The PGCs arise between 3-8 hpf. Embryos were incubated for different periods in EdU and the developing young worms were fixed for Vasa and EdU detection at at 7 dpf. (A) Neither PGCs nor the MPGZ have incorporated EdU between 1 and 3 hpf. (B) Nuclei of the PGCs, but not of the MPGZ, are labeled at 3-7 hpf. (C) When embryos were incubated from 7-9 hpf, only two out of four PGCs are labeled. (D) No labeled PGCs are detectable, when embryos were incubated later than 8 hpf. Asterisks: PGCs, red: incorporation of EdU, arrows: MPGZ, green: Vasa immunohistochemistry as a marker for PGCs and the MPGZ. A-D are overlayed stacks of confocal images. A'-D' show the PGCs at higher magnification. A"-D" are schematic representations. The three larval segments are labeled C = cephalic segment, I, and II. Anterior of the larvae is to the top. Scale bar = 50 μm in A-D and 20 μm in A'-D'.