In vitro delayed senescence of extirpated buds from zooids of the colonial tunicate Botryllus schlosseri

In the colonial growth of botryllid ascidians, blastogenesis (bud formation) is a cyclical and synchronized developmental process characterized by a weekly rhythm of budding and apoptotic events. Very little is known about this cycle regulation and its control. In this study, the in vitro fate of developing buds and regressing zooids extirpated from Botryllus schlosseri colonies at different blastogenic stages were examined, revealing that stages 'B' to 'D' buds (but not stage 'A' buds) developed new structures under in vitro conditions. These were mainly spheres (up to 1 mm in diameter) and epithelial monolayers around the attached buds. We also found that: (1) when attached spheres and epithelial monolayers appeared, the life expectancy of an isolated bud in vitro reached 50-60 days, five times the life expectancy of intact, in vivo developing zooids; (2) the life expectancy of in vitro buds that remained unattached to the substrates was at least 150 days; (3) after attaching to the substrates, buds obeyed a newly imposed developmental clock dictating up to 35 survival days for spheres and up to 14 days for epithelial monolayers; (4) the prevailing mode of death in vitro was necrotic, in contrast to the apoptotic mode of zooidal deterioration at the takeover phase of blastogenesis; (5) under in vitro conditions, degenerating zooids surprisingly produced epithelial monolayers within 3 weeks of culturing. Monolayers survived for up to 10 additional days, extending the lifespan of the degenerating zooids from a few hours to up to 1 month. We conclude that under in vitro conditions, not only are the underlying colonial growth mechanisms replaced by different developmental pathways, but also the internal colonial-level clocks programming death, are replaced by a new biological mechanism with different timetables.