(A) Images illustrating the distribution of MGD cells in Clytia organs (manubrium, gonads, tentacle bulbs), as indicated for each row. MGD cells can be recognized by the orange/brown pigment (DIC images in left panels). Second and third panels: Fluorescent micro-Beads (FB, in red) are ingested by cells residing in the gastroderm. Nuclei are counterstained with Hoechst (in blue), the organization of tissues in shown by phalloidin staining (in white). Right panel: higher magnification images including all three channels showing MGD cell morphology. All confocal images are maximum projection except the close-up image of the manubrium MGD cells that is a single plane of a z-stack. (B) Tracking of FB-labeled cells (following 24 hr feeding) in manubrium-regenerating medusae, cartoon illustrates experimental design. Distribution of FB-labeled MGD cells, 48 hr after manubrium dissection: DIC and fluorescence images (colors as in A) show FB-labeled cells in the gonad digestive cavity (i), in the radial canals (ii) and in the regenerating manubrium (iii). (C) Frames from time lapse movies of the MG-segment of radial canal (rc), showing rapid movement of MGD cells in the lumen. The five top panels show outward movement from the gonad (g), the bottom series shows movement into the regenerating manubrium (rm). (D) Quantification of circulating cells in the MG-segment of all four radial canals in fed, starved and regenerating jellyfish. Starved and dissected animals were last fed 8 hr before experiment (n: 24 for each condition, see Figure 4—source data 1) (E) Grafting of a FB-labeled gonad to a naïve host (cartoon illustrates experimental design) demonstrates untargeted migration of FB-labeled cells into all organs, either in regenerating jellyfish (manubrium dissected, i - top panel) or in non-dissected condition (n.d. ii – bottom panel). Panels show, from left to right: tentacle bulb, gonad, manubrium; FB in red, nuclear counterstain (Hoechst) in blue. (F) Experiment to determine the source of FB-labeled cells during manubrium regeneration (48 hpd), cartoons illustrate the three experimental conditions (from top to bottom): manubrium-dissected jellyfish, manubrium- and gonad-dissected (connection to tentacle bulbs is maintained), manubrium- dissected and canal-disconnected (gonads removed). Images show phalloidin staining (in white, to the left) visualizing the tissue organization around the manubrium site and fluorescent micro-beads (FB, right column, shown in red with Hoechst staining of nuclei in blue). FB-labeled cells are present in the regenerating manubrium in the first two conditions, but if there is no connection to another organ, manubrium regeneration is stalled, and no FB-labeled cells can be detected. Scale bars: 100 µm, except in the right column of (A): 25 µm.