Tools for neuroanatomy and neurogenetics in Drosophila.

We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.

Discovery of selective nonpeptidergic neuropeptide FF2 receptor agonists.

We report the discovery and initial characterization of a novel class of selective NPFF2 agonists. HTS screening using R-SAT, a whole cell based functional assay, identified a class of aryliminoguanidines as NPFF1 and NPFF2 ligands. Subsequent optimization led to molecules exhibiting selective NPFF2 agonistic activity. Systemic administration showed that selective NPFF2 agonists (1 and 3) are active in various pain models in vivo, whereas administration of a nonselective NPFF1 and NPFF2 agonist (9) increases sensitivity to noxious and non-noxious stimuli.

A P element with a novel fusion of reporters identifies regular, a C2H2 zinc-finger gene downstream of the circadian clock.

Elucidating the mechanisms that link the circadian pacemaker to the timing of behaviors it controls is one of the greatest challenges in circadian biology. We report the generation of a P element, Pluc+, containing a novel reporter fusion. Our fusion reporter capitalizes on the use of luciferase bioluminescence to easily analyze temporal expression as well as the strength of myc epitopes and GFP to identify spatial expression. Using Pluc+ we have identified and characterized a novel C2H2 zinc-finger gene, regular (rgr), that cycles circadianly in phase with period (per) gene expression, but shifts to light-dark regulation in Clk(Jrk) mutant flies. By following myc expression of the Pluc+ reporter, we demonstrate that Rgr is expressed in a discrete number of neurons in the brain which overlap with axons expressing pigment-dispersing factor.

Mutation of the photoreceptor specific homeodomain gene Pph13 results in defects in phototransduction and rhabdomere morphogenesis.

The expression and organization of the phototransduction signaling proteins into a specialized light-sensing organelle, the rhabdomere, is required for photoreceptor cells to detect light. We report the characterization of the mutant Pph13(hazy). Pph13 is a homeodomain transcription factor expressed only in photoreceptor cells. Pph13 expression correlates with the differentiation and not specification of photoreceptor cells. In agreement with its expression profile, we find Pph13 is required for both rhabdomere morphogenesis and for the proper detection of light. In addition, we demonstrate that Pph13 exerts its effect by the regulation of photoreceptor specific gene expression.