<i>Drosophila</i> Distal-less and Rotund Bind a Single Enhancer Ensuring Reliable and Robust <i>bric-a-brac2</i> Expression in Distinct Limb Morphogenetic Fields

<div><p>Most identified <i>Drosophila</i> appendage-patterning genes encode DNA-binding proteins, whose cross-regulatory interactions remain to be better characterized at the molecular level, notably by studying their direct binding to tissue-specific transcriptional enhancers. A fine-tuned spatio-temporal expression of <i>bric-a-brac2</i> (<i>bab2</i>) along concentric rings is essential for proper proximo-distal (P-D) differentiation of legs and antennae. However, within the genetic interaction landscape governing limb development, no transcription factor directly controlling <i>bab2</i> expression has been identified to date. Using site-targeted GFP reporter assay and BAC recombineering, we show here that restricted <i>bab2</i> expression in leg and antennal imaginal discs relies on a single 567-bp-long <i>cis</i>-regulatory module (CRM), termed LAE (for <u>l</u>eg and <u>a</u>ntennal <u>e</u>nhancer). We show that this CRM (i) is necessary and sufficient to ensure normal <i>bab2</i> activity in developing leg and antenna, and (ii) is structurally and functionally conserved among Drosophilidae. Through deletion and site-directed mutagenesis approaches, we identified within the LAE essential sequence motifs required in both leg and antennal tissues. Using genetic and biochemical tests, we establish that in the LAE (i) a key TAAT-rich activator motif interacts with the homeodomain P-D protein Distal-less (Dll) and (ii) a single T-rich activator motif binds the C2H2 zinc-finger P-D protein Rotund (Rn), leading to <i>bab2</i> up-regulation respectively in all or specifically in the proximal-most ring(s), both in leg and antenna. Joint ectopic expression of Dll and Rn is sufficient to cell-autonomously activate endogenous <i>bab2</i> and LAE-driven reporter expression in wing and haltere cells. Our findings indicate that accuracy, reliability and robustness of developmental gene expression do not necessarily require <i>cis</i>-regulatory information redundancy.</p>