The function of Groucho proteins in the midbrain development

During early development of the vertebrate brain, a signalling centre is established in the mid/hindbrain boundary (mhb), which controls the formation of neighbouring structures, the midbrain and the cerebellum. Targeted mutagenesis in the mouse identified several genes with key functions in this process. Interestingly, a number of these genes (En1/2, Pax2/5, Tcf/Lef as targets of Wnt1 signalling) interact with Groucho proteins, a family of transcriptional co-repressors. In preliminary experiments we identified two other genes important for mhb development, Otx2 and Gbx2, as interaction partners for Groucho proteins. Thus Groucho is involved in a large number of regulatory interactions in the mhb and therefore has to be assigned a key role in this process. It is nevertheless unlikely that this co-repressor would simply transform all interaction partners into transcriptional repressors. Evidence exists that Groucho activity can be modulated by other factors, generating a complicated network of transcriptional responses in the developing brain. In the presented project we will first characterize in detail the interactions between Otx2, Gbx2 and Groucho. Then we will try to validate the importance of Groucho protein interactions for development of this brain structure and finally identify factors, which are involved in these regulatory pathways.

Groucho/Tle proteins remained highly conserved during evolution. During embryonic development and in adult animals and humans, they fulfil a number of important functions. In this project we analysed their function in midbrain formation. During early brain development, an organising centre forms at the boundary between the midbrain and the hindbrain, regulating the formation of neighbouring brain structures. Such organizing centres are of major importance for brain development, it is therefore critical to understand their formation and genetic regulation. Groucho/Tle proteins act as so called corepressors, binding to other transcription factors. We could show in this project, that Groucho/Tle proteins bind to Otx and Gbx, two factors important for this region and thus affect their genetic regulation. The experiments were performed with Japanese medaka fish, a well established model system for vertebrates. To achieve these experiments, a new inducible promoter was developed. Heat inducible promoters are known for some time, but have the disadvantage of high background activity, making their application difficult. Constructing a new artificial promoter, we could now completely eliminate the background activity. These properties of the promoter are of major importance also for applications in gene therapy and plant biotechnology. A patent for the promoter was filed.