FISH in whole-mount Drosophila embryos. RNA: activation of a transcriptional locus, DNA: gene architecture and expression

Abstract

Using examples of hybridization both to RNA and to DNA sequences we demonstrate that whole-mount Drosophila embryos are excellent objects with which to study questions about nuclear structure and function by combining FISH and confocal laser scanning microscopy. A fluorescently labelled 29-base oligonucleotide was used to probe transcription at a locus known to be strongly induced upon heat shock. The transcript from this locus apparently serves to stabilize and protect nuclear proteins which may be needed for nuclear processes after heat or chemical stress. We found that less than 5% of the protein is colocalized with the transcript under normal growth conditions, but more than 50% is sequestered by the transcript during heat shock. DNA probes to genes in the bithorax complex were used to examine the relationship between homologous pairing and gene expression in late stage gastrulating embryos. Analysis of the disposition of probes cloned in P1 vectors in embryos from mid-blastoderm throughout gastrulation allowed us to conclude that polarized nuclear organization breaks down after the blastoderm stage. Homologous pairing of the bithorax complex genes proceeds during gastrulation so that at the time of germ band retraction the two alleles are always in close proximity independent of expression of the gene or the region along the anterior–posterior axis of the body. Finally, we demonstrate that smaller DNA targets can be visualized in whole mount embryos by enhancement of the FISH signal by tyramide-fluorophore deposition.