Specification of motoneuron fate in Drosophila: integration of positive and negative transcription factor inputs by a minimal eve enhancer.

We are interested in the mechanisms that generate neuronal diversity within the Drosophila central nervous system (CNS), and in particular in the development of a single identified motoneuron called RP2. Expression of the homeodomain transcription factor Even-skipped (Eve) is required for RP2 to establish proper connectivity with its muscle target. Here we investigate the mechanisms by which eve is specifically expressed within the RP2 motoneuron lineage. Within the NB4-2 lineage, expression of eve first occurs in the precursor of RP2, called GMC4-2a. We identify a small 500 base pair eve enhancer that mediates eve expression in GMC4-2a. We show that four different transcription factors (Prospero, Huckebein, Fushi tarazu, and Pdm1) are all expressed in GMC4-2a, and are required to activate eve via this minimal enhancer, and that one transcription factor (Klumpfuss) represses eve expression via this element. All four positively acting transcription factors act independently, regulating eve but not each other. Thus, the eve enhancer integrates multiple positive and negative transcription factor inputs to restrict eve expression to a single precursor cell (GMC4-2a) and its RP2 motoneuron progeny.