UC Santa Barbara

Examples of metazoan regeneration can be seen within all animal phyla; however, the ability to restore lost tissues upon injury varies greatly. Some species can only partially replace tissues and organs, while others can repeatedly regenerate entire limbs, severed spinal cords, and portions of their brain. There are also extraordinary cases of whole-body regeneration (WBR) where animals can produce entire bodies from small fragments of leftover tissue after injury. A few species of botryllids can even utilize circulating blood borne stem cells for WBR. These animals are in the subphylum tunicata, and evolutionarily classified alongside vertebrata in the phylum Chordata. Botryllids are marine invertebrates that grow by repeated rounds of asexual reproduction to form a colony of clonally derived individuals called zooids. Under normal conditions, zooids are regenerated de novo on a weekly basis, and a colony increases in size by expanding the number of individuals. Two distinct processes that differ by the source of new bodies are responsible for zooid regeneration in the botryllids. In the first, called blastogenesis, new zooids arise from a multipotent epithelium of a pre-existing zooid. In the second, termed vascular budding (VB) or WBR, mobile cells in the vasculature are the source of the new zooid. In some botryllid species, blastogenesis and VB occur concurrently. In others, blastogenesis is exclusively used for expansion while WBR only occurs following injury or exit from dormancy. We studied WBR in two related species: Botryllus schlosseri and Botrylloides diegensis. Both have an extracorporeal vasculature that develops outside the bodies to connect all individuals within a colony. We isolated blood vessels to induce WBR by removing all zooids, primary buds, and secondary buds. This surgery was performed under a dissecting microscope with scalpels and extra-fine tip forceps to prevent damage to the vasculature. Regeneration was then analyzed using time-lapse microscopy with the animal being maintained in a temperature-controlled seawater recirculation basin. We compared and characterized early stages of WBR utilizing these days-long videos. Our data suggest that unlike other botryllid species, new zooid growth following injury in B. schlosseri is not due to WBR, but instead through ectopic development of tissues leftover from the blastogenic process. Despite these differences, we did find a common theme in the two species: developmental competition results in only a single zooid reaching maturity. We utilized this phenomenon to control the number of buds and their spatial orientation in B. schlosseri and found that competition is reversible and mediated by circulating factors and/or cells. This represents a new model to study resource allocation and competition within an individual.