Figure 1: Botryllus Schlosseri morphology, gonad formation and expression of integrin alpha-6 and s1pr1 in vasa-positive cells.

(a) Ventral view of a colony of individual adult animals (green boxes and arrows), each of which is connected to asexual propagating primary buds (blue boxes and arrows) and secondary buds (yellow boxes and arrows). During the asexual budding process, new buds form as a thickening of the peribranchial epithelium (stage A), which forms a pocket and eventually closes to form a double vesicle (stage B). Buds undergo invaginations and differentiate into all somatic tissues and organs (stage C). Germ cells (red) enter the newly formed secondary buds at stage B, and differentiate into testes and oocytes, as primary buds develop into the adult form. Individual animals are connected by a common extracorporeal vasculature, which ends in terminal projections known as ampullae a. (b) Representative examples of expression patterns of integrina alpha-6 and s1pr1 in vasa-positive cells by double-labelled fluorescent in situ hybridzation (n=5). Left panel: All vasa-positive (red) germ cell precursors co-express integrin alpha-6 (green). Right panel: S1pr1 (red) is expressed in vasa-positive cells (green). Red and green channels are shown individually together with nuclear counterstaining (blue), and merged images show co-expression of both genes (yellow). Top panels represent stages before homing of vasa-positive cells into the secondary buds. Note that the weak red signal on the secondary bud at this stage is due to slight trapping of the probe inside the secondary bud, and not true signal for s1pr1 mRNA. Bottom panels represent stages after homing of vasa-positive cells into the secondary buds. Nuclei were counterstained with Hoechst 33342 (blue). For better orientation, secondary buds have been outlined with broken white lines. Scale bars, 20 μm.