Expression study of molecular markers involved in staminality and differentiation in the colonial ascidians Botryllus schlosseri

Ascidians are invertebrate chordates, members of the subphylum Tunicata that represents the sister group of vertebrates. They offer the opportunity to investigate and compare the behaviour of both embryonic and adult stem cells. Morphological data suggest the presence of undifferentiated haemocytes (haemoblasts) able to proliferate and give rise to terminally differentiated cells. Relevant studies were also carried out in the neural lineage, in which neural progenitor cells regenerate the brain after extirpation. In B. schlosseri, during the cyclical generation change, bud primordial cells, probably deriving from a pool of long-living stem cells, are able to give rise to the neural complex. We screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic and neural stem cells. Four sequences, orthologous to mammalian transcripts considered markers of haematopoietic progenitor cells, were identified in B. schlosseri. They are: bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. In situ hybridization on haemocyte monolayers and colony sections, resulted in labelling of cells in the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche. Quantitative real time PCR (qRT-PCR) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. During this phase, there is the formation of new secondary buds emerging from the primary buds. The high expression levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for giving rise to the new blastogenetic generation. For the neural lineage, we identified and characterised two transcripts orthologues of vertebrate neural stem cell markers (BsSox2 and BsMsi2). We also studied the expression, during the blastogenetic cycle, of a panel of genes already known to be involved in ascidian larvae neurogenesis, i.e., orthologues of Pax2/5/8, Hox1 and Hox3. ISH with riboprobes for BsSox2, BsMsi2, BsPax2/5/8, BsHox1 and BsHox3 revealed a common labelling in the endostyle niche. The presence of bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 transcripts in the cells of the region known to be a stem cell niche, led us to conclude, not only that our probes identified undifferentiated cells but even that in B. schlosseri are probably present a single population of pluripotent stem cells that could differentiate into haematopoietic or neural cells. The qRT-PCR, showed an high expression level in the mid-cycle phase of all the putative neural markers considered. In this phase new secondary buds are produced from primary buds. Each new bud needs its own neural complex and this requires the proliferation of undifferentiated cells to originate neural gland rudiment and cerebral ganglion. Bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 increased their expression associated with these neurogenesis events and this support their involvement in neural stem cell differentiation.