Paired appendages — the pectoral and pelvic fins of fishes and the forelimbs and hindlimbs of tetrapods — were a major innovation in vertebrate evolution. Their emergence greatly expanded the ways vertebrates could move through their environments and ultimately provided the developmental foundation for the evolution of limbs. Yet how the genetic system responsible for building paired appendages was assembled during evolution remains an unresolved question.
Hox genes are central regulators of vertebrate body patterning. In particular, HoxA and HoxD paralog groups 9–13 are known to control the later patterning of both fish fins and tetrapod limbs. This deeply conserved role has suggested a connection between the Hox code that specifies positions along the main body axis and the developmental program that patterns paired appendages. However, direct functional evidence showing that posterior Hox genes are required at the very earliest stages of hind appendage formation has been limited.
A research group led by Associate Professor Akinori Kawamura of the Graduate School of Science and Engineering, Saitama University, working with researchers from Tottori University, the National Institute of Genetics, the National Institute for Basic Biology, and the Exploratory Research Center on Life and Living Systems, aimed to determine whether posterior Hox genes are required for the initial establishment of hind appendage progenitor cells and whether this function is conserved across distantly related vertebrates. Using zebrafish carrying combinations of Hox gene mutations, fluorescent reporter lines that visualize developing pelvic fin progenitors, skeletal imaging, and CRISPR-Cas9-mediated disruption of Hox genes in the Iberian ribbed newt, the team revealed an evolutionarily conserved requirement for Hox9–10 genes during the earliest stages of hind appendage formation. The study was published online on October 16, 2026, in the Proceedings of the National Academy of Sciences (PNAS).
In zebrafish, the researchers found that simultaneously disrupting five HoxA/D paralog group 9–10 genes — hoxa9a, hoxa9b, hoxa10b, hoxd9a, and hoxd10a — resulted in the complete loss of pelvic fins. Fluorescent reporter analyses using markers associated with pelvic fin progenitor cells further showed that the characteristic bilateral progenitor populations could no longer be detected in these mutants, indicating that HoxA/D PG9–10 genes act before morphologically recognizable pelvic fin buds form. A hoxa10b reporter also marked cells in the presumptive pelvic fin field before fin bud formation, further supporting an early role for these genes.
The researchers then asked whether this function extends beyond ray-finned fishes. In the Iberian ribbed newt, a lobe-finned vertebrate lineage, they simultaneously disrupted all seven Hox9–10 paralogs using CRISPR-Cas9. Animals showing hindlimb loss also displayed corresponding loss or marked reduction of Prdm16 expression in prospective hindlimb-forming regions. Together, the zebrafish and newt experiments indicate that the requirement for Hox9–10 genes in establishing hind appendage progenitors predates the evolutionary divergence of these distantly related vertebrate lineages.
"Our results show that Hox9–10 genes are involved not only in the later patterning of paired appendages, but also at a much earlier step, when the progenitor cells that will form the pelvic fin or hindlimb are first established," says Hidemichi Nakazawa, a PhD student in the Graduate School of Science and Engineering at Saitama University and co-first author of the study. "This provides a developmental connection between the posterior Hox code of the body axis and the Hox system used to build paired appendages, and gives functional support to the idea that an ancestral posterior Hox program contributed to the evolutionary assembly of this system."
The findings are consistent with a stepwise evolutionary model in which an ancestral Hox9–10 program involved in early hind appendage development was subsequently expanded to include additional posterior Hox genes and later redeployed in anterior paired appendages. The study does not directly reconstruct this evolutionary sequence, but provides developmental evidence linking an ancient posterior Hox program to the origin of the broader vertebrate paired appendage-patterning system.
"This is fundamental research into how the vertebrate body plan was assembled through evolution, rather than research aimed at an immediate clinical or industrial application," says Associate Professor Akinori Kawamura. "Over the next five to ten years, however, understanding how Hox genes establish appendage progenitor cells should help us uncover more general principles governing vertebrate development. Combined with advances in genome editing and comparative developmental biology, this knowledge could also provide an important conceptual foundation for future research into limb development, developmental disorders, and regenerative biology."
