Timed collinear activation of Hox genes during gastrulation controls the avian forelimb position

Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the anteroposterior axis remains largely unknown. Hox genes have long been suspected to control limb position, however supporting evidences are mostly correlative and their role in this process remains unclear. Here we show that Hox genes determine the avian forelimb position in a two-step process: first, their sequential collinear activation during gastrulation controls the relative position of their own successive expression domains along the body axis. Then, within these collinear domains, Hox genes differentially activate or repress the genetic cascade responsible for forelimb initiation. Furthermore, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether our results which characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb position in avians, show a direct and early role for Hox genes in this process.