Figure 6. Smooth muscle hubs mark the position of regenerating manubria.

For each experiment, cartoons illustrate the cut (orange dotted lines), and stereomicroscope images the responses of live regenerates, while fluorescent images show phalloidin staining (in white) of the regions outlined by brown squares in the stereomicroscope images. Quantification of phenotypes at the bottom left of each image. (A) Quarter fragments: 24 hpd, a radially arranged hub of muscle fibers (orange arrowhead) is detected away from the wound site, close to the injured end of the remaining radial canal. At 5 dpd, a regenerated manubrium is located at the smooth muscle hub. (B) Umbrella fragments devoid of radial canals observed after 4 dpd (top panel: re-circularized, no manubrium regeneration) and 14 dpd (bottom panel: a tubular manubrium-like structure, unable to feed, rarely forms). (C) Removal of canal segments, between the ablated manubrium and gonads: at 24 hpd (left) a muscle hub - but no manubrium blastema - is visible; regrowing radial canals (blue outline in phalloidin image) have not yet reached the hub. At 5 dpd (right panel) three radial canals (blue dashed line) are connected to the muscle hub and manubrium regeneration has occurred (orange arrowhead). (D) Manubrium dissection, coupled to a long cut through the umbrella: 24 hpd (left panel) two muscle hubs are visible (orange arrowheads). At 5 dpd (right) two manubria have formed (orange arrowheads), at the corresponding locations. Scale bars: A-C: 150 µm; D: 300 µm; for all stereoscope images: 1 mm.

Figure 6—figure supplement 1. Multiple muscle hubs correlate with the regeneration of multiple manubria.

For each experiment, cartoons illustrate the cut (orange dotted lines), and stereomicroscope images the responses of live regenerates, while fluorescent images show phalloidin staining (in white) of the regions outlined by brown squares in the stereomicroscope images. Quantification of phenotypes at the bottom left of each image. (A) Small fragment containing a gonad and a canal section, with a regenerated manubrium visible at 4 dpd. (B) Manubrium dissection: at 24 hpd, radial smooth muscle fibers have joined at the center of the jellyfish, forming a ‘muscle hub’. Right panel is the same shown in Figure 2A. (C) Manubrium dissection, coupled to a long cut through the umbrella and to the removal of radial canal connections to gonads. Fluorescent image shows a double hub forming 24 hpd (phalloidin staining). The stereomicroscope image of a 7 dpd regenerate shows two manubria, formed once the canal connections were reestablished. (D) Manubrium dissection, coupled to a long cut through the umbrella: Hoechst staining images illustrating the possible outcomes at 24 hpd and 5 dpd, quantified below (as indicated by the color code, see: Figure 6—figure supplement 1—source data 1). Scale bars: 1 mm for stereomicroscope images, 200 µm for fluorescent images.