The 5′Hox genes play crucial roles in limb development and specify regions in the proximal-distal axis of limbs. However, there is no direct genetic evidence that Hox genes are essential for limb development in non-mammalian tetrapods or for limb regeneration. Here, we produced single to quadruple Hox13 paralog mutants using the CRISPR/Cas9 system in newts (Pleurodeles waltl), which have strong regenerative capacities, and also produced germline mutants. We show that Hox13 genes are essential for digit formation in development, as in mice. In addition, Hoxa13 has a predominant role in digit formation, unlike in mice. The predominance is probably due to the restricted expression pattern of Hoxd13 in limb buds and the strong dependence of Hoxd13 expression on Hoxa13. Finally, we demonstrate that Hox13 genes are also necessary for digit formation in limb regeneration. Our findings reveal that the general function of Hox13 genes is conserved between limb development and regeneration, and across taxa. The predominance of Hoxa13 function both in newt limbs and fish fins, but not in mouse limbs, suggests a potential contribution of Hoxa13 function in fin-to-limb transition.

Posterior Hox genes play crucial roles in limb development and specify regions in the proximal-distal (PD) axis of limbs. However, there is no direct genetic evidence that Hox genes are essential for limb regeneration. Moreover, if essential, it is totally unknown which Hox genes have the same or distinct functions between development and regeneration. Here, we mutated hox13 using an efficient CRISPR/Cas9 system in newts (Pleurodeles waltl), which have strong regenerative capacities in various tissues. Triple or double mutants of hox13 paralogs lost their digit and metacarpal/metatarsal bones.Limb regeneration progressed but regenerates lacked the same autopod region. These results showed that hox13 paralogs have the same functions in limb development and regeneration.