Defective Hand1 phosphoregulation uncovers essential roles for Hand1 in limb morphogenesis

Abstract

The morphogenesis of the vertebrate limbs is a complex process in which cell signaling and transcriptional regulation coordinate diverse structural adaptations in diverse species. In this study, we examine the consequences of altering Hand1 dimer choice regulation within developing vertebrate limbs. Although <i>Hand1</i> deletion via the limb-specific <i>Prrx1</i><i>-Cre</i> reveals a non-essential role for Hand1 in mouse limb morphogenesis, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of proximal-anterior limb elements. Molecular analysis reveals a non-cell-autonomous mechanism that causes widespread cell death within the embryonic limb bud. In addition, we observe changes in proximal-anterior gene regulation, including a reduction in the expression of <i>Irx3</i>, <i>Irx5</i>, <i>Gli3</i> and <i>Alx4</i>, all of which are upregulated in <i>Hand2</i> limb conditional knockouts. A reduction of <i>Hand2</i> and <i>Shh</i> gene dosage improves the integrity of anterior limb structures, validating the importance of the Twist-family bHLH dimer pool in limb morphogenesis.