Regulation of neuronal and photoreceptor cell differentiation by Wnt signaling from iris-derived stem/progenitor cells of the chick in flat vs. Matrigel-embedding cultures.

Previously we developed a simple culture method of the iris tissues and reported novel properties of neural stem/progenitor-like cells in the iris tissues of the chick and pig. When the iris epithelium or connective tissue (stroma) was treated with dispase, embedded in Matrigel, and cultured, neuronal cells extended from the explants within 24 h of culture, and cells positively stained for photoreceptor cell markers were also observed within a few days of culturing. In ordinary flat tissue culture conditions, explants had the same differentiation properties to those in tissue environments. Previously, we suggested that iris neural stem/progenitor cells are simply suppressed from neuronal differentiation within tissue, and that separation from the tissue releases the cells from this suppression mechanism. Here, we examined whether Wnt signaling suppressed neuronal differentiation of iris tissue cells in tissue environments because the lens, which has direct contact with the iris, is a rich source of Wnt proteins. When the Wnt signaling activator 6-bromoindirubin-3'-oxime (BIO) was administered to Matrigel culture, neuronal differentiation was markedly suppressed, but cell proliferation was not affected. When Wnt signaling inhibitors, such as DKK-1 and IWR-1, were applied to the same culture, they did not have any effect on cell differentiation and proliferation. However, when the inhibitors were applied to flat tissue culture, cells with neural properties emerged. These results indicate that the interaction of iris tissue with neighboring tissues and the environment regulates the stemness nature of iris tissue cells, and that Wnt signaling is a major factor.

BMP-induced reprogramming of the neural retina into retinal pigment epithelium requires Wnt signalling.

In vertebrates, the retinal pigment epithelium (RPE) and photoreceptors of the neural retina (NR) comprise a functional unit required for vision. During vertebrate eye development, a conversion of the RPE into NR can be induced by growth factors in vivo at optic cup stages, but the reverse process, the conversion of NR tissue into RPE, has not been reported. Here, we show that bone morphogenetic protein (BMP) signalling can reprogram the NR into RPE at optic cup stages in chick. Shortly after BMP application, expression of Microphthalmia-associated transcription factor (Mitf) is induced in the NR and selective cell death on the basal side of the NR induces an RPE-like morphology. The newly induced RPE differentiates and expresses Melanosomalmatrix protein 115 (Mmp115) and RPE65. BMP-induced Wnt2b expression is observed in regions of the NR that become pigmented. Loss of function studies show that conversion of the NR into RPE requires both BMP and Wnt signalling. Simultaneous to the appearance of ectopic RPE tissue, BMP application reprogrammed the proximal RPE into multi-layered retinal tissue. The newly induced NR expresses visual segment homeobox-containing gene (Vsx2), and the ganglion and photoreceptor cell markers Brn3α and Visinin are detected. Our results show that high BMP concentrations are required to induce the conversion of NR into RPE, while low BMP concentrations can still induce transdifferentiation of the RPE into NR. This knowledge may contribute to the development of efficient standardized protocols for RPE and NR generation for cell replacement therapies.

RPE specification in the chick is mediated by surface ectoderm-derived BMP and Wnt signalling.

The retinal pigment epithelium (RPE) is indispensable for vertebrate eye development and vision. In the classical model of optic vesicle patterning, the surface ectoderm produces fibroblast growth factors (FGFs) that specify the neural retina (NR) distally, whereas TGFß family members released from the proximal mesenchyme are involved in RPE specification. However, we previously proposed that bone morphogenetic proteins (BMPs) released from the surface ectoderm are essential for RPE specification in chick. We now show that the BMP- and Wnt-expressing surface ectoderm is required for RPE specification. We reveal that Wnt signalling from the overlying surface ectoderm is involved in restricting BMP-mediated RPE specification to the dorsal optic vesicle. Wnt2b is expressed in the dorsal surface ectoderm and subsequently in dorsal optic vesicle cells. Activation of Wnt signalling by implanting Wnt3a-soaked beads or inhibiting GSK3ß at optic vesicle stages inhibits NR development and converts the entire optic vesicle into RPE. Surface ectoderm removal at early optic vesicle stages or inhibition of Wnt, but not Wnt/ß-catenin, signalling prevents pigmentation and downregulates the RPE regulatory gene Mitf. Activation of BMP or Wnt signalling can replace the surface ectoderm to rescue MITF expression and optic cup formation. We provide evidence that BMPs and Wnts cooperate via a GSK3ß-dependent but ß-catenin-independent pathway at the level of pSmad to ensure RPE specification in dorsal optic vesicle cells. We propose a new dorsoventral model of optic vesicle patterning, whereby initially surface ectoderm-derived Wnt signalling directs dorsal optic vesicle cells to develop into RPE through a stabilising effect of BMP signalling.