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

Abstract

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry,PO Box 2841, 37018 Goettingen, GermanySubmitted 24 July 1996, accepted 8 October 1996Abstract. Using examples of hybridization both to RNA and to DNA sequences wedemonstrate that whole-mountDrosophila embryos are excellent objects with which tostudy questions about nuclear structure and function by combining FISH and confocallaser scanning microscopy. A fluorescently labelled 29-base oligonucleotide was usedto probe transcription at a locus known to be strongly induced upon heat shock. Thetranscript from this locus apparently serves to stabilize and protect nuclear proteinswhich may be needed for nuclear processes after heat or chemical stress. We found thatless than 5% of the protein is colocalized with the transcript under normal growthconditions, but more than 50% is sequestered by the transcript during heat shock. DNAprobes to genes in the bithorax complex were used to examine the relationship betweenhomologous pairing and gene expression in late stage gastrulating embryos. Analysis ofthe disposition of probes cloned in P1 vectors in embryos from mid-blastodermthroughout gastrulation allowed us to conclude that polarized nuclear organizationbreaks down after the blastoderm stage. Homologous pairing of the bithorax complexgenes proceeds during gastrulation so that at the time of germ band retraction the twoalleles are always in close proximity independent of expression of the gene or theregion along the anterior–posterior axis of the body. Finally, we demonstrate thatsmaller DNA targets can be visualized in whole mount embryos by enhancement of theFISH signal by tyramide-fluorophore deposition.Keywords: hnRNP-K, 93D locus, omega-n transcript, bithorax complex, homologouspairing, confocal laser scanning microscopy1. IntroductionMost problems in developmental biology can only beunderstood in the context of the whole animal whichprovides the stage whereon the actors, gradients ofactivators and repressors, or signal molecules and receptors,perform their choreographed and complex interplay.Dissolution of the organism into its single cells destroysthe entire program of events, disrupts the intercellularsignalling and may reset the biological clock to an entirelynew situation. Thus, whether one is defining a phenotypeor determining complex gene expression patterns, theobservation of the entire organism or at least wholeorgan systems is essential. Almost 10 years ago thereplacement of radioactive labels [1,2] by non-radioactivenucleotide derivatives, such as digoxigenin (DIG), biotin orfluorochromes [3–6], revolutionized the detection of mRNAin tissues and cells. Subsequently, Tautz and Pfeifle [7]introduced methods to simultaneously permeabilize and fixwhole Drosophila embryos so that the localization of thetranscripts could be observed in whole organisms. Thistechnique was quickly adapted to embryos from Xenopus,sea urchin and other organisms [8,9]. The result was animprovement in resolution of non-radioactive probes formRNA localization, compared with the radioactive labelmethod. This improvement enabled whole animal labelling,compared with sectioning, and the ability to simultaneouslydetect protein expression by immunochemistry. Thesetechniques have produced important data which contributeto the present understanding of basic concepts in