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 approximately 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.
Visel
Axel
A
Department of Genes and Behavior, Max Planck Institute of Biophysical Chemistry, Goettingen, Germany.
Carson
James
J
Oldekamp
Judit
J
Warnecke
Marei
M
Jakubcakova
Vladimira
V
Zhou
Xunlei
X
Shaw
Chad A
CA
Alvarez-Bolado
Gonzalo
G
Eichele
Gregor
G
D000818:Animals; D001665:Binding Sites; D016000:Cluster Analysis; D004247:DNA / Q000235:genetics; D005136:Eye Proteins / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D018398:Homeodomain Proteins / Q000378:metabolism; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008957:Models, Genetic; D020411:Oligonucleotide Array Sequence Analysis; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D013997:Time Factors
D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D004247:DNA