Studies of mammalian brain development by fate mapping and mutant analysis

grantor: University of TorontoMice lacking 'Engrailed-1' or 'Gli3' exhibit severe brain malformations, demonstrating that these molecules play key roles in mammalian brain development. The mouse 'En' genes are expressed in a band around the embryonic midbrain-hindbrain constriction. This region is involved in patterning of the midbrain and cerebellum and can act as an organizer region to induce midbrain and cerebellar development. Fate-mapping experiments in chick indicate that the cerebellar primordium is located anterior and posterior to the midbrain-hindbrain constriction, whereas midbrain precursors lie more anteriorly. Fate mapping in mice is complicated by the inaccessibility of the postimplantation embryo. I report a new 'in vivo' approach using the Cre/'loxP ' recombination system to map the fate of the mouse midbrain-hindbrain constriction: E9-12 cells originating in the dorsal midbrain-hindbrain constriction contribute significantly to the medial cerebellum. The feasibility of using a recombinase-based lineage-tracing system for fate mapping in mouse is demonstrated. Mice homozygous for a loss-of-function 'Gli3' mutation exhibit defective neural-tube closure in midbrain and forebrain regions, and later exencephaly. I show that: (1) 'Gli3-/-' mutants display transient ectopic 'Sonic hedgehog' (' Shh') expression in early dorsal neural folds, suggesting that Gli3 normally acts there to repress 'Shh' expression, (2) Gli3 is involved in the control of cell proliferation or cell death in the dorsal neural tube of rostral brain structures, and (3) Gli3 restricts ventrolateral expansion of 'Shh' and Shh-responsive gene expression at ventral midline in the brains of E9.5-11.5 embryos. Thus, Gli3 plays a role in the development of both dorsal and ventral neural tube at the rostral level. ' Gli3-/-' embryos were also found to exhibit abdominal heterotaxia, indicating that 'Gli3' is essential for the establishment of gut sidedness. Early somite-stage 'Gli3-/-' embryos exhibit a downregulation of 'nodal' but not ' lefty' expression in left lateral-plate mesoderm, suggesting that ' Gli3' is required for 'nodal' activation downstream of 'lefty' or in a separate pathway of left-right specification. Together, these data support the view that Gli3 genetically acts as both a repressor and activator.Ph.D