Sequencing of morphogenetic modules in the central apical ectodermal ridge

Coordinated limb morphogenesis is governed by the serial ordering and priming of patterning modules that specify distinct tissue architectures, a process here termed morphogenetic sequencing. We monitored this process using high-resolution voltage-sensitive dye imaging in ex ovo limb-bud cultures, where a cohort of avian embryos executed outgrowth programmes of graded structural complexity after exposure to a defined morphogen template and a controlled latency period. Transient depolarisation events associated with initial tissue extension and mechano-sensory feedback were observed, but a sustained bioelectric state persisted throughout all experimental epochs, encompassing morphogen presentation, the delay interval and active outgrowth. Critically, maintained depolarisation in a discrete sector of the apical ectodermal ridge—the central AER (cAER)—scaled with template complexity and predicted the latency to outgrowth onset, implicating this region in morphogenetic sequencing. Focal optogenetic perturbation of the cAER produced dysmorphogenic pauses and segmental duplications reminiscent of congenital patterning disorders, whereas adjacent regions showed minimal sensitivity. These findings argue that morphogenetic sequencing is orchestrated by a distributed bioelectric network in which the cAER acts as a pivotal hub, offering new targets for regenerative modulation of limb patterning.