Summary It is currently thought that antennal target genes are activated in Drosophila by the combined action of Distal-less, homothorax, and extradenticle, and that the Hox gene Antennapedia prevents activation of antennal genes in the leg by repressing homothorax. To test these ideas, we analyze a 62 bp enhancer from the antennal gene spineless that is specific for the third antennal segment. This enhancer is activated by a tripartite complex of Distal-less, Homothorax, and Extradenticle. Surprisingly, Antennapedia represses the enhancer directly, at least in part by competing with Distal-less for binding. We show that Antennapedia is required in the leg only within a proximal ring that coexpresses Distal-less, Homothorax and Extradenticle. We conclude that the function of Antennapedia in the leg is not to repress homothorax, as has been suggested, but to directly repress spineless and other antennal genes that would otherwise be activated within this ring.

The death of larval salivary gland cells during metamorphosis in Drosophila melanogaster has been a key system for studying steroid controlled programmed cell death. This death is induced by a pulse of the steroid hormone ecdysone that takes place at the end of the prepupal period. For many years, it has been thought that the ecdysone direct response gene Eip93F (E93) plays a critical role in initiating salivary gland cell death. This conclusion was based largely on the finding that the three “type” alleles of E93 cause a near-complete block in salivary gland cell death. Here, we show that these three mutations are in fact allelic to Idh3b, a nearby gene that encodes the β subunit of isocitrate dehydrogenase 3, a mitochondrial enzyme of the tricarboxylic acid (TCA) cycle. The strongest of the Idh3b alleles appears to cause a near-complete block in oxidative phosphorylation, as mitochondria are depolarized in mutant larvae, and development arrests early during cleavage in embryos from homozygous-mutant germline mothers. Idh3b-mutant larval salivary gland cells fail to undergo mitochondrial fragmentation, which normally precedes the death of these cells, and do not initiate autophagy, an early step in the cell death program. These observations suggest a close relationship between the TCA cycle and the initiation of larval cell death. In normal development, tagged Idh3b is released from salivary gland mitochondria during their fragmentation, suggesting that Idh3b may be an apoptogenic factor that functions much like released cytochrome c in mammalian cells.

The Drosophila spineless (ss) gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH-PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. In this report, we present evidence that Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo). We show that the ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss(−) mutations. The results of yeast two-hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus. Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. This suggests that the interaction of Ss and Tgo does not require additional signals, unlike the ligand-dependent interaction of Ahr and Arnt. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved.

We describe the mapping of 979 randomly selected large yeast artificial chromosome (YAC) clones of Drosophila DNA by in situ hybridization to polytene chromosomes. Eight hundred and fifty-five of the clones are euchromatic and have primary hybridization sites in the banded portions of the polytene chromosomes, whereas 124 are heterochromatic and label the chromocenter. The average euchromatic clone contains about 211 kb and, at its primary site, labels eight or nine contiguous polytene bands. Thus, the extent as well as chromosomal position of each clone has been determined. By direct band counts, we estimate our clones provide about 76% coverage of the euchromatin of the major autosomes, and 63% coverage of the X. When previously reported YAC mapping data are combined with ours, euchromatic coverage is extended to about 90% for the autosomes and 82% for the X. The distribution of gap sizes in our map and the coverage achieved are in good agreement with expectations based on the assumption of random coverage, indicating that euchromatic clones are essentially randomly distributed. However, certain gaps in coverage, including the entire fourth chromosome euchromatin, may be significant. Heterochromatic sequences are underrepresented among the YAC clones by two to three fold. This may result, at least in part, from underrepresentation of heterochromatic sequences in adult DNA (the source of most of the clones analyzed), or from clone instability.