The PGCs do not form during larval development. Larvae were incubated 24 hpf to 7 dpf in EdU and fixed for Vasa protein and EdU detection at 7 dpf. (A) Vasa protein (green) is detectable in the four PGCs (asterisks) and the MPGZ (arrow). (B) Nuclei of proliferating cells have incorporated EdU (red) during development. (C) Overlay. While the cells of the MPGZ exhibit EdU labeled nuclei (arrow), the four PGCs have not divided during the incubation period (asterisks). (D) Single plane image showing more clearly that the nuclei of the PGCs (asterisks) are EdU negative. Note that the other two PGCs lie in a different focal plane. (E) Schematic drawing of the corresponding region in (C) (dashed box). The three larval segments are labeled C = cephalic segment, I, and II. Anterior is to the top. Images are merged stacks of confocal images, except the single plane image in (D). Scale bar corresponds to 50 μm in A-C, and 20 μm in D and E.