Biological events during Nematostella wound healing. (A) Timeline of morphological events during the first six hours after injury. Filamentous actin, a core component of the extracellular matrix and muscle fibers, was labeled with Phallacidin-FL (false-colored black). A comparison of uninjured animals (far left), to injured animals shows that after six hours (far right), the wound is unidentifiable. The yellow box around the aboral side of the uninjured animal designates the zone of injury throughout our study. At one hour, the animal exhibits a deflated collapsed state due to water loss from the gastrovascular cavity, and the mesentery can be seen extending towards the wound (yellow M). (B) By hour two, high concentrations of actin are found in the cells along the margin of the wound (left), with nuclei positioned outside the wounded area (right), suggesting these are actin filopodial projections into the wounded area. (C) In a small percentage of animals, long actin filopodia can be found connecting the two parallel wounds. (D) Punctured live animals (stained with acridine orange) often plug their wound with mesentery structures (left) and secrete a mucus-like material from the injury site (right). (E) Time series of potential apoptotic cells during puncture wound healing, as revealed by the DeadEnd tunel assay (labels cells with DNA damage). Control samples show no signal immediately after injury; the highest concentration of signal is seen immediately (15 minutes) after injury and the signal diminishes over time. (Negative control did not have recombinant terminal deoxynucleotidyl transferase enzyme - as specified from the protocol). Counterstaining with DAPI (image 2) designates that the kit is identifying cells with extensive DNA damage (apoptotic). (All red arrow heads or white stars indicate the position of the injury site).