Extraocular dorsal signal affects the developmental fate of the optic vesicle and patterns the optic neuroepithelium

Dorsal-ventral (DV) specification in the early optic vesicle plays a crucial role in the proper development of the eye. To address the questions of how DV specification is determined and how it affects fate determination of the optic vesicle, isolated optic vesicles were cultured either in vitro or in ovo. The dorsal and ventral halves of the optic vesicle were fated to develop into retinal pigment epithelium (RPE) and neural retina, respectively, when they were separated from each other and cultured. In optic vesicles treated with collagenase to remove the surrounding tissues, the neuroepithelium gave rise to cRax expression but not Mitf, suggesting that surrounding tissues are necessary for RPE specification. This was also confirmed in in ovo explant cultures. Combination cultures of collagenase-treated optic vesicles with either the dorsal or ventral part of the head indicated that head-derived factors have an important role in the fate determination of the optic vesicle: in the optic vesicles co-cultured with the dorsal part of the head Mitf expression was induced in the neuroepithelium, while the ventral head portion did not have this effect. The dorsal head also suppressed Pax2 expression in the optic vesicle. These observations indicate that factors from the dorsal head portion have important roles in the establishment of DV polarity within the optic vesicle, which in turn induces the patterning and differentiation of the neural retina and pigment epithelium.     

The role of bone morphogenetic proteins in the differentiation of the ventral optic cup

The ventral region of the chick embryo optic cup undergoes a complex process of differentiation leading to the formation of four different structures: the neural retina, the retinal pigment epithelium (RPE), the optic disk/optic stalk, and the pecten oculi. Signaling molecules such as retinoic acid and sonic hedgehog have been implicated in the regulation of these phenomena. We have now investigated whether the bone morphogenetic proteins (BMPs) also regulate ventral optic cup development. Loss-of-function experiments were carried out in chick embryos in ovo, by intraocular overexpression of noggin, a protein that binds several BMPs and prevents their interactions with their cognate cell surface receptors. At optic vesicle stages of development, this treatment resulted in microphthalmia with concomitant disruption of the developing neural retina, RPE and lens. At optic cup stages, however, noggin overexpression caused colobomas, pecten agenesis, replacement of the ventral RPE by neuroepithelium-like tissue, and ectopic expression of optic stalk markers in the region of the ventral retina and RPE. This was frequently accompanied by abnormal growth of ganglion cell axons, which failed to enter the optic nerve. The data suggest that endogenous BMPs have significant effects on the development of ventral optic cup structures.     

Role of apoptosis and mitosis during human eye development

The spatial and temporal distribution as well as ultrastructural and biochemical characteristics of apoptotic and mitotic cells during human eye development were investigated in 14 human conceptuses of 4-9 postovulatory weeks, using electron and light microscopy. In the 5th developmental week, apoptotic and mitotic cells were found in the neuroepithelium of the optic cup and stalk, being the most numerous at the borderline between the two layers of the optic cup, and at the place of transition of the optic cup into stalk. They were also found at the region of detachment of the lens pit from the surface ectoderm. In the later developmental stages (the 6th-the 9th week), apoptotic and mitotic cells were observed in the neural retina and the anterior lens epithelium. Throughout all stages examined, mitotic cells were found exclusively adjacent to the lumen either of the intraretinal space or the optic stalk ventricle, or were restricted to the superficial epithelial layer of the lens primordium. Unlike mitotic cells, apoptotic cells occurred throughout the whole width both of the neuroepithelium and the surface epithelium. Ultrastructurally, apoptotic cells were characterised by round- or crescent-shaped condensations of chromatin near the nuclear membrane, while in the more advanced stages of apoptosis by apoptotic bodies. The distribution of caspase-3-positive cells coincided with the location of apoptotic cells described by morphological techniques indicating that the caspase-3-dependent apoptotic pathway operates during the all stages of human eye development. The location of cells positive for anti-apoptotic bcl-2 protein was in accordance with the regions of eye with high mitotic activity, confirming the role of bcl-2 in protecting cells from apoptosis. In the earliest stage of eye development, apoptosis and mitosis might be associated with the sculpturing of the walls of optic cup and stalk, while high mitotic activity along the intraretinal space and optic stalk ventricle indicates its role in the gradual luminal closure. These processes also participate in the detachment of the lens pit epithelium from the surface ectoderm as well as in further closure of the lens vesicle. Later on, both processes seem to be involved in the neural retina differentiation, lens morphogenesis and secondary lens fibre differentiation.     

Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation

The formation of the vertebrate optic cup is a morphogenetic event initiated after the optic vesicle contacts the overlying surface/pre-lens ectoderm. Placodes form in both the optic neuroepithelium and lens ectoderm. Subsequently, both placodes invaginate to form the definitive optic cup and lens, respectively. We examined the role of the lens tissue in inducing and/or maintaining optic cup invagination in ovo. Lens tissue was surgically removed at various stages of development, from pre-lens ectoderm stages to invaginating lens placode. Removal of the pre-lens ectoderm resulted in persistent optic vesicles that initiated neural retinal differentiation but failed to invaginate. In striking contrast, ablation of the lens placode gave rise to optic vesicles that underwent invagination and formed the optic cup. The results suggest that: (1) the optic vesicle neuroepithelium requires a temporally specific association with pre-lens ectoderm in order to undergo optic cup morphogenesis; and (2) the optic cup can form in the absence of lens formation. If ectopic BMP is added, a neural retina does not develop and optic cup morphogenesis fails, although lens formation appears normal. FGF-induced neural retina differentiation in the absence of the pre-lens ectoderm is not sufficient to create an optic cup. We hypothesize the presence of a signal coming from the pre-lens ectoderm that induces the optic vesicle to form an optic cup.     

Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.     

Expression of Bmp ligands and receptors in the developing Xenopus retina

Bone morphogenetic proteins (BMPs) act repeatedly in the development of nervous system tissues. While BMP signaling is critical for the early growth and patterning of the eye, we are interested in possible later functions of BMPs in the morphological development of retinal neurons and formation of synaptic connections. Therefore, we conducted an in situ hybridization analysis of the mRNA expression for the ligands Bmp2, -4 and 7 and the type Ia, Ib and II receptors (BmprIa, BmprIb and BmprII) during development of the retina of Xenopus laevis. Bmp4 mRNA is expressed in the dorsal retina and Bmp7 in the distal peripheral retina during the period of cell differentiation, while Bmp2 is not present in the eye. The type I receptors are expressed predominantly ventrally, from the optic vesicle stage until at least stage 35/36, after most cells have differentiated and many synaptic connections have formed. BmprII mRNA, however, is distributed evenly across the dorsoventral axis, with highest expression in retinal ganglion cell and inner nuclear layers.     

Proper patterning of the optic fissure requires the sequential activity of BMP7 and SHH

The optic disc develops at the interface between optic stalk and retina, and enables both the exit of visual fibres and the entrance of mesenchymal cells that will form the hyaloid artery. In spite of the importance of the optic disc for eye function, little is known about the mechanisms that control its development. Here, we show that in mouse embryos, retinal fissure precursors can be recognised by the expression of netrin 1 and the overlapping distribution of both optic stalk (Pax2, Vax1) and ventral neural retina markers (Vax2, Raldh3). We also show that in the absence of Bmp7, fissure formation is not initiated. This absence is associated with a reduced cell proliferation and apoptosis in the proximoventral quadrant of the optic cup, lack of the hyaloid artery, optic nerve aplasia, and intra-retinal misrouting of RGC axons. BMP7 addition to organotypic cultures of optic vesicles from Bmp7-/- embryos rescues Pax2 expression in the ventral region, while follistatin, a BMP7 antagonist, prevents it in early, but not in late, optic vesicle cultures from wild-type embryos. The presence of Pax2-positive cells in late optic cup is instead abolished by interfering with Shh signalling. Furthermore, SHH addition re-establishes Pax2 expression in late optic cups derived from ocular retardation (or) embryos, where optic disc development is impaired owing to the near absence of SHH-producing RGC. Collectively, these data indicate that BMP7 is required for retinal fissure formation and that its activity is needed, before SHH signalling, for the generation of PAX2-positive cells at the optic disc.     

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

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

Otx genes are required for tissue specification in the developing eye

Patterning of the vertebrate eye appears to be controlled by the mutual regulation and the progressive restriction of the expression domains of a number of genes initially co-expressed within the eye anlage. Previous data suggest that both Otx1 and Otx2 might contribute to the establishment of the different eye territories. Here, we have analysed the ocular phenotype of mice carrying different functional copies of Otx1 and Otx2 and we show that these genes are required in a dose-dependent manner for the normal development of the eye. Thus, all Otx1(-/-); Otx2(+/-) and 30% of Otx1(+/-); Otx2(+/-) genotypes presented consistent and profound ocular malformation, including lens, pigment epithelium, neural retina and optic stalk defects. During embryonic development, optic vesicle infolding was severely altered and the expression of pigment epithelium-specific genes, such as Mitf or tyrosinase, was lost. Lack of pigment epithelium specification was associated with an expansion of the prospective neural retina and optic stalk territories, as determined by the expression of Pax6, Six3 and Pax2. Later in development the presumptive pigment epithelium region acquired features of mature neural retina, including the generation of Islet1-positive neurones. Furthermore, in Otx1(-/-); Otx2(+/-) mice neural retina cell proliferation, cell differentiation and apoptotic cell death were also severely affected. Based on these findings we propose a model in which Otx gene products are required for the determination and differentiation of the pigment epithelium, co-operating with other eye patterning genes in the determination of the specialised tissues that will constitute the mature vertebrate eye.     

Patterning the optic neuroepithelium by FGF signaling and Ras activation.

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.