(A) Dynamic calcium pulses appear near the anterior fold, localized to a region more narrow than the Antp domain. (B) Transient pulses in GCaMP6s intensity occur on the timescale of seconds and increase in amplitude when folding begins (). Red, green, and blue channels of images represent maximum intensity projections of confocal stacks separated in time by 9 seconds, here and below. (C) Integrated transient pulses for the embryo in (B) show calcium pulses are localized near the fold: GCaMP6s signals 20 μm in front (red) or behind the fold (yellow) are less intense. (D) Snapshots of GCaMP6s fluorescence in muscle cells demonstrate calcium activity near constrictions. Each frame is a composite of three subsequent snapshots in red, green, and blue, so that transient pulses appear as colored signal, while background appears gray. Different muscle cells report calcium activity in adjacent frames. (E) Average fluorescent activity during the first 15 min of folding show localized signatures at each constriction, with particularly sharp peaks in the middle and anterior constrictions (, , and for anterior, middle, and posterior folds, respectively). (F) In Antp mutants, GCaMP6s fluorescence is significantly reduced () and is not localized in space. Snapshots of GCaMP6s fluorescence 28 min after posterior fold onset (right) show almost no activity in the anterior region compared to the control (left). (G) Disruption of calcium regulation in muscle cells inhibits constrictions. The probability of forming three folds is reduced under heat-shock induced expression of the dominant negative mutant allele SERCA.R751Q with a muscle-specific driver Mef2-GAL4 (, ), and is likewise reduced under RNA interference of MLCK driven by tub67-GAL4;tub16-GAL4 (, ). (H) Altogether, we infer that hox genes are upstream of patterned calcium pulses, which generate muscle contraction that is mechanically coupled to the endoderm, driving tissue strains and ultimately organ shape.