A conserved Hox9-10 code underlies the early establishment of hind appendage progenitors in zebrafish and the Iberian ribbed newt.

The acquisition of paired appendages was a key innovation in vertebrate evolution. Although HoxA/D paralog groups (PGs) 9-13 play central roles in patterning paired fins and limbs, the evolutionary origin of this paired appendage-patterning system remains unknown. Here, we show that HoxA/D PG9-10 genes are essential for the early establishment of pelvic fin progenitors in zebrafish, and that Hox9-10 function is similarly required for hindlimb progenitor formation in the Iberian ribbed newt. In zebrafish, combined disruption of five HoxA/D PG9-10 genes abolishes the detectable pelvic fin progenitor population, resulting in complete loss of pelvic fins. A Tg(hoxa10b:EGFP) reporter labeled cells in the presumptive pelvic fin field before pelvic fin bud formation. Importantly, simultaneous CRISPR-mediated disruption of all seven Hox9-10 paralogs in the Iberian ribbed newt similarly impaired the establishment of hindlimb progenitors, suggesting that an early requirement for Hox9-10 function in hind appendage development predates the divergence of ray-finned and lobe-finned vertebrates. Together, these findings identify an evolutionarily conserved role for posterior Hox9-10 genes in the initial establishment of hind appendage progenitors. Because overlapping posterior Hox paralog groups are subsequently deployed during paired appendage patterning, this early Hox9-10 requirement provides a developmental link between the posterior axial Hox code and the paired appendage-patterning system. Our results are therefore consistent with the hypothesis that recruitment of a posterior Hox code into hind appendage progenitor formation contributed to the evolutionary assembly of the vertebrate paired appendage-patterning system.