Molecular Characterization of Predatory Stem Cells in a Chimeric Protochordate

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Understanding the regulation of allogeneic stem cell activity in a host is a major challenge of transplantation medicine. Despite extensive investigation in this area, many fundamental issues remain unresolved. In our lab, we have focused our studies on an organism with certain unique features that make it an appealing model for studying chimerism and stem cell functionality in allogeneic environment. This protochordate (Botryllus schlosseri) demonstrates' genetic hierarchies for somatic and germline stem cell parasitism. In this organism, a histocompatibility locus (Fu/HC) with extensive polymorphism allows sharing of vascular systems and creation of chimera among kin. In the chimeras, the circulating pluripotent cells of one partner can parasitize and replace the germline and/or the soma of the other partner (termed germline or somatic stem cell parasitism). This ability to replace host tissues follows genetic hierarchies of "winner strain" that replace "loser strain" tissues. Our recent studies have defined the predatory cell lineages as prospectively isolatable stem cells and showed that the Fu/HC locus and germline competition inheritance operates at the level of injected, purified stem cells. We cloned the Fu/HC gene and found that it has 4 homologues to the human and mouse: Tie 1,Tek, IgSF4, (markers for either mouse blood forming stem cells or blood vessel forming stem cells) and the poliovirus receptor, CD155 (a target for human natural killer cell recognition, potentially implicating NK cells as mediators of tolerance induction versus tissue rejection). In addition by direct visualization of cells that exhibit fundamental and important aspects of mammalian stem cell biology, we have identified a major adult somatic stem cell niche in this organism. We plan to investigate the transmission genetics of stem cell competitive potential, and to identify the genes which determine this phenotype. We will perform systematic breeding experiments and establish genetically defined lines that are homozygous for the trait of stem cell competitive potential (SCP; winner versus loser homozygous strains). The homozygous strains will be further used to construct a genetic map of the SCP region. AFLP's markers which co-segregate with the SCP region (bulk segregation analysis) will be cloned, sequenced and further aligned with the Botryllus genome sequence and EST's (project in process). Based on these data, candidate SCP genes will be selected and their expression profiles in "winner strain" stem cells and their niches, versus "loser strain" stem cells and their niches will be determined via high throughput mRNA sequencing. The involvement of a few selected genes in allogeneic stem cell competitive regulation will be tested directly using functional assays. This comprehensive molecular study in Botryllus will provide novel insights into the pathways that lead to successful allogeneic stem cell competition that will likely be applicable to vertebrates in general.