The Weismann doctrine holds that cells of the germline are sequestered early in development in order to prevent inheritance of mutations. Organisms that remake their bodies through many asexual generations introduce the problem of how to sequester germ cell precursors for bodies that do not yet exist. Botryllus schlosseri is an excellent experimental model for studying stem cells and the question of germ line segregation. Botryllus is a sessile, colonial protochordate which reproduces sexually as well as by continuous and cyclical asexual budding. Bordering colonies with at least one shared allele at a histocompatibility locus undergo vascular fusion and establish a common blood circulation. Colonies acquire partial or complete genetic replacement of gonads, asexual buds, or both, by cells from the parabiosed partner. Genetic parasitism of somatic and germline tissue occurs in repeatable, genetically-heritable hierarchies. Persistence of the parasitic genotype for months following surgical separation of the fused partners lead to the hypothesis that parasitism is caused by transfer of somatic, germ, and possibly totipotent stem cells. Here we describe the development of a radioprotection assay in Botryllus and find that the inability to survive to engraftment is evidence of the high rate of cell turnover required for weekly replacement of all somatic tissues in a colonial organism. We find a role for a novel gene, Athena, in asexual bud development utilizing novel genetic knockdown tools for Botryllus. We demonstrate the presence of telomerase in the maintenance of Botryllus chromosome ends and find that the pattern of its developmental modulation supports the hypothesis that a small pool of self-renewing stem cells in nascent buds gives rise to lineages of decreasingly multipotent progenitors. We find the frequency of these stem cells to be 1:500 in the entire colony by limiting dilution transplant between fusible genotypes. We isolate a population of cells with high telomerase and ALDH activity that inseparably induces somatic and germ line chimerism by transplant of 25 to 50 cells. Taken together, these findings provide evidence for the first totipotent stem cell isolated in an adult metazoan as well as advancing Botryllus schlosseri as a manipulable experimental organism.
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