Project 462507

Pax6 is a key gene in the development of many regions of the brain. When it is mutated it causes a diverse set of abnormalities that include the absence of an iris and autism. To understand how Pax6 regulates brain development we have focused on the cerebellum for multiple reasons. First, it is composed of a limited number of cells that have a very well-detailed development. Second, only two classes of neurons are produced from the neuroepithelium of the cerebellum and one of these sets of cells, the cells that use glutamate as their neurotransmitter, are all affected by mutations of Pax6. Third, the recent ability to assess gene expression of single cells allows us to look at the genes of single cell types that are altered by the elimination of Pax6 and studies with identification of DNA that bind specifically to the Pax6 protein can identify target genes that are regulated by Pax6. Finally, the analysis of large data sets, termed bioinformatics, can permit us to put all these elements together to describe in impressive detail how a single gene can determine the final developmental state of multiple cell types. In this proposal, we do this with three sets of experiments: 1) State of the art single cell gene product analysis will be applied to normal and Pax6 mutant cerebellum to identify the genes that are significantly mis-expressed in the mutant cerebellum at critical points in development; 2) Chromatin immunoprecipitation with an antibody to Pax6 will be used to determine the elements of DNA that are regulated by Pax6; and 3) Focus will be on the validation of Pax6-driven pathways in the granule cells using cell culture and whole animal approaches. These studies will lead to an unprecedented view of how Pax6 orchestrates the development of such a complex organ as the brain and provide insights about how interventions may ameliorate disorders that come from mutations in the Pax 6 gene.