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4H), D. pseudoobscura ( Fig. 4I), D. willistoni ( Fig. 4J), and D. virilis ( Fig. 4K). Thus, the high levels of link midline expression observed in D. melanogaster are likely a recently acquired trait that appeared in two steps: appearance of midline expression MK-4827 clinical trial link tracheal expression was observed in all of the species and has been present for at least ?60 million years ( Fig. 4A�CK). To begin a molecular analysis of link embryonic gene expression, including addressing the questions whether Zld directly regulates link expression and how link midline expression evolved, we sought to identify a link embryonic enhancer. We cloned link flanking sequences into the GFP reporter vector pMintgate ( Jiang et al., 2010) and analyzed reporter expression by GFP in situ hybridization and immunodetection. Two fragments were analyzed that encompass the entire intergenic regions: a 285?bp 5��-flanking sequence fragment, link-5��, and an 1197?bp 3��-flanking sequence fragment, link-3�� ( Fig. 2A). While GFP expression driven by link-3�� did not reflect any obvious aspect of link endogenous expression (data not shown), GFP expression under the control of link-5�� closely matched selleck compound endogenous link expression throughout embryogenesis ( Fig. 2B��CG��). This indicated that all regulatory sequences required for the embryonic expression of link are contained within link-5��. Initially, we took an unbiased approach to identify evolutionarily-conserved over-represented putative transcription factor binding sites in the D. melanogaster link 285?bp link-5�� fragment. Utilizing the PhyloGibbs software program, we identified a conserved sequence motif, AGGTRG (R=A/G), referred to as Motif-T, with four sites in link-5�� ( Fig. 5A and B, S3). Two sites were identical to each DEF6 other, with the sequence CAGGTAG (T1, T3) and were conserved in most sequenced Drosophila species ( Fig. 5B). Two additional Motif-T sites in link-5�� were related to CAGGTAG with either a single mismatch (GAGGTAG; T4) or two mismatches (TAGGTGG; T2) ( Fig. 5A and B). Motif-T sites T1 and T3 match strong sites of the TAGteam heptamers (CAGGTAG, TAGGTAG, CAGGTAA, CAGGCAG) ( ten Bosch et al., 2006), which are recognized by the Zld, Grainyhead (Grh), and Bicoid Stability Factor transcription factors ( De Renzis et al., 2007, Harrison et al., 2010?and?Liang et al., 2008). The link T1 and T3 sites were previously recognized as putative Zld binding sites ( Liang et al., 2008), and Zelda ChIP-seq detects strong binding to the link 5�� region in vivo ( Harrison et al., 2011). The Sim and Trh bHLH-PAS transcription factors are known regulators of midline and tracheal expression, respectively. Both of these proteins form heterodimers with Tgo and bind the CME sequence ACGTG. We identified one CME at the promoter-proximal end of D. melanogaster link-5�� (C; Fig.