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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.

Mutations in the Drosophila class IV POU domain gene, abnormal chemosensory jump 6 (acj6), have previously been shown to cause physiological deficits in odor sensitivity. However, loss of Acj6 function also has a severe detrimental effect upon coordinated larval and adult movement that cannot be explained by the simple loss in odorant detection. In addition to olfactory sensory neurons, Acj6 is expressed in a distinct subset of postmitotic interneurons in the central nervous system from late embryonic to adult stages. In the larval and adult brain, Acj6 is highly expressed in central brain, optic and antennal lobe neurons. Loss of Acj6 function in larval optic lobe neurons results in disorganized retinal axon targeting and synapse selection. Furthermore, the lamina neurons themselves exhibit disorganized synaptic arbors in the medulla of acj6 mutant pupal brains, suggesting that Acj6 may play a role in regulating synaptic connections or structure. To further test this hypothesis, we misexpressed two Acj6 isoforms in motor neurons where they are not normally found. The two Acj6 isoforms are produced from alternatively spliced acj6 transcripts, resulting in significant structural differences in the amino-terminal POU IV box. Acj6 misexpression caused marked alterations at the neuromuscular junction, with contrasting effects upon nerve terminal branching and synapse formation associated with specific Acj6 isoforms. Our results suggest that the class IV POU domain factor, Acj6, may play an important role in regulating synaptic target selection by central neurons and that the amino-terminal POU IV box is important for regulation of Acj6 activity.

We have identified the Drosophila UDP-glucose dehydrogenase gene as being involved in wingless signaling. Mutations in this gene, called kiwi, generate a phenotype identical to that of wingless. UDP-glucose dehydrogenase is required for the biosynthesis of UDP-glucuronate, which in turn is utilized in the biosynthesis of glycosaminoglycans. By rescuing the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan heparan sulfate, we show that kiwi function in the embryo is crucial for the production of heparan sulfate in the extracellular matrix. Further, injection of heparin degrading enzyme, heparinase (and not chondroitin, dermatan or hyaluronic acid degrading enzyme) into wild-type embryos leads to the degradation of heparin-like glycosaminoglycans and a 'wingless-like' cuticular phenotype. Our study thus provides the first genetic evidence for the involvement of heparin-like glycosaminoglycans in signal transduction.

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.