Pax9 governs anterior identity and deployment of sclerotome to the median fins

The caudal fin is an anomaly among vertebrate locomotory appendages: its internal skeleton is as asymmetric as the human hand, but it lacks the Shh-secreting Zone of Polarizing Activity (ZPA) and the Gli3/HoxD/Hand2 programs that govern anterior-posterior patterning in other appendages. As the caudal fin was also the first appendage to evolve, deciphering its alternative patterning program may provide clues to the ancestral state. Pax9 is one of few conserved appendage patterning factors reported to also be active in the caudal fin, specifically in the anterior domain. We report that loss of pax9 function in zebrafish not only disrupts anterior-specific caudal fin anatomy, but also results in a spectacular fusion of the caudal and anal fins along the ventral midline. The dorsal fin is also expanded to a lesser degree, the paired fins not at all. The mutant caudal fin initially forms as an irregularly patterned structure lacking anterior molecular identity, with supernumerary elements spilling out beyond the normal anterior boundary. Unexpectedly, this phenotype is subsequently compounded by neighboring trunk somites erroneously deploying skeletal mesenchyme in the normally finless caudal peduncle region, completing the ectopic skeleton. scRNAseq analysis at caudal fin bud stage indicates that pax9 mutants gain skeletal mesenchyme at the expense of a specialized type of fibroblast involved in the formation of fin fold actinotrichia. Median fin skeletal mesenchyme and fin fold fibroblasts both arise from the sclerotome, a somite compartment that also robustly expresses pax9. We propose that, within the sclerotome, Pax9 pushes progenitors towards fin fold fibroblast fate, limiting how many cells will later be available to make median fin skeleton. Within the fin bud, it drives anterior identity, with the strongest impact on the ZPA-free caudal fin bud. These dual sites of action make Pax9 a uniquely powerful governor of median fin development.