De novo emerged stemness signatures in epithelial monolayers developed from extirpated palleal buds.

In botryllid ascidians, astogeny is executed through blastogenesis, a weekly, highly synchronized phenomenon of growth and death cycles, each constitutes four major developmental stages (A-D), operating simultaneously on three coexisting asexually derived generations, including primary and secondary palleal buds. This study documents the de novo expression of Piwi transcript and protein in extirpated blastogenic stage "D" buds isolated from Botryllus schlosseri colonies that are maintained in vitro, days after the disappearance of corresponding intact zooids in control colonies. Under in vitro conditions, floating buds attach to substrates and develop monolayers of epithelial sheets that live for long periods (compared to intact colonial buds) prior to their deterioration. Here, we further demonstrate that various cell types within floating blastogenic stage "D" buds are labeled as Piwi +, as do other cells that are dispersed over the epithelial sheets (that are Piwi -), representing highly differentiated state), all revealing a surprising new flag for stemness in these tissue fragments that developed exclusively under in vitro conditions. No single permanent cell-line is currently available from colonial tunicates or from other marine invertebrates, since cells stop dividing in vitro within 24-72 h after their isolation and start cellular quiescence. The development of epithelial sheets from isolated Botryllus palleal buds and the recorded molecular stemness flag of various cells, remaining for long periods under in vitro conditions, may pave the way for establishing cell cultures from Botryllus epithelial cells.