Multiple gene knockouts in newts reveal novel functions of 5′ Hox genes in limb development along the anterior–posterior and proximal–distal axes

5' Hox genes play crucial roles in limb patterning along the proximal-distal and anterior-posterior axes in mice. However, their functional conservation across tetrapods remains unclear. We previously found that newt Hox13 is essential for digit formation during both development and regeneration. In contrast, the functions of other 5' Hox genes (Hox9-Hox12) in newts remain u[WLYJ-108]nknown. Therefore, we generated 5' Hox knockout newts (Pleurodeles waltl) using CRISPR-Cas9. Individual knockouts of Hox9, Hox10, and Hox12 disrupted all respective paralogs; however, these newts displayed no apparent abnormalities in limb skeletons. In contrast, Hox11 knockout newts exhibited skeletal defects in the posterior zeugopod and autopod of both the forelimbs and hindlimbs. Moreover, compound knockouts of Hox9 and Hox10 caused substantial loss of stylopod and anterior zeugopod/autopod elements specifically in the hindlimbs. These findings indicate that Hox9 and Hox10 redundantly regulate stylopod formation in the hindlimbs. Furthermore, Hox9/Hox10 and Hox11 contribute to the development of the anterior and posterior regions of the zeugopod/autopod in the hindlimbs, respectively. These novel roles of 5' Hox genes identified in newts suggest the functional diversification of 5' Hox genes in tetrapod limb development.