Patterning of the vertebrate brain depends on signals arising from the notochord as well as signaling operating within the induced neural plate. One such signal is encoded by the protooncogene Wnt-1. Wnt-1 expression demarcates the presumptive midbrain at presomite and early somite stages and its expression has been shown, by mutational analysis in the mouse, to be essential for midbrain development. Our goal is to understand how regional specification of the midbrain is established. To determine how the molecular regulation of midbrain specific gene expression is controlled, we have started to investigate the cis-acting regulatory sequences governing Wnt-1 expression. A 5.5 kb Wnt-1 enhancer has been identified which is capable of driving reporter gene expression within the normal Wnt-1 domain. We propose to use transgenic mice and P19 cell culture to identify the precise cis-acting regulatory elements within this enhancer. Founder transgenic mice injected with reporter constructs containing sub-elements of the enhancer will be assayed for reporter gene expression at 8.5 dpc. Reporter constructs will also be tested for activation in retinoic acid-treated P19 embryonal carcinoma cells which normally activate Wnt-1 on differentiation to neuronal cell types. We will also generate midbrain cell lines from transgenic embryos expressing a temperature sensitive T-antigen to induce conditional immortalization. These cell lines will be incorporated into functional and biochemical analysis of Wnt-1 expression. Cis-acting binding sites responsible for activating Wnt-1 in culture systems will be tested for their ability to activate Wnt-1 in vivo. On the basis of cell culture and transgenic experiments, we will use cDNA expression libraries from 8.5 dpc embryos, affinity chromatography with nuclear extracts from cell lines and a yeast transcriptional activation screen to identify putative regulatory factors. These experiments will further our understanding of how regional patterning in the brain is initiated and consequently how these structures are generated during normal development of the human CNS. They therefore should enhance our understanding of potential brain-related birth defects.
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