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The Drosophila Vestigial protein has been shown to play an essential role in the regulation of cell proliferation and differentiation within the developing wing imaginal disc. Cell-specific expression of vg is controlled by two separate transcriptional enhancers. The boundary enhancer controls expression in cells near the dorsoventral (DV) boundary and is regulated by the Notch signal transduction pathway, while the quadrant enhancer responds to the Decapentaplegic and Wingless morphogen gradients emanating from cells near the anteroposterior (AP) and DV boundaries, respectively. MAD-dependent activation of the vestigial quadrant enhancer results in broad expression throughout the wing pouch but is excluded from cells near the DV boundary. This has previously been thought to be due to direct repression by a signal from the DV boundary; however, we show that this exclusion of quadrant enhancer-dependent expression from the DV boundary is due to the absence of an additional essential activator in those cells. The Drosophila POU domain transcriptional regulator, Drifter, is expressed in all cells within the wing pouch expressing a vgQ-lacZ transgene and is also excluded from the DV boundary. Viable drifter hypomorphic mutations cause defects in cell proliferation and wing vein patterning correlated with decreased quadrant enhancer-dependent expression. Drifter misexpression at the DV boundary using the GAL4/UAS system causes ectopic outgrowths at the distal wing tip due to induction of aberrant Vestigial expression, while a dominant-negative Drifter isoform represses expression of vgQ-lacZ and causes severe notching of the adult wing. In addition, we have identified an essential evolutionarily conserved sequence element bound by the Drifter protein with high affinity and located adjacent to the MAD binding site within the quadrant enhancer. Our results demonstrate that Drifter functions along with MAD as a direct activator of Vestigial expression in the wing pouch.

Organogenesis of the Drosophila tracheal system involves extensive directed cell migrations leading to a stereotypic series of interconnected tubules. Although numerous gene products have been shown to be essential for tracheal morphogenesis, direct functional relationships between participants have not been previously established. Both the breathless gene, encoding a Drosophila fibroblast growth factor receptor tyrosine kinase homologue, and the POU-domain transcription factor gene, drifter, are expressed in all tracheal cells and are essential for directed cell migrations. We demonstrate here that ubiquitously expressed Breathless protein under control of a heterologous heat-shock promoter is able to rescue the severely disrupted tracheal phenotype associated with drifter loss-of-function mutations. In the absence of Drifter function, breathless expression is initiated normally but transcript levels fall drastically to undetectable levels as tracheal differentiation proceeds. In addition, breathless regulatory DNA contains seven high affinity Drifter binding sites similar to previously identified Drifter recognition elements. These results suggest that the Drifter protein, which maintains its own expression through a tracheal-specific autoregulatory enhancer, is not necessary for initiation of breathless expression but functions as a direct transcriptional regulator necessary for maintenance of breathless transcripts at high levels during tracheal cell migration. This example of a mechanism for maintenance of a committed cell fate offers a model for understanding how essential gene activities can be maintained throughout organogenesis.

Transcriptional regulators utilizing the POU domain DNA-binding motif have been shown to form multi-protein complexes dependent on the POU domain itself and its flexible recognition of various octamer sequence elements. We have identified two variant POU domain recognition elements DFRE1 and DFRE2, which are found within a 514-bp autoregulatory enhancer of the Drosophila melanogaster POU domain gene drifter (dfr). Both elements are capable of binding bacterially produced full-length DFR protein with high affinity, although they differ in the 5'-to-3' orientation of POU-specific and POU homeodomain subelements. When placed in dfr loss-of-function genetic backgrounds, all expression of dfr-lacZ fusion genes under control of the autoregulatory enhancer is dependent on DFR activity levels. However, the complete enhancer sequence directs beta-galactosidase expression in only a subset of cells which normally express the endogenous DFR protein, including the middle pair of midline glias of the ventral nerve cord, the oenocyte clusters, and all tracheal cells. In addition, DFRE1 and DFRE2 exhibit separable tissue-specific functions when independently disrupted or deleted. Disruption of DFRE1 function specifically abolishes beta-galactosidase expression in the middle pair of midline glias. Deletion of DFRE causes a specific loss of tracheal expression, leaving oenocyte and midline glia expression intact. These results suggest that dfr cell-specific autoregulation is determined by the context of DFR POU domain binding within the enhancer, which is possibly mediated by the formation of recognition element-specific heteromultimeric complexes containing additional tissue-specific factors.