Image of CCAP expression in Drosophila produced with the conventional CCAP-Gal4 driver courtesy of Dr. Benjamin H. White, National Institute of Mental Health, Bethesda, Maryland.
If you leave the apartment number off a mailing address, your letter may not reach its proper destination. Similarly, current transgene targeting techniques lack precision. Cell-specific promoters target transgene expression to promoter-expressing cells, but many cell populations are defined by the intersection of two genes. Luan et al. target transgene expression to cells that express two gene products by dividing the yeast transcription factor Gal4 in a recent article in Neuron.
The authors separated the DNA-binding domain (DBD) and activation domain (AD) of Gal4. They named the independent constructs containing Gal4DBD and Gal4AD 'hemidrivers.' The authors linked the yeast upstream activating sequence (UAS) to the target gene. Therefore, only cells with both the DBD and AD expressed the target gene.
Drosophila with Gal4 driven by the neuronal promoter elav and a UAS-enhanced green fluorescent protein (EGFP) reporter expressed EGFP throughout the nervous system. The authors generated Drosophila with either the Gal4DBD or Gal4AD driven by elav (elavGal4DBD and elavGal4AD, respectively). They introduced a UAS-EGFP into elavGal4DBDDrosophila and crossed the elavGal4DBD and elavGal4AD lines. Although neither parent line expressed EGFP, their progeny showed EGFP expression that was similar to elavGal4 fruit flies. The authors found a similar pattern of EGFP expression when they substituted the AD from the Herpes Simplex Virus 1 transcription factor VP16, which also drives UAS-gene expression, for Gal4AD.
The authors targeted the subset of neurons expressing the neuropeptide crustacean cardioactive peptide (CCAP) by crossing the elav hemidrivers with hemidrivers driven by the CCAP promoter. They selectively ablated CCAP-expressing neurons by substituting UAS-reaper, which activates apoptosis, for UAS-EGFP. The loss of CCAP neurons can be lethal in the pupal stage, and Drosophila that survive fail to open their wings. Progeny from elavGal4DBD and CCAP Gal4AD crosses survived into adulthood, and 90% had wing expansion problems. In contrast, most of the progeny from CCAPGal4DBD and elavVP16ADDrosophila died in the pupal stage, suggesting that VP16AD, but not Gal4AD, drives reaper expression in the neurons that cause pupal lethality.
To functionally define subsets of CCAP-expressing neurons, the authors did enhancer-trap experiments, in which enhancers near the integration sites of promoterless transgenes target their expression. The authors crossed the N4 line of the VP16AD enhancer trap with CCAPGal4DBDDrosophila expressing UAS-EGFP and found EGFP expression in CCAP neurons in the subesophageal and thoracic ganglia. In contrast, progeny from the N6 enhancer-trap line showed EGFP expression in CCAP neurons in the brain and abdominal ganglia. The authors substituted UAS-reaper for UAS-EGFP and found that progeny from the N4, but not the N6 Drosophila line had wing expansion deficits, suggesting that CCAP neurons in the subesophageal and thoracic ganglia are necessary for wing expansion.
The authors propose that their technique can target transgenes to cells at the intersection of any two gene products, which will be particularly useful in brain structures such as the amygdala and hypothalamus, in which nuclei are defined by their co-expression of several neurotransmitters.