Analysis of the Otx2 midbrain enhancer in medaka fish

During embryonic development the vertebrate central nervous system initially appears as a uniform tube-like structure. Stepwise subdivisions of this neural tube generate increasing complexity, but little is known how the individual domains are positioned along the anteroposterior axis. First the forebrain/midbrain region is separated from the hindbrain and this process critically depends on Otx transcription factors. An enhancer for the mouse Otx2 gene has been isolated, which controls expression in the forebrain and midbrain and is conserved among vertebrates (Otx2FM). In this project we will analyse in depth the upstream regulatory pathways controlling this enhancer and thus orchestrating the first subdivision of the neural tube. For this purpose we will combine two favourable methods available for the fish model system, transgenic lines and transient injection experiments. Thus stable transgenic lines expressing Gfp under control of the Otx2FM enhancer will be used to analyse the effects of candidate genes on this regulatory element by transient misexpression. Wnt signalling is known to play a crucial role in anteroposterior patterning and could be implicated in Otx2 regulation. Binding sites for Tcf/Lef transcription factors, the nuclear targets of this pathway have been identified in the enhancer and mutation experiments suggested a positive effect. Nevertheless, misexpression experiments activating this pathway resulted in a reduced Otx2 domain, leaving controversial results for the function of Wnt signalling. One of the main questions of the project will therefore be to analyse the exact role of this signal transduction pathway in positioning subdomains of the developing brain.

The activity of genes is strictly controlled in an organism and allows activation of different genetic programs in separate regions of the body. This tissue specific expression is the basis for the specification of cells for certain functions by differentiation. The basic mechanisms of this regulation are known. Regulatory elements in promoter and enhancer regions of the gene integrate different signals, resulting in the activation or repression of the gene. The signals are mediated by transcription factors which themselves react to signals from both within the cells or from the outside. We analysed the regulation of the Otx2 gene. Otx2 is active in an early phase of embryonic development in the anterior part of the embryo. Its function in this region is highly conserved and Otx genes appear in insects as well as in humans. However, the signals which direct its expression into this region are not fully understood. We therefore tried to understand the molecular details of this regulation. An enhancer responsible for the expression in the anterior embryo has previously been isolated. Enhancers are regions of a gene which together with a promoter regulate the activity of the gene. Promoters are positioned directly upstream of a gene, whereas enhancers are found in a distance. Within the Otx2 enhancer certain elements have already been identified. Among them is a binding site for Otx2 itself, indicating an autoregulation by the gene product which itself also is a transcription factor. In addition, two highly conserved Wnt responsive sequence elements were identified. These elements are bound by Tcf/Lef transcription factors which react to the Wnt signalling pathway. The Wnt signalling pathway indeed was proposed to be critical for gene expression along the anterior/posterior axis of the embryo and thus very well could direct expression of Otx2 to the anterior part. However, the exact molecular mechanism of this regulation is still an open question. Analysing the elements we indeed found a strong response to Wnt signalling activity for both binding sites. They strongly reacted to ß-catenin which is the main transducer of this pathway within the cell. The two Tcf/Lef binding sites are highly conserved, both in their own sequence, as well as in their context including the exact spacing. Our results indicate that this specific arrangement allows the formation of complexes, which provide additional affinity for DNA and thus enhance Wnt signalling activity. A highly specific regulation of Otx2 expression in the embryo by the Wnt signalling pathway is suggested.