Dorsal and ventral aspects of the eye are distinct from the early stages of development. The developing eye cup grows dorsally, and the choroidal fissure is formed on its ventral side. Retinal axons from the dorsal and ventral retina project to the ventral and dorsal tectum, respectively. Misexpression of the Tbx5 gene induced dorsalization of the ventral side of the eye and altered projections of retinal ganglion cell axons. Thus, Tbx5 is involved in eye morphogenesis and is a topographic determinant of the visual projections between retina and tectum.

During vertebrate embryonic development, a key to unraveling specific functions of gene products is the capability to manipulate expression of the gene of interest at the desired time and place. For this, we developed a ‘microelectroporation’ technique by which DNA can be locally introduced into a targeted site of avian embryos, restricting spatial expression of the protein products during development. This technique involved injection of DNA solution in ovo around the target tissue and pinpoint application of an electric field by tungsten electrodes, allowing efficient and reproducible targeted gene transfer, for example, into an optic vesicle, somites, cranial mesoderm and limb mesenchyme. Because of the locality of gene introduction and its expression, survival rates of the embryos were high: approximately 90% of the embryos injected in optic vesicles were alive for at least 1 day after microelectroporation. The instantaneous gene transfer into embryonic cells allowed rapid expression of protein products such as green fluorescence protein within 2.5 h with fluorescence maintained for 3 days of incubation. This improved technique provides a convenient and efficient way to express transgenes in a spatially and temporally restricted manner in chicken embryos.

Regionalised activation of canonical Wnt signalling via β-catenin stabilisation is a key early step in embryonic patterning in many metazoans, including the basally diverging cnidarians, but the upstream maternal cues appear surprisingly variable. In Clytia, regionalised β-catenin stabilisation defining a presumptive 'oral' territory is determined by two maternally coded Frizzled family Wnt receptors of opposite localisation and function. We have identified a maternally coded ligand, CheWnt3, the RNA of which is localised to the animal cortex (future oral side) of the egg. Antisense morpholino oligonucleotide experiments showed that CheWnt3 is required maternally for regionalised oral β-catenin stabilisation in the early embryo, being only the second clear example of a maternally required Wnt ligand after Xenopus Xwnt11. In line with the determinant role of the maternally localised Frizzleds, CheWnt3 overexpression by RNA injection initially had little effect on establishing the oral domain. Subsequently, however, overexpression had dramatic consequences for axis development, causing progressive expansion of β-catenin stabilisation to yield spherical 'oralised' larvae. Upregulation of both CheFz1 and CheFz3 RNAs in CheWnt3 morpholino embryos indicated that CheWnt3 participates in an active axial patterning system involving reciprocal downregulation of the receptors to maintain oral and aboral territories. Localised introduction of CheWnt3 RNA induced ectopic oral poles in CheWnt3 morpholino embryos, demonstrating its importance in directing oral fate. These findings suggest that the complete ligand-dependent Wnt signalling cascade is involved in axial patterning in ancestral eumetazoans. In Clytia, two variant Frizzled receptors and one Wnt ligand produced from localised RNAs cooperate to initiate regionalised Wnt pathway activation.