Further portrayal of epithelial monolayers emergent de novo from extirpated ascidians palleal buds.

Astogeny in botryllid ascidians is executed by highly synchronized, repeated development and death cycles operating simultaneously on three coexisting asexually derived generations: zooids, primary buds, and secondary buds. In this study, we validated the fact that surgically removed blastogenic stage "D" primary buds cultured under in vitro conditions, away from any discrete colonial regulatory cues, exhibit intrinsic phenomena that are probably masked by astogenic controls. They produce de novo epithelial monolayers (EM), extending their lifespan from a few days to 1 mo and up to 5 mo when floating in the medium. Enhanced EM formation was documented when fibroblast growth factor (FGF) was added after at least 24 h incubation in FGF-free medium. Surprisingly, with no FGF administration, while intact isolated buds did not develop any EM, injured buds developed EM in half of the cases. Working on actin, PL10, FGF-R, P-MEK, MAP-kinase, and cadherin expressions, we documented that extirpated buds and monolayers are very active on the molecular/biochemical levels, revealing various cells and cellular organelle stains and rapid changes in the protein levels along a daily basis. Cells situated in the center of the monolayers stained differently for some proteins than peripheral cells. Cumulatively, results showed that flattened attached monolayers, as well as free-floating stage "D" buds, are highly active, not only exhibiting differential expressions of various proteins along incubation, but are also highly responsive to physical damages. These results establish a novel in vitro model system for epithelial cell development and senescence, revealing surprising rejuvenation and extended lifespan phenomena.