Movie 1.

Foci of the actin fiber organization develop into topological defects localized at the regenerated head and foot regions. Time-lapse movies of a regenerating Hydra spheroid expressing Lifeact-GFP in the ectoderm acquired simultaneously from two opposite sides of the tissue using an up-andunder spinning-disk confocal microscope (Fig. 1C; upper panels: bottom 20x objective; lower panels: top 10x objective). The two regions around the foci of the actin fiber organization were labeled by laser-induced uncaging of Abberior CAGE 552 in the folded spheroid ∼3 hours after excision, allowing us to follow the two focus regions throughout the regeneration process. An aster-shaped +1 defect emerges in the focus region at the future head site (orange labels), while a pair of +1/2 defects emerge in the second focus at the future foot site (cyan labels). The locations of the actin foci are determined manually, and depicted by dots that are replaced by square outlines once well-defined point defects appear. The images show projected views of the ectodermal basal surface (left), the ectodermal apical surface (middle), and the basal layer with an overlay of the photoactivated dye (magenta; right). The images show computationally generated projected views of the ectodermal basal surface (left), the ectodermal apical surface (middle), and the basal layer with an overlay of the photoactivated dye (magenta; right). The images were centered to correct for movements of the whole tissue. The elapsed time from excision is displayed (hh:mm), and the scale bar is 100 μm.