Spatiotemporal relationships between a novel Drosophila stripe expressing gene and known segmentation genes by simultaneous visualization of transcript patterns

We describe the cloning of the Drosophila transcription unit odd-striped (ost), which maps close to the P-element insertion site of the enhancer-trap line 05279. In order rapidly to gain information on its chromosomal localization, and to determine the spatial and temporal expression patterns of ost in whole-mount embryos, we established a double-label in situ hybridization protocol to localize two different DNA or RNA sequences simultaneously. The double-label in situ hybridization method involves digoxigenin- and biotin-labeled DNA probes that are processed to result in blue alkaline phosphatase and brown peroxidase reaction products, respectively. Using reference probes as internal standards, we show that the ost transcription unit is located within the cytogenetic band interval 89A1,2 on the right arm of the third chromosome, and that it exerts diagnostic segmentation gene expression patterns in the embryo. The ost transcripts are initially expressed in an anterior cap at the blastoderm stage, followed by a transient pair-rule gene expression pattern, which eventually changes into a segmental polarity gene pattern at gastrulation. Our results establish detailed spatial and temporal relationships between ost expression and the known patterns of the segmentation genes fushi tarazu and wingless, and demonstrate the potential of the double-label method with differently tagged DNA probes.