The Iroquois (Iro/Irx) homeobox genes are conserved Hox targets involved in motor neuron development

The <i>Iroquois (Iro/Irx)</i> homeobox genes encode transcription factors with fundamental roles in animal development. Despite their link to various congenital conditions in humans, our understanding of <i>Iro/Irx</i> gene expression, function, and regulation remains incomplete. Here, we conducted a systematic expression analysis of all six mouse <i>Irx</i> genes in the embryonic spinal cord. We found that <i>Irx1</i>, <i>Irx2</i>, <i>Irx3</i>, <i>Irx5</i>, and <i>Irx6</i> are expressed in specific groups of motor neurons (MNs). Further, we employed CRISPR-Cas9 gene editing to uncover essential but distinct roles for <i>Irx2</i> and <i>Irx6</i> in MN development. We also found that HOX proteins, which are conserved regulators of MN development across species, control <i>Irx</i> gene expression both in mouse and <i>Caenorhabditis elegans</i> MNs. Altogether, our study provides insights into <i>Iro/Irx</i> expression and function in the developing spinal cord and uncovers an ancient gene regulatory relationship between HOX and <i>Iro/Irx</i> genes.