Meta data to Fig 4.

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 IP3R-mediated Ca2+ 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 Ca2+ signaling.钾离子（K+）通道可通过调控形态发生素（morphogen）以缩放成鱼鳍的尺寸，但目前对于该通道的上游调控机制，以及其如何控制形态发生素的表达，仍知之甚少。本研究以斑马鱼胸鳍芽作为脊椎动物鳍/肢早期发育的模型，发现钾离子通道同样可调控该解剖结构的尺寸缩放，并阐明了一种钾离子漏通道（K+-leak channel）Kcnk5b整合进入发育程序的具体机制。通过基于福斯特共振能量转移（Förster Resonance Energy Transfer，FRET）的钾离子传感器的荧光寿命成像显微镜（Fluorescence Lifetime Imaging Microscopy，FLIM）检测，我们观测到在鳍芽生长过程中，细胞内钾离子水平呈现协同性下降；而体内过表达钾离子漏通道可协同提升鳍芽的比例尺寸。可促进鳍/肢芽生长的视黄酸（Retinoic acid），可降低鳍芽组织内的钾离子水平，并上调钙调神经磷酸酶调控因子（rcan2）的表达。过表达rcan2可促进鳍芽生长并降低细胞内钾离子水平；而通过CRISPR-Cas9靶向敲低rcan2则会抑制鳍芽生长，并提升细胞内钾离子水平。在成鱼尾鳍中，我们也观测到了一致的实验结果。此外，通过CRISPR靶向敲除Kcnk5b的实验显示，Rcan2介导的鳍芽生长依赖于Kcnk5b的功能。我们还发现，Kcnk5b可通过钠离子（Na+）通道增强鳍芽细胞的去极化过程，而这一增强的去极化过程是Kcnk5b促进鳍芽生长所必需的。最后，Kcnk5b诱导的shha转录与鳍芽生长，依赖于三磷酸肌醇受体（IP3R）介导的钙离子（Ca2+）释放以及钙调蛋白激酶激酶（CaMKK）的活性。综上，本研究阐明了视黄酸通过rcan2调控钾离子通道活性，进而通过细胞间钙离子信号通路缩放脊椎动物附肢尺寸的具体分子机制。