BMP-Mediated Functional Cooperation between <em>Dlx5;Dlx6</em> and <em>Msx1;Msx2</em> during Mammalian Limb Development

<div><p>The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of <em>Msx1</em> and <em>Msx2</em>, as well as that of <em>Dlx5</em> and <em>Dlx6,</em> lead to limb patterning defects with anomalies in digit number and shape. <em>Msx1;Msx2</em> double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. <em>Dlx5;Dlx6</em> double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of <em>Msx</em> genes seems to involve the BMP molecules, the mode of action of <em>Dlx</em> genes still remains elusive. Here, examining the limb phenotypes of combined <em>Dlx</em> and <em>Msx</em> mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In <em>Msx1;Dlx5;Dlx6</em> triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of <em>Msx1;Msx2</em> double mutants suggesting an epistatic role of <em>Dlx5</em> and <em>Dlx6</em> over <em>Msx2</em>. In <em>Msx2;Dlx5;Dlx6</em> TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of <em>Msx2</em> by <em>Dlx5</em> and <em>Dlx6</em> occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.</p>