Lhx9 specifically modulates phalanx number, whereas Lhx2 constrains digit length in tetrapods.

Most nonmammalian tetrapods exhibit more than three phalanges in their posterior digits (III-V), whereas mammals are restricted to no more than three. So far, the molecular mechanism underlying this evolutionary reduction and the functions of Lhx2 and Lhx9 in digit development remain unclear. Here we show divergent evolution of Lhx2 and Lhx9 expression patterns in embryonic limbs across tetrapods. We further demonstrate that although ectopic overexpression of each gene in chick embryos delayed limb skeletal development, only Lhx9 reduced the phalanx number in the posterior digits. Transcriptomic analysis showed that Lhx2 regulates pathways for chondrocyte and osteoblast differentiation, and Lhx9 preferentially influences skeletal segmentation via the bone morphogenetic protein (BMP) pathway. The unique function and differential expression of Lhx9 in the limb buds between mammals and nonmammalian tetrapods suggest it may have acquired a role in suppressing phalanx number, driving the evolutionary reduction of phalanx number in mammals.