Identification of an evolutionarily-conserved CRM that recapitulates leg and antennal <i>bab2</i>-expression.

<p>(A) A GFP-reporter assay identified a leg/antennal enhancer (LAE) element situated in the middle of the 150-kb <i>bric-a-brac</i> locus, between the <i>bab1</i> and <i>bab2</i> transcription units. The position of the 26B15 BAC used for phenotypic rescue experiments is shown, as well as the two overlapping genomic fragments (BP42 and BP47) driving GFP-reporter expression in developing legs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003581#pgen.1003581-Williams1" target="_blank">[27]</a>. Beneath, an enlarged view of the 11-kb BP42-47 region shows the positions of the eight genomic sub-fragments (#1–8) tested in our site-directed GFP reporter assay (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003581#s4" target="_blank">Materials and methods</a>). The four fragments (#3, 4, 7 and 8) that faithfully reproduce wild-type leg and antennal (ant) <i>bab2</i> expression (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003581#pgen.1003581.s001" target="_blank">Figure S1</a>) are indicated in green. (B) Conservation of the LAE sequence among Drosophilidae. Identical, highly- or non-conserved LAE sequences among a set of 10 <i>Drosophila</i> species, with respect to the <i>D. melanogaster</i> sequence, are depicted, respectively, by thick vertical black, grey or white lines, whereas thin horizontal grey lines indicate gaps. As determined from the UCSC genome browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>), sequence conservation (from 0 to 100% identity) over a 10-bp sliding window is represented above by the height of the conservation plot, with the three >20-pb-long highly-conserved CR1-3 regions indicated within (A full alignment from LAE sequences of 22 Drosophilidae species is provided in Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003581#pgen.1003581.s002" target="_blank">Figure S2</a>). (C) The 567 bp LAE recapitulates spatial and temporal <i>bab2</i>-expression pattern in developing leg and antenna. The expression of the <i>LAE-GFP</i> reporter construct is shown for early, mid and late third-instar larval as well as pharate adult stages. Expression sites in mature appendages are indicated by arrows. (D) Functional conservation of the <i>D. virilis</i> LAE regulatory activity in developing <i>D. melanogaster</i> appendages. GFP expression (green) alone and in combination with Bab2 immunostaining (red) of leg (E–E′ and G–G′, respectively) and antennal (F–F′ and H–H′, respectively) imaginal discs from late third-instar larvae expressing either the <i>D. melanogaster</i> (<i>D. mel</i>) or <i>D. virilis</i> (<i>D. vir.</i>) <i>LAE-GFP</i> construct (E–F and G–H, respectively), are shown.