The Pax6 gene encodes a transcription factor with a restricted expression in the ventricular zone of the pallium and subpallium. We tested whether the function of Pax6 is necessary for the correct patterning and morphogenesis of the vertebrate telencephalon. Homozygous embryos of the Pax6/Small eye mutant lack functional PAX6 protein because of a point mutation of the gene. In the mutant Small eye embryos we detected a ventralization of the molecular patterning of the telencephalon at two borders, the pallium/subpallium and the lateral/medial ganglionic eminence. The results indicate that Pax6 controls the lateral limit of the expression of Nkx2.1, Shh, and Lhx6 in the prechordal neural tube, the telencephalon. This finding is in agreement with previous studies and supports a model for a common genetic mechanism for modulation of the dorsoventral patterning of the prechordal and epichordal CNS. The pattern defects caused by the loss of Pax6 function result in multiple morphological abnormalities in the Small eye brain: dysgenesis of the piriform, insular, and lateral cortices, the claustrum-endopiriform nucleus, and a failure in the differentiation of a subpopulation of the cortical precursors. Together the results demonstrate that Pax6 has an essential role for the modulation of the dorsoventral patterning of the embryonic telencephalon, influencing thereby the forebrain morphogenesis.

Members of the Pax gene family are expressed in various tissues during ontogenesis. Evidence for their crucial role in morphogenesis, organogenesis, cell differentiation and oncogenesis is provided by rodent mutants and human diseases. Additionally, recent experimental in vivo and in vitro approaches have led to the identification of molecules that interact with Pax proteins.

The novel homeobox-containing gene

The quail-chick chimaera system was used to investigate the origin of the various neuronal cell types of the cerebellum from the mesencephalic and metencephalic brain vesicles at the 11- to 14-somite stage in the avian embryo. We have already demonstrated that the cerebellum is derived from both the mesencephalic and metencephalic brain vesicles. The mesencephalic contribution to the cerebellum is restricted to a mediodorsal territory inserted as a V-shaped area into the primitive metencephalic vesicle through complex morphogenetic movements taking place from day 2 to day 4 of embryonic development. Here we report that the cerebellar presumptive territory extends along the anteroposterior axis, over the caudal half of the mesencephalon and the rostral half of the metencephalon. Along the dorsoventral axis, the cerebellar anlage is located in the alar plates at the exclusion of the roof and basal plates, i.e. lies in the lateral walls of the neuroepithelium in the area included between approximately 25 and 120 degrees with respect to the sagittal plane. We also report that the neuroepithelium corresponding to the cerebellar presumptive territory also yields other brain structures (e.g. part of the optic tectum in the mesencephalon). The external granular layer (EGL) arises only from the rostral metencephalon, undergoing extensive tangential movements which we have analysed in detail: the more ventral the position of cells in the metencephalic alar plates the more rostral and lateral is their position in the EGL. Finally, we discuss the fact that the cerebellar cortex, an integrative structure of the brain, arises from the alar plates of the neural tube. This is consistent with the general spatial organization of the neural anlage of the vertebrate embryo, in which this part of the neuroepithelium is devoted to the production of interneurons, whereas the basal plate and the neural folds yield motor structures and primary sensory neurons respectively.

In mouse embryo, the early induction of the head region depends on signals from the anterior visceral endoderm (AVE) and the anterior primitive streak. Subsequently, node derivatives, including anterior definitive endoderm and axial mesendoderm, are thought to play a role in the maintenance and elaboration of anterior neural character. Foxa2 encodes a winged-helix transcription factor expressed in signaling centers required for head development, including the AVE, anterior primitive streak, anterior definitive endoderm, and axial mesendoderm. To address Foxa2 function during formation of the head, we used conditional mutants in which Foxa2 function is preserved in extraembryonic tissues during early embryonic stages and inactivated in embryonic tissues after the onset of gastrulation. In Foxa2 conditional mutants, the anterior neural plate and anterior definitive endoderm were initially specified. In contrast, the axial mesendoderm failed to differentiate. At later stages, specification of the anterior neural plate and anterior definitive endoderm was shown to be labile. As a result, head truncations were observed in Foxa2 conditional mutants. Our results therefore indicate that anterior definitive endoderm alone is not sufficient to maintain anterior head specification and that an interaction between the axial mesendoderm and the anterior definitive endoderm is required for proper specification of the endoderm. Foxa2 therefore plays an integral role in the formation of axial mesendoderm, which is required to maintain the specification of the forebrain and the anterior definitive endoderm.

Here we report the use of double-stranded RNA (dsRNA) and morpholino technologies to specifically 'knock down' gene expression in early postimplantation mouse embryos. Sequence specific interference mediated by either dsRNA or by morpholino has been a useful tool for studying gene function in several organisms. However, specifically for the dsRNA, doubts have been raised about whether it could successfully be applied on vertebrate embryos. We demonstrate that electroporation of dsRNA directed against Otx2 or Foxa2 into postimplantation mouse embryos results in specific knock down of the expression of the respective endogenous genes in a region- and germ-layer specific manner. We also show that electroporation of morpholino directed against Foxa2 into the node of mouse embryos leads to a specific down regulation of Foxa2 expression in the floor plate. Our results demonstrate for the first time that dsRNA and morpholino technologies can be successfully applied in early postimplantation mouse embryos to specifically knock down gene expression.

The vertebrate forebrain is formed at the rostral end of the neural plate under the regulation of local and specific signals emanating from both the endomesoderm and neuroectoderm. The development of the rostral and ventral forebrain in particular was difficult to study, mainly because no specific markers are available to date. Here, we report the identification of Vax1, a novel homeobox-containing gene identified in mouse, Xenopus and human. It is closely related to members of the Not and Emx gene families, all of which are required for the formation of structures where they are expressed. In mouse and Xenopus, Vax1 expression first occurs in the rostral neural plate, in the medial anterior neural ridge and adjacent ectoderm. Later, at midgestation in the mouse and tadpole stage in Xenopus, the expression remains confined in the derivatives of this territory which differentiate into rostromedial olfactory placode, optic nerve and disc, and anterior ventral forebrain. Together, these observations suggest that Vax1 could have an early evolutionary origin and could participate in the specification and formation of the rostral and ventral forebrain in vertebrates. Comparison of the limits of the expression territory of Vax1 with that of Dlx1, Pax6 and Emx1 indicates that the corticostriatal ridge is a complex structure with distinct identifiable genetic compartments. Besides, the study of Vax1 expression in Pax6-deficient homozygous brains indicates that its regulation is independent of Pax6, although the expression patterns of these two genes appear complementary in wild-type animals. Vax1 chromosomal location is mapped at the distal end of the mouse chromosome 19, linked with that of Emx2. These two genes may have arisen by tandem duplication. The Vax1 gene is thus an interesting new tool to study the rostral ventral forebrain patterning, morphogenesis and evolution as well as the terminal differentiation of the forebrain in mouse and Xenopus.

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.