Figure 4: Mobile vasa- and s1p1r-positive germ cells migrate to secondary buds.

(a) Schematic representation of the in situ hybridization data presented in b–g. Small, round cells (7–10 μm diameter, green) expressing vasa and s1pr, are present in the peripheral blood vessels, particularly in ampullae. At early stages of secondary bud development, these small vasa/s1pr1-positive germline stem cells (GSCs) are also present inside the primary buds, in the vicinity of the developing gonads. We hypothesize that these small GSC that are present in the peripheral blood vessels enter into the primary bud by migration (red arrow), but the exact site and time point of entry is unknown. Larger germ cell precursors (15–30 μm, green), which also express vasa, s1pr1, likewise cluster around the gonads. When a secondary bud develops and closes to form a double vesicle, germ cell precursors (green) migrate into the secondary bud. As secondary bud development proceeds, more and larger germ cells are visible inside, indicating proliferation and differentiation. (b–g) Representative images showing expression patterns of vasa and s1p1r by whole mount fluorescent in situ hybridization (green signal) merged with nuclear counterstaining (blue; n=10). (b) Round, small vasa (green)-positive GSC (arrows) inside an ampullae. (c) Vasa-positive germ cells (arrowhead) next to the developing secondary bud (broken white line). (d) Vasa-positive germ cells (arrowhead) inside closed double vesicle secondary bud. (e) Round, small s1pr (green)-positive GSC (arrows) inside an ampullae. (f) s1pr-positive germ cells (arrowheads) next to the developing secondary bud (broken white line). (g) s1pr-positive germ cells (arrowhead) inside closed double vesicle secondary bud. Nuclei were counterstained with Hoechst 33342 (blue). Note that small GSC in b and e have a high nuclear-to-cytoplasmic ratio. The blue signal from the nuclear counterstain overlying the green signal from the FISH appears turquoise. Scale bars, 20 μm.