Pax2 function in midbrain development

During the early development of the vertebrate brain, a signalling centre is established in the mid/hindbrain boundary, which controls the formation of the midbrain and the cerebellum. Although targeted mutagenesis in the mouse identified several genes as key components in this process, among them Pax2, little is still known about regulatory interactions. I will use hormone-inducible fusion proteins to identify target genes of Pax2 and thus clarify the position of the transcriptional activator Pax2 within this genetic hierarchy. Furthermore, I present a model in which Pax2 acts as a repressor of Otx2 function, and thereby regulates positioning of the mhb. This model is supported by cell culture experiments, as well as by injection experiments in Xenopus embryos. In both cases Pax2 strongly suppresses the activity of Otx2. The effects of this trans-repression, a novel aspect of Pax2 function, will be studied during midbrain development.

Signalling centres are of key importance for embryonic development. During formation of the vertebrate brain, a signalling centre is established in the mid/hindbrain boundary and Pax2 is one of the genes known to be involved in this process. In order to understand the genetic basis of this important structure, we started the attempt to define the position of Pax2 within the regulatory network. After testing fish and frog embryonic systems, a chick system finally turned out to be suitable for this purpose. First experiments with inducible versions of Pax2 gave promising results and should allow us to make a step towards the understanding of a genetic pathway of a signalling centre. Results During the early development of the vertebrate brain, a signalling centre is established in the mid/hindbrain boundary (mhb), which controls the formation of the neighbouring structures, the midbrain and the cerebellum. The critical importance of this signalling centre is demonstrated by transplantation, where the organizing function can be transferred to other parts of the brain. Several genes, among them Pax, Engrailed, Fgf and Wnt family members are essential for its formation, unfortunately little is known about the gene regulatory network organizing this structure. Pax2 is a member of the Pax gene family expressed in the mhb. In order to identify genes regulated by this transcription factor we generated inducible versions, which can be switched on or off by external addition of drugs. One system is induced by glucocorticoid, the other by rapamycin. We then examined different experimental systems suitable for misexpression of our inducible proteins. We tested mRNA injection techniques in embryos of the small Japanese Killifish (medaka) and frogs (Xenopus). Both systems turned out to be of limited success due to a high toxicity of Pax2 during gastrulation. Electroporation of chick embryos, a recently established method, eliminates problems during early development and therefore allowed efficient overexpression of Pax2 during mhb development. The next step will be to apply the inducible Pax2 versions in order to define downstream targets of this gene.