JCS02522 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. kop RNA is expressed in the germ plasm and the encoded protein is localized to the nucleus. (A) The expression pattern of kop RNA during the first day of zebrafish development. kop RNA is localized to the cleavage planes at the 1 hpf (4-cell stage) and is then expressed in PGCs. The inset included in the image depicting a 22 hpf embryo shows a magnification of the region where the PGCs are normally found. (B) Graphical illustration of the open reading frame of the Kop protein, which contains an ATP/GTP-binding site A. (C, upper panels) PGCs in a 10.5 hours old (1-somite stage) embryo co-injected with DsRedex-nos1-3’UTR (red channel) and kop-gfp-nos1-3’UTR (green channel). The merged picture shows that the Kop-GFP fusion protein is localized to the nuclei of PGCs. (C, lower panels) PGCs in a 10.5 hours old (1-somite stage) embryo co-injected with DsRedex-nos1-3’UTR (red channel), kop-gfp-nos1-3’UTR (green channel) and kop-specific morpholino antisense oligonucleotides (kop-MO). Translation of Kop-GFP fusion protein is inhibited, whereas DsRed expression is not affected. Despite the efficient inhibition in kop mRNA translation, injection of kop-MO did not affect PGC development (data not shown).

• Supplemental Figure 2 - Fig. S2. PGCs do not activate caspases before undergoing cell death and PGCs lacking Dead end function die during somitogenesis stages. (A) A clone of somatic cells which expresses diphtheria toxin shows positive caspase activity at 4.5 hpf. (B) In a-amanitin treated PGCs activated caspases can not be detected at 5 hpf or 9 hpf (insets show negative PGCs (green) and a positive somatic cell (red)) and dnd-MO treated PGCs do not show caspase activity at 6 hpf. (C) Low magnification snapshots from a movie showing Dead end depleted PGCs from 7 hpf to 16 hpf. At 14 hpf (about 8 hours after PGCs normally become motile), first signs of PGC death can be observed as cell fragmentation (see magnified boxes in the lower panels).

• Movie 1 -

  All supplementary movies were generated by observing cells at 28 degrees. A 10x objective and a 63x or 40x objective were used to record low-magnification and high magnification views, respectively. The stamped time for low-magnification movies refers to hours and minutes whereas for high-magnification movies it refers to minutes and seconds.

  Movie 1. Early behaviour of PGCs from 3 hpf to 7 hpf. A low-magnification movie showing PGCs labelled with EGFP-F. The movie starts at 3 hpf, spans 4 hr of development and is played at a speed of 10 min per second.

• Movie 2 - Movie 2. The behaviour of PGCs after their formation. At 3 hpf, PGCs show simple cell morphology and do not migrate. A high-magnification view of a PGC expressing EGFP-F. The movie starts at 3 hpf, spans 5 min of development and is played at a speed of 1 min per second.

• Movie 3 - Movie 3. The behaviour of PGCs between high and dome stages (3.3 hpf-4.3 hpf). At these stages the PGCs show small as well as long protrusions but do not migrate. A high-magnification view of a PGC expressing EGFP-F. The movie starts at 3.7 hpf, spans 5 min of development and is played at a speed of 1 min per second.

• Movie 4 - Movie 4. During cell division the PGCs transiently assume simple cell morphology. A high-magnification view of a PGC expressing EGFP-F. The movie starts at 4 hpf, spans 22 min of development and is played at a speed of 1 min per second.

• Movie 5 - Movie 5. Polarization of PGCs and active migration at 30%-epiboly stage. A high-magnification view of a PGC expressing EGFP-F. The movie starts at 4.7 hpf, spans 5 min of development and is played at a speed of 1 min per second.

• Movie 6 - Movie 6. Transplanted PGCs from a 12 hpf donor embryo into a 3 hpf host embryo are motile. A low-magnification movie showing PGCs labelled with GFP. The movie starts at 3 hpf, spans 1 hr of development and is played at a speed of 10 min per second.

• Movie 7 - Movie 7. PGCs derived from a 12 hpf donor embryo and transplanted into a 3 hpf host embryo show polarization and active migration. A high-magnification view (40x) of a PGC expressing GFP. The movie starts at 3 hpf, spans 5 min of development and is played at a speed of 1 min per second.

• Movie 8 - Movie 8. a-amanitin treated  EGFP-F expressing PGCs show behaviour similar to wild-type cells between high and dome stages (3.3 hpf-4.3 hpf). At these stages the PGCs exhibit small as well as long protrusions but do not migrate. The movie starts at 3.5 hpf, spans 3 min of development and is played at a speed of 1 min per second.

• Movie 9 - Movie 9. a-amanitin treated EGFP-F expressing PGCs do not polarize and do not show active migration at 5.5 hpf. At this stage the treated PGCs appear to behave similar to cells before the transition. The movie starts at 5.5 hpf, spans 3 min of development and is played at a speed of 1 min per second.

• Movie 10 - Movie 10. PGC in dnd-MO injected embryos exhibit normal early behaviour. A high-magnification view of a PGC expressing EGFP-F. The movie starts at 4.3 hpf, spans 3 min of development and is played at a speed of 1 min per second.

• Movie 11 - Movie 11. PGCs in dnd-MO injected embryos fail to show polarized morphology, do not form broad pseudopodia and do not migrate.

  A high-magnification view of a PGC expressing EGFP-F. The movie starts at 4.7 hpf, spans 10 min of development and is played at a speed of 1 min per second.

• Movie 12 - Movie 12. Lack of response to SDF-1a by PGCs early in their development. A low-magnification view of PGCs (green) and the SDF-1a overexpressing explant (red). The movie starts at 3 hpf, spans 2 hr of development and is played at a speed of 10 min per second. Images from the green channel were captured every minute and images from the red channel were captured at 3 min intervals. For the red channel, image duplications were performed to fill the missing 2 min.

• Movie 13 - Movie 13. PGCs respond to SDF-1a after dome stage. A low-magnification view of PGCs (green) and the SDF-1a overexpressing explant (red). The movie starts at 5.3 hpf, spans 2 hr of development and is played at a speed of 10 min per second. Images from the green channel were captured every minute and images from the red channel were captured at 3 min intervals. For the red channel, image duplications were performed to fill the missing 2 min.

• Movie 14 - Movie 14. PGCs do not respond to an explant that does not express SDF-1a. A low-magnification view of PGCs (green) and an explant (red). The movie starts at 5.3 hpf, spans 2 hr of development and is played at a speed of 10 min per second. Images from the green channel were captured every minute and images from the red channel were captured at 3 min intervals. For the red channel, image duplications were performed to fill the missing 2 min.