A multitude of signalling pathways are involved in the process of forming an eye. neural retinal cell classes surrounded buy 99533-80-9 by an RPE. Unlike the RPE cells, the neural retinal cells could be -catenin-negative revealing that differentiation of the neural retinal cell classes is -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, -catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina. Introduction 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 . The Vav1 lateral walls of the diencephalon then evaginate resulting in the optic vesicle . 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 . 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 . Lens morphogenesis, establishment of dorsoventral polarity and specification of the neural retina, retinal pigment epithelium (RPE) and optic stalk occurs concurrently with the transformation buy 99533-80-9 of the optic vesicle to optic cup . 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 . There is evidence to suggest that RPE cells are specified by the transforming growth factor (TGF) family member Activin A which is secreted by the extraocular mesenchyme . The RPE cell is a versatile cell type and is involved in many important aspects of eye physiology , 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 ((is required for expression and activates genes important for pigmentation in co-operation with Mitf , . In contrast to Mitf, Otx2 is also important for formation of specific cell populations in the neural retina such as photoreceptor cells . During the transformation of the optic vesicle into the optic cup, the opposing actions of bone morphogenetic protein (Bmp) and hedgehog buy 99533-80-9 signalling are thought to generate dorsoventral patterning. Hedgehog signalling has been implicated in the specification of ventral structures such as the optic stalk , whereas Bmp signalling has also been shown to be involved in optic vesicle development and lens placode induction , , , . The gene is expressed in the dorsal part of buy 99533-80-9 the optic vesicle and is involved in dorsal patterning , . The establishment of dorsoventral identity in the neural retina is manifested by the transcription factors and in early eye progenitor cells prior to cellular commitment in the optic vesicle. This approach revealed that -catenin is essential for eye development since the optic vesicle is unable to transform into buy 99533-80-9 the optic cup. This phenotype is most likely due to that the RPE layer is not specified at the correct time during eye development. Materials and Methods Ethics Statement The mice were maintained at the animal facility at Ume? University and all experiments involving animals were approved by the Animal Review Board at the Court of Appeal of Northern Norrland in Ume?. Generation and Maintenance of Mice The transgenic mouse, mice (exons 2C6 were flanked by loxp sites) and mice has been described previously   . The germ-line null allele was generated by crossing with transgenic mice which inactivate genes in the female germ-line . The and the and transgene: Lhx2CreF: and Lhx2CreR: and the and RM43: 5-TACACTATTGAATCACAGGGACTT- 3. mice, IMR8052: and IMR8546: Hybridisation and Immunohistochemistry Embryos were isolated and fixed in 4% (w/v) paraformaldehyde (PFA) in PBS at 4C. Embryos used for hybridisation were fixed between 30 minutes to 2 hours. After fixation the embryos were transferred to 30% (w/v) sucrose in PBS for 24 hours at 4C, mounted in Tissue-tek (Sakura) and stored at ?80C. Sectioning (8C10 m) was performed on.