Molecular and genetic analysis of DNA regulatory sequences controlling mouse Engrailed-2 embryonic brain expression

grantor: University of TorontoThe vertebrate 'Engrailed' ('En') genes, homologues of the 'Drosophila' segment polarity gene ' engrailed' ('en'), encode a class of highly conserved homeodomain-containing transcription factors. Two murine 'En' genes, 'En-1' and 'En-2', have been identified and characterized. The 'En' genes display spatially and temporally restricted expression in the developing midbrain and cerebellum that is essential for development of these brain regions. This thesis has been conducted to elucidate the molecular and genetic mechanisms controlling 'En-2' embryonic brain expression. Minimal 'En-2' DNA regulatory regions were identified by analyzing 'lacZ' reporter genes in transgenic mice. A 1.0 kb enhancer fragment, which contains multiple positive and negative regulatory elements, was shown to be sufficient to direct 'En-2'-like ' lacZ' expression from embryonic day (E) 8.5 to 11.5. Cross species sequence comparison and transgenic analyses revealed that the 'En-2 ' mid-hindbrain regulatory sequences have been conserved in mice and humans. The vertebrate 'Pax' genes, homologues of the ' Drosophila' paired box-containing gene 'paired' (' prd'), encode a family of transcription factors. Nine murine ' Pax' genes have been identified. 'Pax'-'2'/' 5'/'8' show overlapping expression with 'En ' in the developing midbrain and hindbrain. Two DNA-binding sites specific for Pax-2/5/8 proteins were identified in the 1.0 kb 'En-2 ' enhancer fragment. Mutation of the two Pax-binding sites in the 1.0 kb-'En-2' enhancer-'lacZ' reporter constructs abolished 'En-2'-like mid-hindbrain expression in transgenic mice. A targeted deletion of the two Pax-binding sites in the 'En-2 ' locus was generated using homologous recombination in mouse embryonic stem cells. Embryos homozygous for this mutation were found to have a significant reduction in 'En-2' expression in the mid-hindbrain region at E8.5 but not later stages, showing that the two Pax-binding sites are required for proper initiation of the endogenous 'En-2' brain expression. Results from these studies provide direct molecular and genetic evidence that the 'Pax' genes are upstream regulators of 'En-2' in a genetic cascade controlling early development of the midbrain and cerebellum.Ph.D