Identification of genes expressed with temporal-spatial restriction to developing cerebellar neuron precursors by a functional genomic approach.

Hedgehog pathway activation is required for proliferation of cerebellar granule cell neuron precursors during development and is etiologic in certain cerebellar tumors. To identify genes expressed specifically in granule cell neuron precursors, we used oligonucleotide microarrays to analyze regulation of 13,179 genes/expressed sequence tags in heterogeneous primary cultures of neonatal mouse cerebellum that respond to the mitogen Sonic hedgehog. In conjunction, we applied experiment-specific noise models to render a gene-by-gene robust indication of up-regulation in Sonic hedgehog-treated cultures. Twelve genes so identified were tested, and 10 (83%) showed appropriate expression in the external granular layer (EGL) of the postnatal day (PN) 7 cerebellum and down-regulation by PN 15, as verified by in situ hybridization. Whole-organ profiling of the developing cerebellum was carried out from PN 1 to 30 to generate a database of temporal gene regulation profiles (TRPs). From the database an algorithm was developed to capture the TRP typical of EGL-specific genes. The "TRP-EGL" accurately predicted expression in vivo of an additional 18 genes/expressed sequence tags with a sensitivity of 80% and a specificity of 88%. We then compared the positive predictive value of our analytical procedure with other widely used methods, as verified by the TRP-EGL in silico. These findings suggest that replicate experiments and incorporation of noise models increase analytical specificity. They further show that genome-wide methods are an effective means to identify stage-specific gene expression in the developing granule cell lineage.