Eye organogenesis: A hierarchical view of ocular development.

This chapter provides an overview of the early developmental origins of six ocular tissues: the cornea, lens, ciliary body, iris, neural retina, and retina pigment epithelium. Many of these tissue types are concurrently specified and undergo a complex set of morphogenetic movements that facilitate their structural interconnection. Within the context of vertebrate eye organogenesis, we also discuss the genetic hierarchies of transcription factors and signaling pathways that regulate growth, patterning, cell type specification and differentiation.

Ciliary margin-derived BMP4 does not have a major role in ocular development.

Heterozygous Bmp4 mutations in humans and mice cause severe ocular anterior segment dysgenesis (ASD). Abnormalities include pupil displacement, corneal opacity, iridocorneal adhesions, and variable intraocular pressure, as well as some retinal and vascular defects. It is presently not known what source of BMP4 is responsible for these defects, as BMP4 is expressed in several developing ocular and surrounding tissues. In particular, BMP4 is expressed in the ciliary margins of the optic cup which give rise to anterior segment structures such as the ciliary body and iris, making it a good candidate for the required source of BMP4 for anterior segment development. Here, we test whether ciliary margin-derived BMP4 is required for ocular development using two different conditional knockout approaches. In addition, we compared the conditional deletion phenotypes with Bmp4 heterozygous null mice. Morphological, molecular, and functional assays were performed on adult mutant mice, including histology, immunohistochemistry, in vivo imaging, and intraocular pressure measurements. Surprisingly, in contrast to Bmp4 heterozygous mutants, our analyses revealed that the anterior and posterior segments of Bmp4 conditional knockouts developed normally. These results indicate that ciliary margin-derived BMP4 does not have a major role in ocular development, although subtle alterations could not be ruled out. Furthermore, we demonstrated that the anterior and posterior phenotypes observed in Bmp4 heterozygous animals showed a strong propensity to co-occur, suggesting a common, non-cell autonomous source for these defects.

Neonatal and Juvenile Ocular Development in Sprague-Dawley Rats: A Histomorphological and Immunohistochemical Study.

As in many altricial species, rats are born with fused eyelids and markedly underdeveloped eyes. While the normal histology of the eyes of mature rats is known, the histomorphological changes occurring during postnatal eye development in this species remain incompletely characterized. This study was conducted to describe the postnatal development of ocular structures in Sprague-Dawley (SD) rats during the first month of age using histology and immunohistochemistry (IHC). Both eyes were collected from 51 SD rats at 13 time points between postnatal day (PND)1 and PND30. Histologic examination of hematoxylin and eosin-stained sections was performed, as well as IHC for cleaved-caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to evaluate apoptosis, and IHC for Ki-67 and phospho-histone-H3 to evaluate cell proliferation. Extensive ocular tissue remodeling occurred prior to the eyelid opening around PND14 and reflected the interplay between apoptosis and cell proliferation. Apoptosis was particularly remarkable in the maturing subcapsular anterior epithelium of the lens, the inner nuclear and ganglion cell layers of the developing retina, and the Harderian gland, and was involved in the regression of the hyaloid vasculature. Nuclear degradation in the newly formed secondary lens fibers was noteworthy after birth and was associated with TUNEL-positive nuclear remnants lining the lens organelle-free zone. Cell proliferation was marked in the developing retina, cornea, iris, ciliary body and Harderian gland. The rat eye reached histomorphological maturity at PND21 after a rapid phase of morphological changes characterized by the coexistence of cell death and proliferation.

A mouse model of aniridia reveals the in vivo downstream targets of Pax6 driving iris and ciliary body development in the eye.

The Pax6 transcription factor is essential for development of the brain, eye, olfactory and endocrine systems. Haploinsufficiency of PAX6 in humans and mice causes the congenital condition aniridia, with defects in each of these organs and systems. Identification of the PAX6 transcription networks driving normal development is therefore critical in understanding the pathophysiology observed with loss-of-function defects. Here we have focused on identification of the downstream targets for Pax6 in the developing iris and ciliary body, where we used laser capture microdissection in mouse eyes from E12.5-E16.5, followed by chromatin immunoprecipitation, promoter-reporter assays and immunohistochemistry. We identified 6 differentially expressed genes between wildtype and Pax6 heterozygous mouse tissues and demonstrated that Bmp4, Tgfß2, and Foxc1 were direct downstream targets of Pax6 in developing iris/ciliary body. These results improve our understanding of how mutations in Bmp4, Tgfß2, and Foxc1 result in phenocopies of the aniridic eye disease and provide possible targets for therapeutic intervention.

Profiling of DNA and histone methylation reveals epigenetic-based regulation of gene expression during retinal differentiation of stem/progenitor cells isolated from the ciliary pigment epithelium of human cadaveric eyes.

Millions of people around the world suffer from retinal degenerative diseases at varying degrees of vision loss including, complete blindness that are caused by the damage to cells of the retina. The cell replacement therapy could be a promising tool in treating these conditions, since the stem/progenitor cells could be isolated form adult ciliary pigment epithelial cells and could be differentiated into retinal phenotypes in vitro and could be of great importance. The present study aims to identify the role of epigenetic regulators during cellular differentiation, which involves loss of pluripotency and gain of lineage and cell type-specific characteristics. We analyzed DNA methylation and Histone methylation-H3K4me3 and H3K27me3 in ciliary body derived lineage committed progenitor to terminally differentiated cells. Our results demonstrate that several promoters including pluripotency and lineage specific genes become methylated in the differentiated population, suggesting that methylation may repress the pluripotency in this population. On the other hand, we detect bivalent modifications that are involved in the process of differentiation of stem/progenitor cells. Therefore, this data suggest a model for studying the epigenetic regulation involved in self renewal, pluripotency and differentiation potential of ciliary stem/progenitor cells. This work presents the first outline of epigenetic modifications in ciliary derived stem/progenitor cells and the progeny that underwent differentiation into retinal neurons/glial cells and shows that specific DNA methylation and histone methylations are extensively involved in gene expression reprogramming during differentiation.

Ciliary Muscle Cell Changes During Guinea Pig Development.

PURPOSE: Guinea pig ciliary muscle (CM) increases robustly in volume, length, and thickness with age. We wanted to characterize CM cells during development to determine the contributions of hypertrophy (cell size increase) and hyperplasia (cell number increase) during development. METHODS: Six pigmented guinea pig eyes were collected at each of five ages: 1, 10, 20, 30, and 90 days. Refractive errors and axial lengths were determined. Eyes were temporally marked, enucleated, hemisected, and fixed. Nasal and temporal eye segments were embedded and 30-µm serial sections were collected; the two most central slides from each hemisection were analyzed with an epifluorescence microscope and Stereo Investigator software to determine normal morphologic parameters. RESULTS: Refractive errors became less hyperopic (P = 0.0001) while axial lengths and CM lengths, cross-sectional areas, volumes, and cell sizes all increased linearly with log age (all P < 0.00001). Ciliary muscle cell numbers increased only during the first 20 days of life (P = 0.02). Nasal and temporal CM lengths (P = 0.07), cross-sectional areas (P = 0.18), and cell numbers (P = 0.70) were not different, but CM cell sizes were initially larger temporally and became larger nasally after age 30 days. CONCLUSIONS: The mechanism of guinea pig CM cell growth during the first 90 days of life was characterized by early hyperplasia combined with hypertrophic cell growth throughout development that results in larger CM lengths, cross-sectional areas, and volumes. Nasal-temporal CM development was generally symmetric, but there was more CM hypertrophy nasally at older ages.

Guinea pig ciliary muscle development.

PURPOSE: The purpose of this study was to develop a method for quantifying guinea pig ciliary muscle volume (CMV) and to determine its relationship to age and ocular biometric measurements. METHODS: Six albino guinea pigs' eyes were collected at each of five ages (n = 30 eyes). Retinoscopy and photography were used to document refractive error, eye size, and eye shape. Serial sections through the excised eyes were made and then labeled with an α-smooth muscle actin antibody. The ciliary muscle was then visualized with an Olympus BX51 microscope, reconstructed with Stereo Investigator (MBF Bioscience), and analyzed using Neurolucida Explorer (MBF Bioscience). Full (using all sections) and partial (using a subset of sections) reconstruction methods were used to determine CMV. RESULTS: There was no significant difference between the full and partial volume determination methods (p = 0.86). The mean (±SD) CMV of the 1-, 10-, 20-, 30-, and 90-day-old eyes was 0.40 (±0.16) mm, 0.48 (±0.13) mm, 0.67 (±0.15) mm, 0.86 (±0.35) mm, and 1.09 (±0.63) mm, respectively. Ciliary muscle volume was significantly correlated with log age (p = 0.001), ocular length (p = 0.003), limbal circumference (p = 0.01), and equatorial diameter (p = 0.003). It was not correlated with refractive error (p = 0.73) or eye shape (p = 0.60). Multivariate regression determined that biometric variables were not significantly associated with CMV after adjustment for age. CONCLUSIONS: Three-dimensional reconstruction was an effective means of determining CMV. These data provide evidence that ciliary muscle growth occurs with age in tandem with eye size in normal albino guinea pigs. Additional work is needed to determine the relationship between CMV and abnormal ocular growth.

The ciliary marginal zone (CMZ) in development and regeneration of the vertebrate eye.

The ciliary marginal zone (CMZ) is a circumferential ring of cells found at the extreme periphery of the maturing and mature neural retina that consists of retinal stem and progenitor cells. It functions to add retinal neurons to the periphery of the neural retina in larval and adult fish, larval frogs, and birds. Additionally, the CMZ may contribute to regeneration of the damaged retina in frogs and fish. In mammals, cells from the ciliary epithelium can be induced to express retinal stem cell-like characteristics in culture but may not comprise a classically defined CMZ.

Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye.

The ciliary body (CB) of the mammalian eye is responsible for secreting aqueous humor to maintain intraocular pressure, which is elevated in the eyes of glaucoma patients. It contains a folded two-layered epithelial structure comprising the nonpigmented inner ciliary epithelium (ICE), the pigmented outer ciliary epithelium (OCE), and the underlying stroma. Although the CB has an important function in the eye, its morphogenesis remains poorly studied. In this study, we show that conditional inactivation of the Jagged 1 (Jag1)-Notch2 signaling pathway in the developing CB abolishes its morphogenesis. Notch2 is expressed in the OCE of the CB, whereas Jag1 is expressed in the ICE. Conditional inactivation of Jag1 in the ICE or Notch2 in the OCE disrupts CB morphogenesis, but neither affects the specification of the CB region. Notch2 signaling in the OCE is required for promoting cell proliferation and maintaining bone morphogenetic protein (BMP) signaling, both of which have been suggested to be important for CB morphogenesis. Although Notch and BMP signaling pathways are known to cross-talk via the interaction between their downstream transcriptional factors, this study suggests that Notch2 maintains BMP signaling in the OCE possibly by repressing expression of secreted BMP inhibitors. Based on our findings, we propose that Jag1-Notch2 signaling controls CB morphogenesis at least in part by regulating cell proliferation and BMP signaling.

Expression and role of VEGF--a in the ciliary body.

PURPOSE: The role of VEGF-A in the normal ciliary body is largely unexplored. The ciliary body is similar in many respects to the choroid plexus of the brain, and we demonstrated previously the importance of VEGF-A in maintenance of choroid plexus vasculature and ependymal cells. Therefore, the role of VEGF-A in ciliary body homeostasis was explored. METHODS: Swiss-Webster mice (VEGF-LacZ) were used to determine VEGF-A expression during ciliary body development and in the adult. VEGFR2 expression was determined in adult wild type C56BL/6J mice. Systemic VEGF-A neutralization in vivo was achieved with adenovirus-mediated overexpression of soluble VEGFR1 (sFlt1). Following VEGF-A neutralization, the ciliary epithelium was analyzed by light microscopy and transmission electron microscopy (TEM). The effect of VEGF-A blockade on ciliary body function also was assessed by measuring intraocular pressure. RESULTS: VEGF-A expression was detected at embryonic day 18.5 (E18.5), the onset of ciliary process formation. In the adult ciliary body, VEGF-A was expressed by the pigmented epithelium, whereas VEGFR2 was localized primarily to the capillary endothelium and nonpigmented epithelium. Systemic VEGF-A neutralization led to a thinning of the nonpigmented epithelium, vacuolization of the pigmented epithelium, loss of capillary fenestrations, and thrombosis. These changes were associated with impaired ciliary body function, as evidenced by decreased intraocular pressure in sFlt1-overexpressing animals (15.31 ± 2.06 mm Hg) relative to controls (18.69 ± 1.49 mm Hg). CONCLUSIONS: VEGF-A has an important role in ciliary body homeostasis. Potential for undesired off-target effects should be considered with the chronic use of anti-VEGF-A therapies.

Identification of gene transcripts expressed by postsynaptic neurons during synapse formation encoding cell surface proteins with presumptive synaptogenic activity.

Synapse formation is a well-programmed developmental process involving a variety of cell-cell interactions carried out by distinct groups of molecules. Various molecules that contribute to the assembly of synaptic contacts have been characterized; however, the repertoire of identified proteins expressed by postsynaptic neurons capable of inducing presynaptic differentiation is quite limited. To identify gene transcripts encoding cell surface proteins expressed by postsynaptic cells with molecular features suggestive of synaptogenic activity, this study carried out a genome-wide expression analysis in the chick ciliary ganglion during the different phases of synapse formation. It was found that from the 21,493 gene-probes detected throughout development, 302 protein-coding transcripts were upregulated during the initiation of synapse formation. Analysis of this pool of transcripts showed that 51 of them encoded cell surface proteins (27 membrane-bound and 24 secreted) with protein-protein interacting domains. This includes twelve cell adhesion molecules, six ligand-receptors, six proteins with ligand-like domains, three membrane bound enzymes, eight components of the extracellular matrix, three neuropeptides, three cytokines and growth factors, five extracellular modulators of cell signaling, and five unrelated secreted proteins. Furthermore, the role of synaptic transmission during the initiation of synapse formation was evaluated by assessing the effect of synaptic activity blockade with d-tubocurarine on the expression levels of the pool of 51 transcripts encoding cell surface proteins. Treatment with d-tubocurarine reduced the expression levels of 22% of the selected genes, while the expression levels of 78% of the genes was not affected or was enhanced.

Pax6 dosage requirements in iris and ciliary body differentiation.

Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.

Molecular events in early development of the ciliary body: a question of folding.

Ciliary body morphogenesis is a complicated, multi-step process requiring coordinated changes in cell shape, flexure of epithelial sheets and dynamic shifts in mitotic rates. Very little is known of how these cellular events are triggered or regulated. This review summarises current models of ciliary body morphogenesis. The role of intraocular pressure as a driver of morphogenesis is re-evaluated in the light of new information. An update on the role of the lens in ciliary body morphogenesis is presented. In the second part of the review current gene expression data is related to ciliary body morphogenesis. In particular the role of Bmp4 and its downstream target genes are discussed, with novel gene expression patterns of Bmp4 and Tgfbeta1i4 being presented.

Wnt2b/beta-catenin-mediated canonical Wnt signaling determines the peripheral fates of the chick eye.

Wnt signaling orchestrates multiple aspects of central nervous system development, including cell proliferation and cell fate choices. In this study, we used gene transfer to activate or inhibit canonical Wnt signaling in vivo in the developing eye. We found that the expression of Wnt2b or constitutively active (CA) beta-catenin inhibited retinal progenitor gene (RPG) expression and the differentiation of retinal neurons. In addition, Wnt signal activation in the central retina was sufficient to induce the expression of markers of the ciliary body and iris, two tissues derived from the peripheral optic cup (OC). The expression of a dominant-negative (DN) allele of Lef1, or of a Lef1-engrailed fusion protein, led to the inhibition of expression of peripheral genes and iris hypoplasia, suggesting that canonical Wnt signaling is required for peripheral eye development. We propose that canonical Wnt signaling in the developing optic vesicle (OV) and OC plays a crucial role in determining the identity of the ciliary body and iris. Because wingless (wg) plays a similar role in the induction of peripheral eye tissues of Drosophila, these findings indicate a possible conservation of the process that patterns the photoreceptive and support structures of the eye.

Expression and activation of STAT proteins during mouse retina development.

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT antibodies were used to detect the expression and activation of STATs in embryonic and postnatal neuronal retina, ciliary margin, and retinal pigment epithelium (RPE). In situ hybridization and Western blot were also employed. In embryonic stages, all STAT proteins were expressed in the neuronal retina in distinct cell populations at different embryonic stages. For example, Stat3 expression and activation gradually increased in the inner neuroblast layer and ciliary margin during development. In adult retina, Stat3 was detected in the inner nuclear layer and ganglion cells layers. Stat1 was strongly expressed in both outer and inner plexiform layers. Stat5a was clearly expressed in the outer/inner nuclear layer, the ganglion cell layer, and the inner plexiform layer. Strong expression of Stat3, Stat5a, and Stat6 was observed in the RPE. Activated Stat3 and Stat5a were found in the neural retina and the RPE. Distinct STAT proteins were present in different cell populations in neuronal retina and RPE suggesting multiple functions of STATs in mammalian eye development. Studies of STAT signal pathways in the eye may contribute to the understanding of molecular mechanisms in control of ocular development and pathogenesis.

Pigment epithelium-derived factor expression in the developing mouse eye.

PURPOSE: Pigment Epithelium-Derived Factor (PEDF) is a 50 kDa secretable protein with neuroprotective, neurotrophic, and antiangiogenic properties. Expression patterns in the human eye suggest that modulation of this protein over time and place may play a role in development of normal ocular vasculature. Because of the potential importance of normal PEDF expression patterns in controlling ocular blood vessel growth in health and disease, we characterized these patterns over the period of retinal vascular development in the mouse. METHODS: Eyes from CD1 mice (embryonic days E 14.5, 18.5, P0, P4, P7, P14, and Adult) were cryosectioned and examined. A polyclonal PEDF antibody was used to locate PEDF protein using immunohistochemical techniques while a PEDF RNA probe was used to localize PEDF mRNA by in situ hybridization. RESULTS: Immunohistochemical and in situ hybridization showed initial expression in the ciliary body and choroid during mid-gestation. Near term, relative protein levels increased in the ganglion cell layer and remained high through the first two weeks postnatal. Levels qualitatively decreased after this point but persisted through adulthood. Relative levels of expression in the inner retina were much higher at all timepoints than in the outer retina. CONCLUSIONS: These expression patterns likely maintain the vitreous and aqueous humors as avascular spaces and may also control vascular development in the inner/outer retina.

The mineralocorticoid receptor in rodent retina: ontogeny and molecular identity.

PURPOSE: Mineralocorticoid hormones contribute to ion-water balance in all cell types. In this study, we investigated the presence of mineralocorticoid receptors in rat and bovine ocular tissues and during retinal development. METHODS: Isolated photoreceptors and/or intact retina, retinal pigment epithelium (RPE) cells, and ciliary body were analyzed for the expression of MR (Mineralocorticoid Receptor) using the polymerase chain reaction (PCR) technique. Since aldosterone can stimulate the expression of epithelial Na+ channels (ENaC), expression of this gene in RPE was measured under basal and aldosterone-induced level. RESULTS: MR was expressed in rat photoreceptors and in the inner retina (inner nuclear layer and ganglion cell layer) even one day after birth, almost hundred percent identity was observed between rat retina and kidney MR gene products. The expression was also present in the RPE and in the ciliary body. ENaC gene was expressed in RPE and generated a predicted band at 520 bp following RT-PCR amplification which was 95% homology to that of ENaC mRNA from bovine kidney. The incubation of RPE cells in vitro with aldosterone increased the mRNA level of ENaC. CONCLUSIONS: MR expression in the mammalian retina, RPE, and ciliary body extends the potential field of action for mineralocorticoid hormones. Results on RPE cells are consistent with the idea that steroid hormones may regulate the physiology of these tissues by modulating ENaC expression. This study provides new light on the potential effect of mineralocorticoid in this area of the nervous system.

Immunocytochemical characterization of cysts in the peripheral retina and pars plana of the adult primate.

PURPOSE: To better characterize the cellular constituents of cysts in the peripheral retina and pars plana of the adult monkey. METHODS: Frozen sections of the peripheral retinal margin and pars plana from monkeys (Macaca nemestrina) between 1 and 15 years of age were stained with toluidine blue or immunolabeled with a variety of glia- and neuron-specific antibodies. RESULTS: In animals 1 to 2 years of age, the nonpigmented inner layer of the pars plana is a pseudostratified columnar epithelium. In these young animals, the peripheral retina had distinct layers and did not contain cysts. In animals 6 years of age or older, there were numerous cysts in the pars plana and in the peripheral retina. In the peripheral retina, neurons were randomly distributed and did not have a laminar organization. Cells surrounding cysts were immunoreactive for different types of markers for retinal neurons. Some of the cells surrounding cysts in the pars plana were also unexpectedly immunoreactive for antigens normally expressed only in retinal neurons and glia. CONCLUSIONS: Cysts form in the peripheral retina and pars plana in adult monkeys. The peripheral retinal cysts disrupt the normal lamination of the cells, but all types of retinal neurons are still present in the cysts. In an unexpected finding, cysts in the pars plana also contained cells immunoreactive for a few of the markers of retinal cells, suggesting that neurogenesis may occur in the pars plana of the adult primate.