Identification of genes expressed with temporal-spatialrestriction to developing cerebellar neuron precursors by afunctional genomic approach

摘要

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 predictedexpression in vivo of an additional 18 genes/expressedsequence tags with a sensitivity of 80% and a specificity of 88%. Wethen compared the positive predictive value of our analytical procedurewith other widely used methods, as verified by the TRP-EGL insilico. These findings suggest that replicate experiments andincorporation of noise models increase analytical specificity. Theyfurther show that genome-wide methods are an effective means toidentify stage-specific gene expression in the developing granule celllineage.