Analysis of transcription factors expressed at the anterior mouse limb bud

<div><p>Limb bud patterning, outgrowth, and differentiation are precisely regulated in a spatio-temporal manner through integrated networks of transcription factors, signaling molecules, and downstream genes. However, the exact mechanisms that orchestrate morphogenesis of the limb remain to be elucidated. Previously, we have established EMBRYS, a whole-mount <i>in situ</i> hybridization database of transcription factors. Based on the findings from EMBRYS, we focused our expression pattern analysis on a selection of transcription factor genes that exhibit spatially localized and temporally dynamic expression patterns with respect to the anterior-posterior axis in the E9.5–E11.5 limb bud. Among these genes, <i>Irx3</i> showed a posteriorly expanded expression domain in <i>Shh</i>^<i>-/-</i> limb buds and an anteriorly reduced expression domain in <i>Gli3</i>^<i>-/-</i> limb buds, suggesting their importance in anterior-posterior patterning. To assess the stepwise EMBRYS-based screening system for anterior regulators, we generated <i>Irx3</i> transgenic mice in which <i>Irx3</i> was expressed in the entire limb mesenchyme under the <i>Prrx1</i> regulatory element. The <i>Irx3</i> gain-of-function model displayed complex phenotypes in the autopods, including digit loss, radial flexion, and fusion of the metacarpal bones, suggesting that <i>Irx3</i> may contribute to the regulation of limb patterning, especially in the autopods. Our results demonstrate that gene expression analysis based on EMBRYS could contribute to the identification of genes that play a role in patterning of the limb mesenchyme.</p></div