DEV026922 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -
• Supplemental Table S2 -
• Supplemental Figure S1 -

  Fig. S1. Coordination of movement among iYSN and among mesendoderm cells. (A,B) Quantification of the movement similarity among iYSN and among mesendoderm cells in wild-type embryos at mid-gastrulation stages (7-8 hpf). Histograms of the similarity values were generated separately for each embryo. The box plots show the distribution of the bin heights among the embryos. (A) Among iYSN, 53% of the similarity values are higher than 0.5 (n=6 embryos). (B) Among mesendoderm cells, 82% of the similarity values are higher than 0.5 (n=6 embryos).

• Supplemental Figure S2 -

  Fig. S2. Cortical flow within the YSL is altered in MZoep mutant embryos. (A,B) Trajectories of iYSN (arrowheads) and 0.5 µm diameter fluorescent microspheres (beads; arrows) in the YSL in 70% epiboly (7 hpf; A) and bud stage (10 hpf; B) MZoep mutant embryo. Images are z-projections. Some nuclear and bead trajectories obtained using Motion Tracking Software are shown. Circles indicate the endpoint of each track. White line indicate the dorsal midline of the embryo. Animal is towards the top. Scale bars: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. Overexpression of Oep within the YSL does not rescue defective iYSN convergence movements in MZoep mutant embryos. (A-C) Oep-FLAG antibody staining in MZoep embryos overexpressing oep in the YSL during gastrulation. The YSL of MZoep embryos were injected with 400 pg oep-FLAG and 100 pg membrane-bound gap43-GFP. Confocal transversal sections showing expression of Oep-FLAG within the YSL (A), which colocalize with membrane-bound GFP (B,C; arrows). (A′) Oep-FLAG antibody staining in an MZoep embryo injected with 200 pg oep-FLAG at the one-cell stage, showing expression of Oep on the cell membranes of blastoderm cells. (D,E) MZoep embryo injected with 100 pg membrane bound-GFP into the YSL. Confocal transversal section showing no detection of the FLAG tag (D), whereas membrane-bound GFP is strongly expressed in the YSL (E,F, arrows). (A,D) Flag antibody staining. (B,E) Membrane-bound GFP. (C,F) Merged image of FLAG and GFP channels. (G,H) iYSN trajectories in MZoep mutant injected with oep-FLAG into the YSL at 80% epiboly (8 hpf; G, startpoint of tracks) and two-somite stage (11 hpf; H, endpoint of tracks). Images are z-projections. Some nuclear trajectories obtained using Motion Tracking Software are shown. Circles indicate the endpoint of each track. White line marks the dorsal midline of the embryo. Animal is towards the top. Blast, blastoderm. Scale bars 40 µm in A-F; 50 µm in G,H.

• Supplemental Figure S4 -

  Fig. S4. Endoderm progenitors are not required for iYSN convergence movements. (A,B) iYSN trajectories in cas mutant embryos at 70% epiboly (7 hpf; A, startpoint of tracks) and bud stage (10 hpf; B, endpoint of tracks). iYSN undergo normal convergence movements. Images are z-projections. Some nuclear trajectories obtained using Motion Tracking Software are shown. Circles indicate the endpoint of each track. Dorsal views. White line marks the dorsal midline of the embryo. Animal is towards the top. Scale bars: 50 µm.

• Movie 1 -

  Movie 1. iYSN and mesendoderm trajectories in a wild-type embryo from 70% to 80% epiboly, obtained using 3D tracking software. Dorsal is towards the right, the animal pole is towards the top. z-projection of 30 slices. Orange tracks represent mesendoderm cells and blue tracks represent iYSN. Circles indicate the current timepoint in each track. Scale bar: 50 µm.

• Movie 2 -

  Movie 2. Two-photon excitation timelapse movie of Tau-GFP-labeled microtubule motion within the paraxial iYSL at 90% epiboly. Dorsal is towards the right, the animal pole is to the top. Each frame is a single slice. The time between frames is 3 seconds. iYSN is marked with an asterisk. Scale bar: 10 µm.

• Movie 3 -

  Movie 3. Two-photon excitation timelapse movie of EB3-GFP labeled microtubule plus end motion within the paraxial iYSL at bud stage. Dorsal is towards the right, the animal pole is towards the top. Each frame is a single slice. The time between frames is 1 second. iYSN is marked with an asterisk. Scale bar: 10 µm.

• Movie 4 -

  Movie 4. Two-photon excitation timelapse movie of Lifeact-GFP-labeled actin motion within the paraxial iYSL at 90% epiboly. Dorsal is towards the right, the animal pole is towards the top. Each frame is a single slice. The time between frames is 3 seconds. iYSN is marked with an asterisk. Scale bar: 10 µm.

• Movie 5 -

  Movie 5. Two-photon excitation timelapse movie of fluorescently labeled iYSN and polystyrene microsphere movements in a wild-type embryo during gastrulation (dorsal view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Several nuclear (marked by asterisks) and microsphere trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. White line marks the dorsal midline of the embryo. Animal is towards the top. Scale bar: 50 µm.

• Movie 6 -

  Movie 6. Two-photon excitation timelapse movie of fluorescently labeled iYSN movements in a wild-type embryo during gastrulation (dorsal view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. White line indicates the dorsal midline of the embryo. Animal is towards the top. Scale bar: 50 µm.

• Movie 7 -

  Movie 7. Two-photon excitation timelapse movie of fluorescently labeled iYSN movements in an MZoep mutant embryo during gastrulation (dorsal view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. White line indicates the dorsal midline of the embryo. Animal is towards the top. Scale bar: 50 µm.

• Movie 8 -

  Movie 8. Two-photon excitation timelapse movie of fluorescently labeled iYSN movements in an MZoep mutant embryo during gastrulation (paraxial view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. White line marks the dorsal midline of the embryo. Animal is towards the top. Scale bar: 50 µm.

• Movie 9 -

  Movie 9. Two-photon excitation timelapse movie of fluorescently labeled iYSN movements in an MZoep mutant embryo during gastrulation (lateral view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. Dorsal is towards the right and animal towards the top. Scale bar: 50 µm.

• Movie 10 -

  Movie 10. Two-photon excitation timelapse movie of fluorescently labeled iYSN movements in an MZoep mutant embryo during gastrulation (ventral view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. The embryo is slightly tilted, so the dorsal region is closer to the right side of the image. Animal is towards the top. Scale bar: 50 µm.

• Movie 11 -

  Movie 11. Two-photon excitation timelapse movie of fluorescently labeled iYSN and polystyrene microsphere movements in an MZoep embryo during gastrulation (lateral view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Several nuclear and microsphere trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. Dorsal is towards right and animal towards the top. Scale bar: 50 µm.

• Movie 12 -

  Movie 12. Two-photon excitation timelapse movie of fluorescently labeled iYSN movements in an MZoep mutant embryo overexpressing Oep in the YSL. The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. White line marks the dorsal midline of the embryo. Animal is towards the top. Scale bar: 50 µm.

• Movie 13 -

  Movie 13. Two-photon excitation timelapse movie of fluorescently labeled iYSN movements in a cas mutant embryo during gastrulation. The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint in each track. White line marks the dorsal midline of the embryo. Animal is towards the top. Scale bar: 50 µm.

• Movie 14 -

  Movie 14. Two-photon excitation timelapse movie of fluorescently labeled iYSN and membrane-labeled transplanted mesendoderm progenitors in an MZoep embryo during gastrulation. The images are maximum projections of 30 z-slices. The time between frames is 1 minute. Dorsal is towards the right, animal towards the top. Orange tracks represent transplanted cells and blue tracks iYSN. Circles indicate the current timepoint in each track. Scale bar: 50 µm.

• Movie 15 -

  Movie 15. Two-photon excitation timelapse movie of fluorescently labeled iYSN movements in an MZoep embryo containing transplanted mesendoderm cells during gastrulation (dorsal view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint of each track. White line marks the dorsal midline of the embryo. Red line indicates the position of the transplanted cells. Animal is towards the top. Scale bar: 50 µm.

• Movie 16 -

  Movie 16. Two-photon excitation timelapse movie of histone labeled iYSN and membrane labeled transplanted mesendoderm cells in an MZoep embryo during gastrulation (paraxial view). The images are maximum projections of 35 z-slices. The time between frames is 2.5 minutes. Some of the trajectories obtained using Motion Tracking Software are shown. Circles indicate the current timepoint of each track. White line marks the dorsal midline of the embryo. Animal is towards the top. Scale bar: 50 µm.

• Movie 17 -

  Movie 17. iYSN and mesendoderm trajectories in an e-cadherin morphant embryo from 70% to 80% epiboly, obtained using 3D tracking software. Dorsal is towards the right, the animal pole is towards the top. Z-projection of 10 slices. Orange tracks represent mesendoderm cells and blue tracks iYSN. Circles indicate the current timepoint in each track. Scale bar: 50 µm.

• Movie 18 -

  Movie 18. iYSN and mesendoderm trajectories in a weg/e-cadherin mutant embryo from 70% to 80% epiboly, obtained using 3D tracking software. Dorsal is towards the right, the animal pole is towards the top. Z-projection of 10 slices. Orange tracks represent mesendoderm cells and blue tracks iYSN. Circles indicate the current timepoint in each track. Scale bar: 50 µm.

• Movie 19 -

  Movie 19. Two-photon excitation timelapse movie of Lifeact-GFP-labeled actin motion within the paraxial iYSL in a cytochalasin-treated embryo at 90% epiboly. Dorsal is towards the right, the animal pole is towards the top. Each frame is a single slice. The time between frames is 3 seconds. iYSN is marked with an asterisk. Scale bar: 10 µm.

• Movie 20 -

  Movie 20. iYSN and mesendoderm trajectories in a cytochalasin-treated embryo from 70% to 80% epiboly, obtained using 3D tracking software. Dorsal is towards the right, the animal pole is towards the top. Z-projection of 10 slices. Orange tracks represent mesendoderm cells and blue tracks iYSN. Circles indicate the current timepoint in each track. Scale bar: 50 µm.

• Movie 21 -

  Movie 21. Two-photon excitation timelapse movie of Lifeact-GFP-labeled actin motion within the paraxial iYSL in an embryo previously injected with phalloidin into the YSL at 80% epiboly. Dorsal is towards the right, the animal pole is towards the top. Each frame is a single slice. The time between frames is 3 seconds. iYSN is marked with an asterisk. Scale bar: 10 µm.

• Movie 22 -

  Movie 22. iYSN and mesendoderm trajectories in an embryo injected with phalloidin into the YSL from 70% to 80% epiboly, obtained using 3D tracking software. Dorsal is towards the right, the animal pole is towards the top. Z-projection of 10 slices. Orange tracks represent from mesendoderm cells, blue tracks from iYSN. Circles indicate the current timepoint in each track. Scale bar: 50 µm.