The scale of zebrafish pectoral fin buds is determined by intercellular K+ levels and consequent Ca2+-mediated signaling via retinoic acid regulation of Rcan2 and Kcnk5b

K + channels regulate morphogens to scale adult fins, but little is known about what regulates the channels and how they control morphogen expression. Using the zebrafish pectoral fin bud as a model for early vertebrate fin/limb development, we found that K + channels also scale this anatomical structure, and we determined how one K + -leak channel, Kcnk5b, integrates into its developmental program. From FLIM measurements of a Förster Resonance Energy Transfer (FRET)-based K + sensor, we observed coordinated decreases in intracellular K + levels during bud growth, and overexpression of K + -leak channels in vivo coordinately increased bud proportions. Retinoic acid, which can enhance fin/limb bud growth, decreased K + in bud tissues and up-regulated regulator of calcineurin ( rcan2 ). rcan2 overexpression increased bud growth and decreased K + , while CRISPR-Cas9 targeting of rcan2 decreased growth and increased K + . We observed similar results in the adult caudal fins. Moreover, CRISPR targeting of Kcnk5b revealed that Rcan2-mediated growth was dependent on the Kcnk5b. We also found that Kcnk5b enhanced depolarization in fin bud cells via Na + channels and that this enhanced depolarization was required for Kcnk5b-enhanced growth. Lastly, Kcnk5b-induced shha transcription and bud growth required IP 3 R-mediated Ca 2+ release and CaMKK activity. Thus, we provide a mechanism for how retinoic acid via rcan2 can regulate K + -channel activity to scale a vertebrate appendage via intercellular Ca 2+ signaling.