Figure 5: S1P signalling is essential for homing of germ cells into secondary buds.

Animals were treated with Sphingosine Kinase inhibitors or S1PR1-antagonist for 3 days, starting at stage A1, and fixed at stage B2, when the secondary bud forms a closed double vesicle (n=4). Controls were left untreated. Vasa-FISH was performed on fixed animals, and localization of germ cells was analysed by confocal microscopy. (a–c) Vasa-positive germ cells (green, arrows) homed into the double-vesicle-stage secondary bud (broken line) in a control animal. (d) Ampulla of a control animal with vasa-positive GSC in the circulation. (e–h) In animals treated with the sphingosine kinase inhibitor A (SK1-I), vasa-positive germ cells do not home to secondary buds (broken lines), but migrate randomly to other locations in the primary bud (e, arrow) and in the blood vessels (h, arrow). (i–l) In animals treated with the sphingosine kinase inhibitor B (CAS 1177741-83-1), vasa-positive germ cells do not home to secondary buds (broken lines). Some larger vasa-positive germ cells remain outside of the closed double vesicle (i and k, arrows), whereas others are found in the blood vessels (l). (m–p) In animals treated with S1PR1-antagonist, vasa-positive germ cells do not home to secondary buds (broken lines). Some larger vasa-positive germ cell precursors randomly migrate in the vasculature. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 20 μm.