduced cell proliferation and abnormal pigmentation of the retina are associated with a preferential upregulation of H- and D-Mitf. This retinal phenotype is corrected when H- and D-Mitf are missing in double Mitf/Chx10 mutants. The results suggest that Mitf regulation in the developing eye is isoform-selective, both temporally and spatially, and that some isoforms, including H- and D-Mitf, are more crucial than others in effecting normal retina and pigment epithelium development.

Background: Nanophthalmos is a genetic disorder characterized by very small, hyperopic eyes that are without gross structural defects. Recessive nanophthalmos is caused by severe mutations in the MFRP gene, which encodes a Frizzled-related transmembrane protein that is selectively expressed in the retinal pigment epithelium (RPE) and ciliary body., Results: For two MFRP -/- adults, we have obtained records of refraction that begin in early childhood. At the age of 6 months, one patient's eyes already had a refractive error of +12.25 D, and over the next 20 years this slowly increased to +17.50 D. Adults homozygous for null mutations in MFRP have eyes with axial lengths shorter than those of normal newborns. Furthermore, the unusually high curvature of their corneas is consistent with eyes that had been smaller than normal during late fetal development. MFRP protein was first detected at 14 weeks of gestation, when it was restricted to the posterior pole RPE. By 20 weeks gestation, MFRP expression had spread laterally, and was found throughout the RPE. MFRP protein was detected in both posterior and lateral RPE of the adult eye., Conclusions: Embryonic function of the MFRP gene appears necessary for the eye to reach its full size at birth. Its onset of expression in the RPE during mid-gestation suggests that MFRP does not participate in early formation of the optic cup, and is consistent with a role in later growth and development of the eye. Patients without MFRP gene function exhibit no correction of refractive error during childhood, which suggests that this gene is essential for emmetropization, a complex process by which vision regulates axial growth of the eye.

The iris plays a key role in visual function. It regulates the amount of light entering the eye and falling on the retina and also operates in focal adjustment of closer objects. The iris is involved in circulation of the aqueous humor and hence functions in regulation of intraocular pressure. Intriguingly, iris pigmented cells possess the ability to transdifferentiate into different ocular cell types of retinal pigmented epithelium, photoreceptors and lens cells. Thus, the iris is considered a potential source for cell-replacement therapies. During embryogenesis, the iris arises from both the optic cup and the periocular mesenchyme. Its interesting mode of development includes specification of the peripheral optic cup to a non-neuronal fate, migration of cells from the surrounding periocular mesenchyme and an atypical formation of smooth muscles from the neuroectoderm. This manner of development raises some interesting general topics concerning the early patterning of the neuroectoderm, the specification and differentiation of diverse cell types and the interactions between intrinsic and extrinsic factors in the process of organogenesis. In this review, we discuss iris anatomy and development, describe major pathologies of the iris and their molecular etiology and finally summarize the recent findings on genes and signaling pathways that are involved in iris development.

Microphthalmia-associated transcription factor (Mitf) is expressed in neural crest cell-derived melanocytes, and in the retinal pigment epithelium (RPE) during ocular development. Mutations in Mitf are associated with auditory/visual/pigmentary syndromes in humans. Mitf(mi/mi) mouse mutants lack pigmentation, and are microphthalmic, while Mitf(vit/vit) mouse mutants display abnormal RPE pigmentation, and progressive retinal degeneration. Microarray analysis was used to identify novel downstream gene targets/pathways in the RPE that are altered by mutations in the transcription factor Mitf. Using the Affymetrix platform, gene expression profiles were generated using the eyes of E13.5 mouse fetuses that were wildtype, heterozygous, or homozygous for the Mitf(mi) mutation. In a separate experiment, eyes from E13.5 mouse fetuses homozygous for the Mitf(vit) mutation were compared to eyes from the C57BL/6 control background strain. Statistical analyses were performed using robust multiarray average, mixed-effects ANOVA and random-variance t-tests. Altered expression of genes involved in pigment formation, melanosome biogenesis/transport, and redox homeostasis were observed. Twelve genes were commonly mis-regulated in the eyes of both Mitf mutants: 10 of these genes were downregulated in both mutants relative to controls, while 2 of the genes (Nramp1 (Slc11a1) and epoxide hydrolase) were downregulated in Mitf(mi/mi) mutants, and conversely, upregulated in Mitf(vit/vit) mutants. Quantitative RT-PCR and immunohistochemistry were used to confirm altered gene/protein expression. RPE expression of the Fe(+2) iron transporter Nramp1 (Slc11a1) has not previously been reported. Fe(+2) is an important co-factor utilized by the iron-dependent isomerohydrolase RPE65 in the retinoid visual cycle. However, excess accumulation of Fe(+2) in the RPE has recently been associated with oxidative damage and age-related macular degeneration. Abnormal pigmentation and increased activity of Slc11a1 in the RPE of Mitf(vit) mice may contribute to the pathology and progressive retinal degeneration observed in these mutants.

In vertebrates, the neuroepithelium of the optic vesicle is initially multipotential, co-expressing a number of transcription factors that are involved in retinal pigment epithelium (RPE) and neural retina (NR) development. Subsequently, extrinsic signals emanating from the surrounding tissues induce the separation of the optic vesicle into three domains: the optic stalk/nerve, the NR and the RPE. Here, we show that bone morphogenetic proteins (BMPs) are sufficient and essential for RPE development in vivo. Bmp4 and Bmp7 are expressed in the surface ectoderm overlying the optic vesicle, the surrounding mesenchyme and/or presumptive RPE during the initial stages of eye development. During the initial stages of chick eye development the microphthalmia-associated transcription factor (Mitf), important for RPE development, is expressed in the optic primordium that is covered by the BMP-expressing surface ectoderm. Following BMP application, the optic neuroepithelium, including the presumptive optic stalk/nerve and NR domain, develop into RPE as assessed by the expression of Otx2, Mitf, Wnt2b and the pigmented cell marker MMP115. By contrast, interfering with BMP signalling prevents RPE development in the outer layer of the optic cup and induces NR-specific gene expression (e.g. Chx10). Our results show that BMPs are sufficient and essential for RPE development during optic vesicle stages. We propose a model in which the BMP-expressing surface ectoderm initiates RPE specification by inducing Mitf expression in the underlying neuroepithelium of the optic vesicle.

Otx2 and Crx are vertebrate orthologs of the orthodenticle family of homeobox genes, which are involved in retinal development. In this study, the temporal expression patterns of Otx2 and Crx in the rat retina during embryonic and postnatal stages of development were analyzed in detail. This confirmed the presence of Otx2 mRNA in both the embryonic retinal pigment epithelium and the developing neural retina. During development, the expression of Otx2 persists in the pigment epithelium, whereas Otx2 expression of the neural retina becomes progressively restricted to the outer nuclear layer and the outer part of the inner nuclear layer. Immunohistochemistry revealed that Otx2 protein is also present in cell bodies of the ganglion cell layer, which does not contain the Otx2 transcript, suggesting that Otx2 protein is synthesized in cell bodies of the bipolar neurons and then transported to and taken up by cells in the ganglion cell layer. Crx is also highly expressed in the outer nuclear layer starting at E17 and postnatally in the inner nuclear layer. The onset of expression of Crx lags behind that of Otx2 consistent with evidence that Otx2 activates Crx transcription. These expression patterns are consistent with evidence that Otx2 and Crx function during retinal development and extend the period of probable functionality to the adult. In this regard, these results provide an enhanced and expanded temporal and spatial framework for understanding the multiple roles of Otx2 and Crx in the developing and mature mammalian retina.

Retinal mitosis takes place at the interface between the retinal pigment epithelium (RPE) and the neural retina. Multiple studies have highlighted the essential role that gap junction-mediated communication plays in the regulation of retinal organogenesis. Here, the localization pattern and function of the gap junction protein connexin 43 were examined in vivo in the rat at the interface between the retina and RPE during the main phases of retinal cell production. Connexin 43 was expressed at this site from E15 onward, and levels were subsequently temporally regulated. When Cx43 protein levels were reduced experimentally, by using antisense oligodeoxynucleotides, mitotic activity in the retina decreased significantly. Conversely, in the hypopigmented eye elevated mitotic levels were associated with a significant increase of connexin 43. Both excess protein levels and elevated mitosis were corrected by the in vivo administration of L-DOPA (a dopamine precursor and intermediary compound in the melanin synthesis pathway). These findings suggest that connexin 43-mediated communication between the retina and RPE is essential for the correct pacing of retinal organogenesis. Furthermore, this pathway may be gated by levels of ocular catecholamines.

Comparative analysis of the early transformations of differentiated cells of the pigment epithelium, ciliary fold epithelium, and Muller glia in the eye of lower vertebrates and mammals during retina regeneration and cultivation was performed for the first time. Dedifferentiation and proliferation of cells and formation of progenitor multipotent cells, which are a source of retina regeneration in adult newts, were characterized using cell, molecular, and genetic markers. Neurospheres were formed during cultivation of the differentiated cells, in which progenitor multipotent cells were found that transformed into neurons of retina and brain and into glial cells. Comparative analysis of changes in the pigment epithelium cells during retina regeneration and during cultivation of differentiated cells of the pigment and ciliary epithelia and Muller glia suggests similar cell transformations at the early stages of transdifferentiation.

Purpose: Eye development and photoreceptor maintenance is dependent on the retinal pigment epithelium (RPE), a thin layer of cells that underlies the neural retina. Despite its importance, development of RPE has not been studied by a genomic approach. In this study, a microarray expression-profiling methodology was established for studying RPE development., Methods: The intact retina with RPE attached was dissected from developing embryos, and differentially expressed genes in RPE were inferred by comparing the dissected tissues with retinas without RPE, in microarray and statistical analyses., Results: Of the probesets used, 8810 were significantly expressed in RPE at 52 hours postfertilization (hpf), of which 1443 may have biologically meaningful expression levels. Further, 78 and 988 probesets were found to be significantly over- or underexpressed in RPE, respectively, compared with retina. Also, 79.2% (38/48) of the known overexpressed probesets were independently validated as RPE-related transcripts., Conclusions: The results strongly suggest that this methodology can obtain in vivo RPE-specific gene expression from the zebrafish embryos and identify novel RPE markers.

Purpose: To elucidate the early cellular events that take place during induction of retina regeneration in the embryonic chick, focusing on the relationship between fibroblast growth factor (FGF) signaling and the regulation of Pax6 and Mitf., Methods: The retina of embryonic day 4 (E4) chicks was removed and a heparin coated bead soaked in fibroblast growth factor 2 (FGF2) was placed into the optic cup. The pharmacological inhibitor PD173074 was used to inhibit FGF receptors, PD98059 was used to inhibit MAP kinase-kinase/extracellular signal-regulated kinase (MEK/Erk) signaling. Retroviral constructs for paired box 6 (Pax6), MEK, and microphthalmia (Mitf) were also used in overexpression studies. Immunohistochemistry was used to examine pErk, Pax6, Mitf, and melanosomal matrix protein 115 (MMP115) immunoreactivity and bromodeoxyuridine (BrdU) incorporation at different time points after removing the retina., Results: The embryonic chick has the ability to regenerate a new retina by the process of transdifferentiation of the retinal pigment epithelium (RPE). We observed that during the induction of transdifferentiation, downregulation of Mitf was not sufficient to induce transdifferentiation at E4 and that FGF2 was required to drive Pax6 protein expression and cell proliferation, both of which are necessary for transdifferentiation. Furthermore, we show that FGF2 works through the FGFR/MEK/Erk signaling cascade to increase Pax6 expression and proliferation. Ectopic Mitf expression was able to inhibit transdifferentiation by acting downstream of FGFR/MEK/Erk signaling, likely by inhibiting the increase in Pax6 protein in the RPE., Conclusions: FGF2 stimulates Pax6 expression during induction of transdifferentiation of the RPE through FGFR/MEK/Erk signaling cascade. This Pax6 expression is accompanied by an increase in BrdU incorporation. In addition, we show that Mitf is spontaneously downregulated after removal of the retina even in the absence of FGF2. This Mitf downregulation is not accompanied by Pax6 upregulation, demonstrating that FGF2 stimulated Pax6 upregulation is required for transdifferentiation of the RPE. Furthermore, we show that ectopic Mitf expression is able to protect the RPE from FGF2 induced transdifferentiation by inhibiting Pax6 upregulation.

The outer blood-retinal barrier is formed by the retinal pigment epithelium. In any epithelial monolayer, the tight junctions enable the epithelium to form a barrier by joining neighboring cells together and regulating transepithelial diffusion through the paracellular spaces. Tight junctions are complex, dynamic structures that regulate cell proliferation, polarity, and paracellular diffusion. The specific properties of tight junctions vary among epithelia, according to the physiological role of the epithelium. Unlike other epithelia, the apical surface of the retinal pigment epithelium interacts with a solid tissue, the neural retina. Secretions of the developing neural retina regulate the assembly, maturation, and tissue-specific properties of these tight junctions. The slow time course of development allows investigators to dissect the mechanisms of junction assembly and function. These studies are aided by culture systems that model different stages of development.

Purpose: Mutations in the membrane frizzled-related protein (MFRP) gene cause nanophthalmos in humans, and a splice site mutation causes recessive retinal degeneration in the rd6 mouse. In human and mouse genomes, the MFRP gene lies adjoining to the complement 1q tumor necrosis factor-related protein 5 (CTRP5/C1QTNF5) gene involved in causing retinal degeneration and abnormal lens zonules in human. The purpose of this study was to characterize the spatial and temporal expression of the mouse Mfrp gene, determine tissue and subcellular localization of MFRP protein, and study its interaction with CTRP5., Methods: Expression of the Mfrp gene in the mouse was studied by quantitative (q)RT-PCR. MFRP protein expression and distribution were studied by Western blot analysis, immunohistochemistry, and immunoelectron microscopy. Interaction with CTRP5 was studied by immunoprecipitation and immunoblot analysis, using mouse eye and human retinal pigmented epithelium (RPE) choroid extracts and by expressing full-length CTRP5 and MFRP in a heterologous system., Results: The Mfrp gene is specifically expressed in RPE and ciliary body (CB), and its expression starts during early stages of embryogenesis. In the albino mouse eye, MFRP is localized to the apical and basal membranes of RPE and ciliary epithelium (CE). In addition, MFRP and CTRP5 were found to colocalize in RPE, CE, and MDCK cells, a general model of polarized epithelia. These proteins interact with each other in ocular tissues and also in a heterologous system., Conclusions: MFRP is localized to the plasma membrane of CE and RPE, and colocalizes and interacts with CTRP5 indicating a functional relationship between these two proteins.

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest-derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell-type-specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin-synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.

Cadherins are crucial for tissue cohesion, separation of cell layers and cell migration during embryogenesis. To investigate the role of classical type II Xcadherin-6 (Xcad-6), we performed loss-of-function studies by morpholino oligonucleotide injections. This resulted in severe eye defects which could be rescued with murine cadherin-6. In the absence of Xcadherin-6, morphological alterations and a decrease in cell proliferation were observed with eye cup formation. Eye field transplantations of Xcadherin-6 depleted donors yielded grafts that failed to form a proper neuroepithelium in a wildtype environment. At later developmental stages Xcadherin-6 deficient eyes showed lamination defects in the outer neural retina, a reduced thickness of the ganglion cell layer (GCL) and a fragmented retina pigment epithelium (RPE). Thus, Xcadherin-6 is essential early in eye development for structural organization and growth of the neuroepithelium before it differentiates into neural retina and RPE.

Purpose: To study the possibility of generating photoreceptors through programming RPE transdifferentiation by examining cell differentiation after transplantation into the developing chick eye., Methods: RPE was isolated, and the cells were dissociated, cultured, and guided to transdifferentiate by infection with retrovirus expressing neuroD (RCAS-neuroD), using RCAS-green fluorescence protein (GFP) as a control. The cells were then harvested and microinjected into the developing eyes of day 5 to day 7 chick embryos, and their development and integration were analyzed., Results: Cells from the control culture integrated into the host RPE. When grafted cells were present in large number, multilayered RPE-like tissues were formed, and the extra tissues consisted of grafted cells and host cells. None of the cells from the control culture expressed photoreceptor-specific genes. In contrast, most cells from RCAS-neuroD-infected culture remained depigmented. A large number of them expressed photoreceptor-specific genes, such as visinin and opsins. Antibodies against red opsin decorated the apical tips and the cell bodies of the grafted, transdifferentiating cells. In the subretinal space, visinin(+) cells aligned along the RPE or an RPE-like structure. When integrated into the host outer nuclear layer, grafted cells emanated elaborate, axonal arborization into the outer plexiform layer of the host retina., Conclusions: Cultured RPE cells retained their remarkable regenerative capabilities. Cells guided to transdifferentiate along the photoreceptor pathway by neuroD developed a highly ordered cellular structure and could integrate into the outer nuclear layer. These data suggest that, through genetic programming, RPE cells could be a potential source of photoreceptor cells.

Shroom family proteins have been implicated in the control of the actin cytoskeleton, but so far only a single family member has been studied in the context of developing embryos. Here, we show that the Shroom-family protein, Shroom2 (previously known as APXL) is both necessary and sufficient to govern the localization of pigment granules at the apical surface of epithelial cells. In Xenopus embryos that lack Shroom2 function, we observed defects in pigmentation of the eye that stem from failure of melanosomes to mature and to associate with the apical cell surface. Ectopic expression of Shroom2 in naïve epithelial cells facilitates apical pigment accumulation, and this activity specifically requires the Rab27a GTPase. Most interestingly, we find that Shroom2, like Shroom3 (previously called Shroom), is sufficient to induce a dramatic apical accumulation of the microtubule-nucleating protein gamma-tubulin at the apical surfaces of naïve epithelial cells. Together, our data identify Shroom2 as a central regulator of RPE pigmentation, and suggest that, despite their diverse biological roles, Shroom family proteins share a common activity. Finally, because the locus encoding human SHROOM2 lies within the critical region for two distinct forms of ocular albinism, it is possible that SHROOM2 mutations may be a contributing factor in these human visual system disorders.

Vertebrate retinal pigment epithelium (RPE) cells are derived from the multipotent optic neuroepithelium, develop in close proximity to the retina, and are indispensible for eye organogenesis and vision. Recent advances in our understanding of RPE development provide evidence for how critical signaling factors operating in dorso-ventral and distal-proximal gradients interact with key transcription factors to specify three distinct domains in the budding optic neuroepithelium: the distal future retina, the proximal future optic stalk/optic nerve, and the dorsal future RPE. Concomitantly with domain specification, the eye primordium progresses from a vesicle to a cup, RPE pigmentation extends towards the ventral side, and the future ciliary body and iris form from the margin zone between RPE and retina. While much has been learned about the molecular networks controlling RPE cell specification, key questions concerning the cell proliferative parameters in RPE and the subsequent morphogenetic events still need to be addressed in greater detail.

Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.

We compared aged adult and fetal retinal pigment epithelium (RPE) migration and proliferation in cell culture. Aged adult RPE resurfaced on dishes coated with human or mouse laminin, human or rat collagen I, human or mouse collagen IV, or human fibronectin. Resurfacing was best on bovine corneal endothelial cell extracellular matrix. Resurfacing was worst on mouse laminin. Fetal RPE resurfacing was robust on all surfaces studied except human and mouse laminin. In general, fetal RPE resurfacing was much greater than that seen with aged RPE on comparable surfaces. The degree of cell proliferation (labeled by Ki-67) was much greater in fetal than adult RPE cells.

Purpose: Previous studies have demonstrated a role for the retinal pigment epithelium (RPE) in the development and maintenance of the choroidal vasculature, suggesting that RPE serves a trophic role for the choroidal vessels. The goal of this study was to determine the expression pattern of vascular endothelial growth factor (VEGF) and its receptors and their activation status in embryonic and adult choroid, with the purpose of providing cues regarding the role of VEGF in development and stabilization of the choroidal vasculature., Methods: Transgenic VEGF-LacZ mice were used to examine VEGF expression in embryonic and adult eyes. Expression of VEGF isoforms and receptors in the RPE-choroid complex was assessed by RT-PCR and real-time PCR. VEGF receptor 2 expression was assessed by immunohistochemistry and its activation state was examined by immunoprecipitation followed by phosphotyrosine blot., Results: VEGF is expressed by RPE throughout the choroidal vascular development and in the adult. The major VEGF isoforms detected in adult RPE were VEGF120 and VEGF164, with almost no detectable VEGF188. RT-PCR analysis showed expression of VEGF receptors and coreceptors in the RPE-choroid complex. VEGFR2 was detected in the choriocapillaris underlying the RPE. Immunoprecipitation and phosphotyrosine blot of this receptor revealed that VEGFR2 is activated in adult mouse and bovine choroids., Conclusions: The observations suggest that VEGF signaling is involved, not only in choroidal vessel formation, but perhaps also in the maintenance of the choriocapillaris.

Retinal pigment epithelial (RPE) cells possess the potential to transdifferentiate into myofibroblasts after stimulation with transforming growth factor beta (TGFbeta) and are implicated in the pathogenesis of proliferative vitreoretinopathy. In this study we evaluated how TGFbeta2 and various extracellular matrix (ECM) proteins modulate the transdifferentiation of human fetal retinal pigment epithelial cells (RPE) cells into myofibroblast-like cells. Furthermore, we investigated whether hepatocyte growth factor (HGF) can suppress this transdifferentiation. RPE cells were cultured on ECM coated or uncoated surfaces in the presence or absence of TGFbeta2. HGF was added to certain cultures only once or on a daily basis during the treatment. Transdifferentiation of RPE cells into myofibroblasts was assessed by the quantitation of alpha-smooth muscle actin (alpha-SMA) using immunocytochemistry, flow cytometry, real-time PCR and Western blotting. TGFbeta2 induced a significant increase of alpha-SMA expression in a dose-dependent manner. Compared with growth on uncoated surfaces, RPE cultured on fibronectin (FN)-coated surfaces and stimulated with TGFbeta2 showed a significantly higher alpha-SMA expression than untreated cells. This upregulation of alpha-SMA could be markedly reduced by daily treatment with HGF; however, a single HGF administration did not significantly reduce alpha-SMA. These findings are important for further understanding the interaction of cytokines, RPE cells and their environment in mesenchymal transformation as well as its possible modulation. Continuous or long-term treatment with HGF should be further investigated for its potential to prevent mesenchymal transdifferentiation of RPE cells, and ultimately, PVR in vivo.

foxd3 encodes a winged helix/forkhead class transcription factor expressed in the premigratory neural crest cells of many vertebrates. We have investigated the function of this gene in zebrafish neural crest by a loss of function approach using antisense morpholino oligonucleotides and immunostaining for Foxd3 protein. Knockdown of Foxd3 expression produces deficits in several differentiated neural crest derivatives, including jaw cartilage, peripheral neurons, and glia, and iridophore pigment cells. Other derivatives, such as melanophore and xanthophore pigment cells are not affected. Reduction in the expression of several lineage-specific markers becomes evident soon after the onset of neural crest migration, suggesting that Foxd3 knockdown affects these lineages at early stages in their development. In contrast, analysis of the expression of early neural crest markers indicates little effect on neural crest induction or initial emigration. Finally, cell transplantation suggests that with respect to dorsal root ganglia neurons the Foxd3 requirement is cell autonomous, although Foxd3 itself is not detectable in differentiated DRG neurons. These results suggest that in zebrafish Foxd3 may not be required for induction of neural crest identity but is necessary for the differentiation of a subset of neural crest cell fates, perhaps in precursors of particular neural crest lineages.

p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53.

Regressive evolution of morphological features is a common evolutionary event. However, the relationship between structural degeneration and loss of physiological function is often unclear because the ancestral and derived states of a character are usually not available for comparison. Here, we report studies on retinomotor rhythms during development of the blind cavefish Astyanax mexicanus, a single teleost species consisting of a sighted surface-dwelling form (surface fish) and several blind cave-dwelling (cavefish) forms. The eyed and blind forms of Astyanax diverged from a common sighted ancestor within the past million years. Despite the absence of functional eyes in cavefish adults, optic primordia are formed in embryos, but then gradually arrest in development, degenerate, and sink into the orbits. Although a layered retina is formed in cavefish embryos, it is deficient in photoreceptor cells, and in some cases the retinal pigment epithelium has lost its pigmentation. We show that the capacity to exhibit light-entrained retinomotor rhythms has been conserved in the degenerating embryonic eyes of two different Astyanax cavefish populations. The results indicate that loss of circadian retinal function does not precede and is therefore not required for eye degeneration in the blind cavefish.

Despite the importance of the retinal pigment epithelium (RPE) for vision, the molecular processes involved in its specification are poorly understood. We identified two new mutant alleles for the zebrafish gene chokh (chk), which display a reduction or absence of the RPE. Unexpectedly, the neural retina (NR) in chk is specified and laminated, indicating that the regulatory network leading to NR development is largely independent of the RPE. Genetic mapping and molecular characterization revealed that chk encodes Rx3. Expression analyses show that otx2 and mitfb are not expressed in the prospective RPE of chk, indicating that the retinal homeobox gene rx3 acts upstream of the molecular network controlling RPE specification. Cellular transplantations demonstrate that rx3 function is autonomously required to specify the prospective RPE. Though rx2 is also absent in chk, neither rx2 nor rx1 is required for RPE development. Thus, our data provide the first indication that, in addition to controlling optic lobe evagination and proliferation, chk/rx3 also determines cellular fate.

Melanocytes are specialized cells that produce melanin, the pigment responsible for skin, hair and retina color. They derive during embryogenesis from the precursor cells melanoblasts, which are neural crest cells committed to the pigment cell lineage. The differentiation of melanoblasts into melanocytes involves the expression of melanocyte-specific genes, particularly those responsible for melanin production, such as Tyr, Tyrp-1 and Dct, the expression of which depends on the melanocyte-specific transcription factor microphthalmia (Mitf). We have developed and executed a functional screen on melanocytes, with the aim of identifying genes involved in pigment cell biology. We have found Emx1 and Emx2, two highly related homeobox genes that when overexpressed in melanocytes can downregulate Mitf, Tyrp1, Dct and Tyr. Constitutive expression of Emx alters pigment cell morphology and growth properties: it confers TPA independence but not the ability to grow in soft agar. Spatial and temporal expression of Emx and Mitf during embryonic development suggests that Emx could be one factor that regulates correct expression of Mitf by inhibiting its activation in neuroepithelial derivatives other than melanocytes.

Heterozygous mutations in the homeobox gene, PITX2, result in ocular anterior segment defects and a high incidence of early-onset glaucoma. Pitx2 is expressed in both the neural crest and the mesoderm-derived precursors of the periocular mesenchyme. Complete loss of function in mice results in agenesis or severe disruption of periocular mesenchyme structures and extrinsic defects in early optic nerve development. However, the specific requirements for Pitx2 in neural crest versus mesoderm could not be determined using these mice, and only roles in the initial stages of eye development could be assessed due to early embryonic lethality. To determine the specific roles of Pitx2 in the neural crest precursor pool, we generated neural crest-specific Pitx2 knockout mice (Pitx2-ncko). Because Pitx2-nkco mice are viable, we also analyzed gene function in later eye development. Pitx2 is intrinsically required in neural crest for specification of corneal endothelium, corneal stroma and the sclera. Pitx2 function in neural crest is also required for normal development of ocular blood vessels. Pitx2-ncko mice exhibit a unique optic nerve phenotype in which the eyes are progressively displaced towards the midline until they are directly attached to the ventral hypothalamus. As Pitx2 is not expressed in the optic stalk, an essential function of PITX2 protein in neural crest is to regulate an extrinsic factor(s) required for development of the optic nerve. We propose a revised model of optic nerve development and new mechanisms that may underlie the etiology of glaucoma in Axenfeld-Rieger patients.

Mitf is a central regulator of pigment cell development that is essential for the normal development of the melanocyte and retinal pigment epithelium (RPE) lineages. To understand better the role of Mitf, we have used the Xenopus laevis experimental system to allow a rapid examination of the role of Mitf in vivo. Here, we report the function of XlMitfalpha-M on melanophore development and melanization compared with that of Slug that is expressed in neural crest cells. Overexpression of XlMitfalpha-M led to an increase in melanophores that was partly contributed by an increase in Slug-positive cells, indicating that XlMitfalpha-M is a key regulator of melanocyte/melanophore development and melanization. Moreover, overexpression of a dominant-negative form of XlMitfalpha led to a decrease in the number of melanophores and induced abnormal melanoblast migration. We also observed an induction of ectopic RPE and extended RPE by overexpression of XlMitfalpha-M and possible interactions between XlMitfalpha and several eye-related genes essential for normal eye development., (Developmental Dynamics 234:523-534, 2005. (c) 2005 Wiley-Liss, Inc.)

The morphology and distribution of normally placed and displaced A horizontal cells were studied in the retina of a diurnal hystricomorph rodent, the agouti Dasyprocta aguti. Cells were labeled with anti-calbindin immunocytochemistry. Dendritic-field size reaches a minimum in the visual streak, of about 9,000 microm(2), and increases toward the retinal periphery both in the dorsal and ventral regions. There is a dorsoventral asymmetry, with dorsal cells being larger than ventral cells at equal distances from the streak. The peak value for cell density of 281 +/- 28 cells/mm(2) occurs in the center of the visual streak, decreasing toward the dorsal and ventral retinal periphery, paralleling the increase in dendritic-field size. Along the visual streak, the decline in cell density is less pronounced, remaining between 100-200 cells/mm(2) in the temporal and nasal periphery. Displaced horizontal cells are rare and occur in the retinal periphery. They tend to be smaller than normally placed horizontal cells in the ventral region, whilst no systematic difference was observed between the two cell groups in the dorsal region. Mosaic regularity was studied using nearest-neighbor analysis and the Ripley function. When mosaic regularity was determined removing the displaced horizontal cells, there was a slight increase in the conformity ratio, but the bivariate Ripley function indicated some repulsive dependence between the two mosaics. Both results were near the level of significance. A similar analysis performed in the capybara retina, a closely related hystricomorph rodent bearing a higher density of displaced horizontal cells than found in the agouti, suggested spatial independence between the two mosaics, normally placed versus displaced horizontal cells.

Transcription factor genes governing pigment cell development that are associated with spotting mutations in mice include members of several structural transcription factor classes but not members of the basic helix-loop-helix (bHLH) class, important for neurogenesis and myogenesis. To determine the effects of bHLH factor expression on pigment cell development, the neurogenic bHLH factor Mash1 was expressed early in pigment cell development in transgenic mice from the dopachrome tautomerase (Dct) promoter. Dct:Mash1 transgenic founders exhibit variable microphthalmia and patchy coat color hypopigmentation. Transgenic F1 mice exhibit microphthalmia with complete coat color dilution. Marker analysis demonstrates that Mash1 expression in the retinal pigmented epithelium (RPE) initiates neurogenesis in this cell layer, whereas expression in remaining neural crest-derived melanocytes alters their differentiation, in part by profoundly downregulating expression of the p (pink-eyed dilution) gene, while maintaining their cell fate. The effects of transcriptional perturbation of pigment cell precursors by Mash1 further highlight differences between pigment cells of distinct developmental origins, and suggest a mechanism for the alteration of melanogenesis to result in marked coat color dilution.

During embryogenesis, multiple developmental processes are integrated through their precise temporal regulation. Hes1 is a transcriptional repressor that regulates the timing of mammalian retinal neurogenesis. However, roles for Hes1 in early eye development have not been well defined. Here, we show that Hes1 is expressed in the forming lens, optic vesicle, cup, and pigmented epithelium and is necessary for proper growth, morphogenesis, and differentiation of these tissues. Because Hes1 is required throughout the eye, we investigated its interaction with Pax6. Hes1-Pax6 double mutant embryos are eyeless suggesting these genes are coordinately required for initial morphogenesis and outgrowth of the optic vesicle. In Hes1 mutants, Math5 expression is precocious along with retinal ganglion cell, amacrine, and horizontal neuron formation. In contrast to apparent cooperativity between Pax6 and Hes1 during morphogenesis, each gene regulates Math5 and RGC genesis independently. Together, these studies demonstrate that Hes1, like Pax6, simultaneously regulates multiple developmental processes during optic development.

Located between vessels of the choriocapillaris and light-sensitive outer segments of the photoreceptors, the retinal pigment epithelium (RPE) closely interacts with photoreceptors in the maintenance of visual function. Increasing knowledge of the multiple functions performed by the RPE improved the understanding of many diseases leading to blindness. This review summarizes the current knowledge of RPE functions and describes how failure of these functions causes loss of visual function. Mutations in genes that are expressed in the RPE can lead to photoreceptor degeneration. On the other hand, mutations in genes expressed in photoreceptors can lead to degenerations of the RPE. Thus both tissues can be regarded as a functional unit where both interacting partners depend on each other.

Background/aims: Intraocular bone is seen in a wide spectrum of ocular disorders. The pathogenetic mechanisms of bone formation in the eye are unclear. Growth differentiation factor-5 (GDF-5), bone morphogenic protein-7 (BMP-7), and transforming growth factor beta-1 (TGF beta1) are multifunctional cytokines that have important roles in bone formation. Immunohistochemistry was used to localise GDF-5, BMP-7, and TGF beta1 in the human eye to determine their role in intraocular bone formation., Methods: Paraffin embedded sections from human eyes included fetal eyes (n = 5), normal adult eyes (n = 4), eyes with osseous metaplasia (n = 8), and eyes with focal fibrous metaplasia of the retinal pigment epithelium (RPE) without osseous metaplasia (n = 2). Immunohistochemistry was performed using indirect immunofluorescence with antibodies to GDF-5, BMP-7, and TGF beta1. The staining intensity was evaluated semiquantitatively in the RPE, retina, ciliary epithelium, and cornea; and analysed statistically., Results: When compared with normal adult eyes, which showed no RPE immunoreactivity, the RPE metaplasia surrounding areas of osseous metaplasia showed mild GDF-5 and moderate BMP-7 (p = 0.004) intracytoplasmic immunoreactivity. In contrast, trace GDF-5 and mild BMP-7 staining was seen in zones of RPE fibrous metaplasia in areas not associated with osseous metaplasia. Mild intracytoplasmic TGF beta1 expression was seen in the RPE metaplasia surrounding the bone when compared with adult eyes. Both fetal and adult eyes showed trace to mild GDF-5 and BMP-7 labelling of the non-pigmented ciliary epithelium which was increased in the eyes with osseous metaplasia. In eyes with osseous metaplasia, a significant decrease in GDF-5 and BMP-7 labelling was noted in fetal keratocytes (p = 0.0159 for both antibodies) when compared to adult eyes. Also, a significant decrease in BMP-7 labelling was seen in keratocytes in eyes with osseous metaplasia (p = 0.0162)., Conclusions: The increase in GDF-5, BMP-7, and TGF beta1 immunoreactivity in zones of RPE metaplasia in eyes with osseous metaplasia suggests that these proteins have an important role in intraocular ectopic bone formation.

Human retinal pigment epithelium (HRPE) cells are important in maintaining the normal physiology within the neurosensory retina and photoreceptors. Recently, transplantation of HRPE has become a possible therapeutic approach for retinal degeneration. By negative immunoselection (CD45 and glycophorin A), in this study, we have isolated and cultivated adult human bone marrow stem cells (BMSCs) with multilineage differentiation potential. After a 2- to 4-week culture under chondrogenic, osteogenic, adipogenic, and hepatogenic induction medium, these BMSCs were found to differentiate into cartilage, bone, adipocyte, and hepatocyte-like cells, respectively. We also showed that these BMSCs could differentiate into neural precursor cells (nestin-positive) and mature neurons (MAP-2 and Tuj1-positive) following treatment of neural selection and induction medium for 1 month. Furthermore, the plasticity of BMSCs was confirmed by initiating their differentiation into retinal cells and photoreceptor lineages by co-culturing with HRPE cells. The latter system provides an ex vivo expansion model of culturing photoreceptors for the treatment of retinal degeneration diseases.

The aim of this study was to establish synapses between a transplant and a degenerated retina. To tackle this difficult task, a little-known but well-established CNS method was chosen: trans-synaptic pseudorabies virus (PRV) tracing. Sheets of E19 rat retina with or without retinal pigment epithelium (RPE) were transplanted to the subretinal space in 33 Royal College of Surgeons (RCS) and transgenic s334ter-5 rats with retinal degeneration. Several months later, PRV-BaBlu (expressing E. colibeta-galactosidase) or PRV-Bartha was injected into an area of the exposed superior colliculus (SC), topographically corresponding to the transplant placement in the retina. Twenty normal rats served as controls. After survival times of 1-5 days, retinas were examined for virus by X-gal histochemistry, immunohistochemistry and electron microscopy. In normal controls, virus was first seen in retinal ganglion cells and Müller glia after 1-1.5 days, and had spread to all retinal layers after 2-3 days. Virus-labeled cells were found in 16 of 19 transplants where the virus injection had retrogradely labeled the topographically correct transplant area of the host retina. Electron microscopically, enveloped and nonenveloped virus could clearly be detected in infected cells. Enveloped virus was found only in neurons. Infected glial cells contained only nonenveloped virus. Neurons in retinal transplants are labeled after PRV injection into the host brain, indicating synaptic connectivity between transplants and degenerated host retinas. This study provides evidence that PRV spreads in the retina as in other parts of the CNS and is useful to outline transplant-host circuitry.

Pattern formation of biological structures involves organizing different types of cells into a spatial configuration. In this study, we investigate the physical basis of biological patterning of the Drosophila retina in vivo. We demonstrate that E- and N-cadherins mediate apical adhesion between retina epithelial cells. Differential expression of N-cadherin within a sub-group of retinal cells (cone cells) causes them to form an overall shape that minimizes their surface contact with surrounding cells. The cells within this group, in both normal and experimentally manipulated conditions, pack together in the same way as soap bubbles do. The shaping of the cone cell group and packing of its components precisely imitate the physical tendency for surfaces to be minimized. Thus, simple patterned expression of N-cadherin results in a complex spatial pattern of cells owing to cellular surface mechanics.

The aim of retinal transplantation is to prevent blindness and to restore eyesight, i.e. to rescue photoreceptors or to replace damaged photoreceptors with the hope of re-establishing neural circuitry. A promising experimental paradigm is the sub-retinal transplantation of sheets of fetal retina, with or without its attached retinal pigment epithelium (RPE), into recipient rats with retinal degeneration. Sheets of fetal retina have already developed their primordial circuitry. Such transplants can develop lamination resembling a normal retina dependent on the presence of healthy RPE either from the host or from the graft. In several retinal degeneration models, transplants have been shown to restore visually evoked responses in an area of the superior colliculus corresponding to the placement of the transplant in the retina. The functional effect of transplants may be due to transplant/host connectivity and/or rescue of host photoreceptors. In summary, sheets of fetal retina can morphologically repair an area of a degenerated retina, and there is evidence to suggest that transplants form synaptic connections with the host and restore visual responses in rats with retinal degeneration., (Copyright 2004 Elsevier Ltd.)

Photoreceptors in the vertebrate retina are light-sensitive neurons, and their degeneration results in irreversible visual loss. Understanding how photoreceptor fate is determined is a prerequisite for developing photoreceptor replacement therapies. Previous studies identified two basic helix-loop-helix genes, neurogenin2 (ngn2) and neuroD, participating in a genetic pathway leading to photoreceptor genesis. Here we present experimental data suggesting that ath5, which is known for its critical role in retinal ganglion cell development, may also lead to photoreceptor production. In the developing retina, ath5 expression was detected in two zones of cells, and coexpression with neuroD was observed in the zone adjacent to young photoreceptor cells accumulating on the retinal pigment epithelial side. Retroviral-driven misexpression of ath5 in retinal cells increased the population of photoreceptor cells, as well as ganglion cells, in a developmental stage-dependent manner that is consistent with ath5 being involved in the development of multiple types of retinal neurons. Ectopic ath5 expression in cultures of non-neural retinal pigment epithelial cells elicited transdifferentiation into cells that expressed photoreceptor-specific genes and displayed photoreceptor-like morphologies. Gene expression analysis showed that ngn2 did not induce ath5, and ath5 did not induce ngn2, but both induced neuroD and RaxL. These data suggest a pathway of "ath5 --> neuroD --> photoreceptor genes" separate from yet convergent with the ngn2 pathway.

The distribution as well as the ultrastructural and biochemical characteristics of proliferating cells in the human eye were investigated in five conceptuses of 5-9 postovulatory weeks, using morphological techniques and Ki-67 immunostaining. The Ki-67 nuclear protein was used as a proliferation marker because of its expression in all phases of the cell cycle except the resting phase (G0). The labelling indices of Ki-67-positive cells were analysed by means of the Kruskal-Wallis ANOVA test and the Wilcoxon matched-pairs test. In the 5th week, mitotic cells were the most numerous between the two layers of the optic cup, the optic cup and stalk, and between the lens pit and the surface ectoderm. During the 6th week, cells were observed in the lens epithelium covering the whole cavity of the lens vesicle as well as in the neuroblast zone and the pigmented epithelium of the retina. At later stages (7th-9th weeks), Ki-67-positive cells were restricted to the anterior lens epithelium, the outer neuroblast zone, and the pigmented retina. Throughout all stages examined, mitotic figures were found lying exclusively adjacent to the intraretinal space. Early in the lens pit, they were confined to the free epithelial surface, and later were facing the cavity of the lens vesicle. The proliferative activity was the most intensive in the 6th week, whereas it decreased significantly in the later stages. Additionally, when proliferative activities were compared, the peripheral retina appeared to be less mature than the central before the 9th week. In the earliest analysed stage, cell proliferation might be associated with the sculpturing of the optic cup and stalk, the cornea, and the lens. In the 6th week, the most intensive proliferation seems to be involved not only in the further morphogenesis of the optic cup and the lens vesicle but also in the retinal neurogenesis. At later stages, the decreased proliferation might participate in the neurogenesis of the outer neuroblast zone and the secondary lens fibre formation., (Copyright 2004 Springer-Verlag)

The retina pigment epithelium (RPE) is a highly specialised epithelium that serves as a multifunctional and indispensable component of the vertebrate eye. Although a great deal of attention has been paid to its transdifferentiation capabilities and its ancillary functions in neural retina development, little is known about the molecular mechanisms that specify the RPE itself. Recent advances in our understanding of the genetic network that controls the progressive specification of the eye anlage in vertebrates have provided some of the initial cues to the mechanisms responsible for RPE patterning. Here, we have outlined many recent findings that suggest that a limited number of transcription factors, including Otx2, Mitf and Pax6 and a few signalling cascades, are the elements required for the onset of RPE specification in vertebrates. Furthermore, using this information and the data available on the specification of the pigmented cells of primitive chordates, we have ventured some hypotheses on the origin of RPE cells during evolution., (Copyright 2004 Wiley Periodicals, Inc.)