The myc oncogene and transdifferentiation of the retinal pigment epithelium]

The retinal pigment epithelium (RPE) develops from the same sheet of neuroepithelium as the neuroretina. When infected with MC29, a v-myc expressing virus, the RPE cells can be induced to transdifferentiate and to take a neuroretinal epithelium fate. After a PCR-based differential screening from these cells we have identified three genes of interest. Qath5, a quail basic helix-loop-helix (bHLH) gene that is closely related to the Drosophila atonal, and whose expression is found in the developing neuroretina. A Chx10-related homeobox gene also expressed in the developing neuroretina and HuD, a RNA-binding protein not expressed in the RPE but expressed during neurogenesis. Beside these genes whose function is involved in regulating neuronal differentiation myc also induced a transient Mitf expression. Mitf is expressed in the entire optic cup, later restricted to the pigmented retina. Mitf is involved in the regulation of the pigmented differentiation. We conclude that v-myc can reverse the RPE to the bipotential retinal primordia.     

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.     

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.     

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.     

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.     

Ocular tissue interactions during the development of human fetal neuroretina grafted into nude mice

To determine the roles of different ocular tissues in the development of the human fetal neuroretina, a study ethically and technically impossible in human subjects, human embryonic and fetal retinas were heterotopically implanted into nude mice. Ninety-five eyeballs were obtained from legally aborted 6- to 7-week-old embryos or 8- to 10-week-old fetuses. Ten isolated neuroretinas with vitreous but without pigment epithelium, 20 half-eyeballs and 70 intact eyeballs, of which 12 had a thick layer of periocular tissue, were microsurgically grafted. Five intact eyeballs were used for reference. Over a period of 1-245 days, all of the grafts were removed for light and electron microscopy observations. All of the isolated neuroretinas had disappeared by the second day after transplantation. Grafts of the posterior section of the eyeball contained only some clusters of pigment epithelium, occasionally covered with undifferentiated neuroretinal cells. Grafts of the retrolental section of the eyeball contained small areas of dysplasic neuroretina with folds and rosettes. Grafts of the 70 intact eyeballs were successful, but only 26 showed normal histological organization of the choriocapillaris, the retinal pigment epithelium and the neuroretina in the posterior part of the posterior chamber. Photoreceptor differentiation was evident in these retinas after approximately 80 days of transplantation and was complete after 166 days. Their anterior part was always dysplasic, with occasional ciliary differentiation. Twenty-three grafted eyeballs had a dysplasic neuroretina with folds, rosettes and necrotized areas. Twenty-one were atrophic, 12 of which were the eyeballs grafted with periocular tissue. These results demonstrate the role of the fetal mesenchyme and pigment epithelium in the rapid revascularization, and subsequent survival and tissue organization, of the neuroretina. The stratified development of the neuroretina required a thin mesenchymal environment for revascularization of the graft by human vasculogenesis or neoangiogenesis and a normal retinal pigment epithelium for normal neuroretinal differentiation. When these conditions were not satisfied, the neuroretina disappeared or was dysplasic, partly necrotized or atrophic. This model might prove useful for a number of therapeutic or clinical studies.