Canonical Wnt/β-Catenin Signalling Is Essential for Optic Cup Formation

Citation: Hagglund A-C, Berghard A, Carlsson L ( Canonical Wnt/b-Catenin Signalling Is Essential for Optic Cup Formation Anna-Carin Ha gglund 0 Anna Berghard 0 Leif Carlsson 0 Anand Swaroop, National Eye Institute, United States of America 0 1 Umea Center for Molecular Medicine, Umea University, Umea , Sweden, 2 Department of Molecular Biology, Umea University , Umea , Sweden A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that b-catenin is essential for eye development as inactivation of b-catenin prior to cellular specification in the optic vesicle caused anophthalmia in mice. By achieving this early and tissue-specific b-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/b-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape b-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be bcatenin-negative revealing that differentiation of the neural retinal cell classes is b-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, b-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina. - The vertebrate eye develops through a series of co-ordinated interactions between tissues of different embryonic origin. The eye field is specified in the anterior neural plate immediately following gastrulation [1]. The lateral walls of the diencephalon then evaginate resulting in the optic vesicle [2]. The distal portion of the optic vesicle makes contact with the surface ectoderm which initiates the formation of the lens placode. Reciprocal interactions between the lens placode and the optic vesicle promote the formation of the optic cup [3]. However, such inductive interactions might not be strictly necessary since it has been shown recently that the optic vesicle can form the optic cup by a self-organising mechanism that is independent of external cues from the lens placode [4]. Lens morphogenesis, establishment of dorsoventral polarity and specification of the neural retina, retinal pigment epithelium (RPE) and optic stalk occurs concurrently with the transformation of the optic vesicle to optic cup [3]. Specification of different cell types in the eye is mediated by a number of key paracrine signalling molecules. Early neural retina specification is mediated by fibroblast growth factor (FGF) emanating from the surface ectoderm in the prospective lens placode leading to expression of the transcription factor Vsx2 (also Chx10) in the lateral part of the optic vesicle [5]. There is evidence to suggest that RPE cells are specified by the transforming growth factor b (TGFb) family member Activin A which is secreted by the extraocular mesenchyme [6]. The RPE cell is a versatile cell type and is involved in many important aspects of eye physiology [7], although whether RPE cells, once specified, influence the development of other cell types within the eye is unclear. The RPE cells are specified in the optic vesicle before pigmentation. The Microphthalmia-associated transcription factor (Mitf) is essential for RPE cell development but is expressed throughout the early optic vesicle where it marks undifferentiated bipotential neuroepithelial precursor cells and following cellular commitment becomes restricted to RPE cells [8]. The Orthodenticle homolog 2 (Otx2) transcription factor is expressed in the eye field before RPE specification but later becomes restricted to the RPE cells [9,10]. Otx2 is required for Mitf expression and activates genes important for pigmentation in co-operation with Mitf [8,10]. In contrast to Mitf, Otx2 is also important for formation of specific cell populations in the neural retina such as photoreceptor cells [11]. During the transformation of the optic vesicle into the optic cup, the opposing actions of bone morphogenetic protein (Bmp) and hedgehog signalling are thought to generate dorsoventral patterning. Hedgehog signalling has been implicated in the specification of ventral structures such as the optic stalk [12], whereas Bmp signalling has also been shown to be involved in optic vesicle development and lens placode induction [13,14,15,16]. The Bmp4 gene is expressed in the dorsal part of the optic vesicle and is involved in dorsal patterning [15,17]. The establishment of dorsoventral identity in the neural retina is manifested by the transcription factors Tbx5 and Vax2, expressed dorsally and ventrally, respectively [18]. The dorsoventral patterning of the neural retina is critical for correct topographic projection of retinal ganglion cell axons to the optic tectum in the brain, and for establishing a cone opsin gradient in the neural retina [19,20]. In addition to dorsoventral patterning the neural retina is composed of functionally distinct cell types organised into a laminar structure. The different cellular layers are referred to as follows (from outside-in), the outer nuclear layer, the inner nuclear layer and the ganglion cell layer. Photoreceptor proteins (opsins) are activated by light (photons) in the rods and cones that are in the outer nuclear layer. The inner nuclear layer consists of amacrine cells, bipolar cells and horizontal cells that essentially transfer and modulate information from the outer nuclear layer to the ganglion cell layer. The ganglion cell layer consists of retinal ganglion cells which are projection neurons and convey visual input information from the retina along the optic nerve to the brain [21]. The different cell classes in the neural retina are formed from one progenitor cell type during embryonic development, in a specific developmental order that is evolutionary conserved. For example, the retinal ganglion cells are generated first, and rods and bipolar cells are generated last [22,23,24,25,26]. b-catenin has been shown to be involved in different aspects of eye development. The b-catenin molecule has two different cellular functions; to regulate cellular adhesion by interacting with cadherins, and to mediate the canonical Wnt signalling pathway [27]. In the absence of a Wnt ligand and if not bound to cadherins, b-catenin is phosphorylated and actively degraded by a multiprotein destruction complex [28]. Binding of Wnt ligands to Frizzled (Fz) transmembrane receptors and Lrp5/6 co-receptors starts a series of events leading to inhibition of phosphorylatio (...truncated)