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The ventral veinless (vvl) and trachealess (trh) genes are determinants of the Drosophila trachea. Early in development both genes are independently activated in the tracheal primordia by signals that are ill defined. Mutants blocking JAK/STAT signaling at any level do not form a tracheal tree suggesting that STAT92E may be an upstream transcriptional activator of the early trachea determinants. To test this hypothesis we have searched for STAT92E responsive enhancers activating the expression of vvl and trh in the tracheal primordia. We show that STAT92E regulated enhancers can be rapidly and efficiently isolated by focusing the analysis on genomic regions with clusters of putative STAT binding sites where at least some of them are phylogenetically conserved. Detailed analysis of a vvl early tracheal enhancer shows that non-conserved sites collaborate with conserved sites for enhancer activation. We find that STAT92E regulated enhancers can be located as far 60 kb from the promoters. Our results indicate that vvl and trh are independently activated by STAT92E which is the most important transcription factor required for trachea specification. (C) 2010 Elsevier Inc. All rights reserved.