Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients

The entire functional even-skipped locus of Drosophila melanogaster is cont ained within a 16 kilobase region. As a transgene, this region is capable o f rescuing even-skipped mutant flies to fertile adulthood. Detailed analysi s of the 7.7 kb of regulatory DNA 3' of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blast oderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is re stricted anteriorly by Kruppel and posteriorly by giant, the same repressor s that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the po sterior border of stripe 6 are set by zygotic hunchback, and the region bet ween the two stripes is 'carved out' by knirps. Thus the boundaries of stri pes 4 and 6 are set through negative regulation by the same gap gene domain s that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3' region al so contains a single element for neurogenic expression in ganglion mother c ells 4-29 and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were f ound for expression in EL neurons, U/CQ neurons and the mesoderm. The evert -skipped 3' untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels r ather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3' end of the locus, but was not found to con tribute to efficient functional rescue.