Glycolysis regulates Hedgehog signalling via the plasma membrane potential

Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator—the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady‐state levels of ATP and stabilizes Smoothened (Smo), the 7‐pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full‐length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N‐acylethanolamides—lipoprotein‐borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo—a key step in pathway activation—in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential. Synopsis How changes in energy metabolism and plasma membrane potential regulate tissue development remains unclear. Here, glycolysis‐derived ATP is shown to spur the plasma membrane potential and uptake of Hedgehog (Hh) signaling inhibitors to control patterning in Drosophila . Ecdysoneless is autonomously required for wing disc growth and energy metabolism. Glycolytic ATP supports the generation of the plasma membrane potential. Plasma membrane depolarisation blocks N‐acylethanolamide uptake stabilising Smoothened. Loss of Ecdysoneless sensitizes cells to Hedgehog. Graphical Abstract Ecdysoneless‐dependent ATP generation controls developmental patterning in the fly wing disc by altering membrane depolarization and lipid uptake.