DEFINING THE NEURONAL MIGRATION REGULATORY NETWORK

University Of California San Diego, La Jolla CA

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Abstract

DESCRIPTION (Applicant's abstract): To understand the complex regulatory network responsible for neuronal migration, it is necessary to identify the genes of that network. We hypothesize that regulatory networks of genes important for a biological process, such as neuronal migration, can be defined and examined by determining the relative expression of genes in the network when it is perturbed by mutation of a key element in the pathway. We have elected to examine regulator networks for neuronal migration in mouse mutants for Lis1. Lis1 is the mouse homolog of a human gene (LIS1) mutated in isolated lissencephaly sequence (ILS), a human neuronal migration defect. We have made several different Lis1 mutant mice that have varying levels of LIS1 activity, and have used these alleles to produce mice with graded reduction of Lisl activity. We have also produced mice with conditional inactivation of Lis1 (floxed Lis1 allele), as well as transgenic mice that produce Cre in defined regions of the brain during development. Matings between the Cre mice with mice containing the floxed Lisl allele will allow us to inactivate Lisl in defined temporal and spatial patterns in mice. RNA will be isolated from these mice containing graded reduction of Lis1 in the whole brain from birth or in distinct spatial and temporal patterns of the brain. These RNA samples will be used as probes against cDNA microarrays representing 8700 independent mouse genes. The patterns of expression of these genes will be compared throughout development in brain regions in vivo and in granule cell cluster cultures in vitro. Patterns of global gene expression will be compared with those in brains from other mouse mutants with neuronal migration defects, such as reeler and cdk5. This is will allow us to distinguish the importance of LIS1 to the neuronal migration regulatory network. The specific aims of this proposal are: 1) to collect RNA samples from developing and adult brains, subregions of the brains and migrating neurons in vitro from wildtype mice, Lisl mutants with varying dosage of LIS1, conditional mutants with regional specific deletion of the Lisl gene, and other murine neuronal migration mutants, 2) to determine relative levels of global gene expression in these various mutant samples compared with wildtype and other control samples using cDNA microarrays of the UniGene set of 8700 mouse cDNAs; 3) to cluster gene expression profiles based on similar patterns of expression, dose dependent changes in expression, and by mouse mutant by comparing results of these hybridization experiments during development and adulthood in brains and brain regions, untreated cerebellar cluster cultures and cultures treated with PAF from wildtype and different Lisl mutant mice.