Identification and expression studies of putative stem/progenitor cell markers in the urochordate Botryllus schlosseri

In the colonial ascidian Botryllus schlosseri, a cyclical generation change guarantees the recurrent (weekly at 20°C) renewal of the zooids. During the blastogenetic cycle (i.e., the interval of time between a generation change and the following one), buds progressively grow to the adult size before replacing the old zooids. With the aim of better elucidating the process stem cell differentiation, with particular reference to the genesis of haemocytes during the of the colonial ascidian, we screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic stem/progenitor cells. On these sequences, after an in silico translation, we performed the phylogenetic reconstruction that, always, returned us the tunicate relevant position, within the protochordates cluster, of vertebrate sister group. The four mammalian orthologous genes, used as markers for the recognition of haematopoietic stem/progenitor cells, identified in B. schlosseri, are bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. The ISH assay, performed by antisense specific riboprobes, on haemocyte monolayers and colony sections, resulted in a labelling of the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche, strengthening our idea to use bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes for the identification of haematopoietic stem/progenitor cells in B. schlosseri. 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 higher transcription levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for the formation of the new blastogenetic generation.