The four PGCs form a cluster anterior to the MPGZ. (A) In the trochophore larva (24 hpf), four EdU-positive PGCs are located postero-ventral at the basis of the mesodermal bands. Inset: single plane image of the posterior region in B at higher magnification (B) PGCs double labeled with anti-Vasa antibody (green) and EdU (red) are positioned anterior to the Vasa-positive MPGZ in the metatrochophore larva (48 hpf). Inset: Single plane image of the posterior region in B at higher magnification. (C) At 72 hpf, the four double-labeled PGCs are still forming a cluster anterior to the Vasa positive MPGZ. In the developing gut, the large EdU-positive nuclei of the four macromeres are visible. C': single plane image at higher magnification, two PGCs are located at a different focal plane (asterisk). (D) At 96 hpf, four single PGCs leave the MPGZ and start migrating anteriorly towards the primary gonad. D': Single plane image at higher magnification. One of the PGCs lies at a different focal plane (asterisk). Cells which incorporated EdU between 3-7 hpf exhibit red fluorescence in the nuclei. Bristles (b) and spinning glands (sg) are non-specifically stained by the EdU-detection reagent. Green: Vasa expression, asterisks: PGCs, red: incorporation of EdU, arrow: MPGZ, green dashed line: mesodermal bands, g = gut. The three larval segments are labeled C = cephalic segment, I, and II. Anterior of the larvae is to the top. A-D are overlays of confocal images. Insets C'-D' are single focal plane images, therefore not all the structures are always visible. Scale bar = 50 μm in A-D and 20 μm in C'-D'.