Regulatory Pathway Analysis by High-Throughput In Situ Hybridization

Automated in situ hybridization enables the construction of comprehensive atlases of gene expression patterns in mammals. Such atlases can become Web-searchable digital expression maps of individual genes and thus offer an entryway to elucidate genetic interactions and signaling pathways. Towards this end, an atlas housing ∼1,000 spatial gene expression patterns of the midgestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising >90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This process ordered hundreds of complex expression patterns into a matrix that reflects the embryonic architecture and the relatedness of patterns of expression. Clustering yielded 12 distinct groups of expression patterns. Because of the similarity of expression patterns within a group, members of each group may be components of regulatory cascades. We focused on the group containing Pax6, an evolutionary conserved transcriptional master mediator of development. Seventeen of the 82 genes in this group showed a change of expression in the developing neocortex of Pax6-deficient embryos. Electromobility shift assays were used to test for the presence of Pax6-paired domain binding sites. This led to the identification of 12 genes not previously known as potential targets of Pax6 regulation. These findings suggest that cluster analysis of annotated gene expression patterns obtained by automated in situ hybridization is a novel approach for identifying components of signaling cascades.: Signaling pathways drive biological processes with high specificity. Reductionist approaches such as mutagenesis provide one strategy to identity components of pathways. We used high throughput in situ hybridization to systematically map the spatiotemporal expression pattern of ∼1,000 developmental genes in the mouse embryo. The rich information collectively contained in these patterns was captured in annotation tables that were systematically mined using hierarchical clustering, resulting in 12 groups of genes with related expression patterns. We show that this process generates biologically meaningful, high-content information. The expression pattern of developmental master regulator Pax6 is found in a cluster together with that of 81 other genes. The paired DNA binding domain of Pax6 can bind to regulatory sequences in 14 of the 81 genes. We also found that the expression pattern of all these 14 genes is up- or downregulated in Pax6 mutant mice. These results emphasize that determining the expression pattern of many genes in a systematic way followed by an application of integrative tools leads to the identification of novel candidate components of signaling pathways. More generally, when complemented with appropriate data-mining strategies, transcriptome-scale in situ hybridization can be turned into a powerful instrument for systems biology.