Description of study: To characterize the effect of Chiron on the shape transition, we analyzed samples treated with Chiron as well as samples without Chiron, at day 2, 3 and 4 after aggregation (3 of each: 15 samples in total). To make the nuclei visible, they were stained with Sytox DeepRed and imaged in far red light. The samples treated with Chiron were also imaged in the red and green channel to visualize the mesoderm and endoderm germ layers. Figure 1 shows typical gastruloid shapes imaged in 2D brightfield microscopy.
Figure 1. Brightfield images of mouse gastruloids at 48, 72, 96 and 120h (2,3,4, 5 days) after aggregation (2,3,4, 5 days). Chiron pulse between 48 and 96h. Credit: Sergei Ponomartcev.
Figure 2. 2D Fluorescence images of mouse gastruloids at 48, 72, 96 and 120 h (2,3,4 and 5 days after aggregtion, dpa). Chiron pulse between 48 and 96h. Credit: Sergei Ponomartcev. Size of 3-day gastruloid ~300 um.
Figure 3. 3D MIP projections 3 and 4 day-old gastruloid imaged in 3D with the OsloSPIM, our home-built light-sheet microscope. Gastruloids were made from MP1 cell line. Imaged by Joachim Mossige. Rendered by Xian Hu (Edna). Sample provided by Sergei Ponomartcev.
3D light-sheet images of cleared samples
Figure 4. 3D surface visualization of mouse gastruloids with and without Chiron. 2 and 3 day-old samples without Chi have sessile drop shape, 3 day-olds with Chi and 4 day-olds without Chi are spherical, and 4 day-olds with Chi are elongated. Scroll down to bottom for videos. Scalebar: 100 um.
2D images – optical cross sections
Figure 5. 2D (optical) cross sections of Sytox DeepRed-stained nuclei, visualized in the far red channel in the middle of gastruloids. The most spherical/elongated nuclei shape is seen in 3/4 day-old Chiron samples. Scalebar: 100um.
Note that while both 3-day-old samples with Chiron and 4-day-old samples are spherical, the shapes of the cell nuclei and their polarization differ. In the 3-day-olds with Chiron, the nuclei are more or less round, and the cells do not form polarized structures. This is in stark contrast to the 4-day-olds, where the cells are slightly elongated and form so-called neural rosette structures. The 3-day-olds without Chiron also have polarized structures, seen as neural rosettes, but fewer than the 4-day-olds.
Segmentation data
We used the “cyto” segmentation model in the 3D version of Cellpose (cellpose --Zstack), with the following settings:
- Diameter: 18 (pixels)
- Stitch threshold: 0.25
- Anisotropy: 1.0
- min_size: 2000
Figure 6. Segmentation results using the the 3D version of Cellpose. Top left: 2dpa no Chiron, top middle: 3dpa no Chiron, top right: 3dpa with Chiron, bottom left: 4dpa no Chiron, bottom right: 4dpa with Chiron.
Figure 7. Segmentation results using the the 3D version of Cellpose. Top left: 2dpa no Chiron, top middle: 3dpa no Chiron, top right: 3dpa with Chiron, bottom left: 4dpa no Chiron, bottom right: 4dpa with Chiron.
Multichannel cross section images
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Figure 8. 3d_Chiron_s03_373_decon_stitched. Plane 318. Left: mesoderm, Middle: Sytox Deep red (nuclei), right: endoderm. Diameter ~ 310um.
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Figure 9. 3d_Chiron_s04_decon_stitched. Frame 325. Note: Much less red signal compared to s03 and s05. Left: mesoderm, middle Sytox Deep red (nuclei), right: endoderm. Diameter ~ 330um.
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Figure 10. 3d_Chrion_s05_846_decon_stitched_aligned. Plane 323. Left: mesoderm, middle: nuclei, right: endoderm. Diameter ~330 um.
Figure 11. 4dpa with Chiron. Sample: 4d_Chiron_s02_179. frame365.
Figure 12. 4dpa with Chiron. Sample: 4d_Chiron_s03_2024_753_decon_stitched_aligned. frame432
Figure 13. 4d_Chiron_S04. frame517
Figure14. 4d_Chiron_S05. frame503.
Figure 15. 4d_Chiron_S05_multichannel. frame503.
Cross sections of all samples - nuclei
2-day-old gastruloids without Chiron
Figure 16. 2dpa. Left: 2d_s01_413_decon-1-c0_frame238, middle: 2d_s02_626_decon-c1_frame265, right: 2d_s04_994_decon-c0_frame229. Scalebar: 100 um.
3-day-old gastruloids without Chiron
Figure 17. 3dpa noChiron: Left: 3d_noChiron_s03_882_decon_stitched-c0_frame363, middle: 3d_noChiron_s04_806_decon_stitched_aligned-c0_frame293, right: 3d_noChiron_s05_977_decon_stitched_aligned-c0_frame344. Scalebar: 100 um.
3-day-old gastruloids with Chiron
Figure 18. 3dpa +Chiron. Left: 3d_Chiron_s03_373_decon_stitched-c0_cropped_frame318, middle 3d_Chiron_s04_decon_stitched-c0_cropped_frame325, right: 3d_Chiron_s05_846_decon_stitched_aligned-c0_slice. Scalebar: 100 um.
4-day-old gastruloids without Chiron
Figure 19. Cross-sections of nuclei on 4-day-old samples without Chiron. Left: 4d_noChiron_S01_711_decon_stitched_aligned-c0, middle: 4d_noChiron_S02_813_decon_stitched_aligned-c0, right: 4d_noChiron_S03_839_decon_stitched-c0. Scalebar: 100um.
4-day-old gastruloids with Chiron
Figure 20. 4dpa + Chiron. Top left: 4d_Chiron_s02_179_decon_stitched_aligned-c0_frame365, top right: 4d_Chiron_s03_753_decon_stitched_aligned-c0_frame432, bottom left: 4d_Chiron_S04_547_decon_stitched_aligned-c0-1 (frame510), bottom right: 4d_Chiron_s05_442_decon. Scalebar: 100 um.
Our samples were imaged, deconvolved and rendered in 3D using a Zeiss LS7 light-sheet microscope by Jana Harizanova and Sunny Dai at CFIM at Univ. Copenhagen. Clearing: EZ Clear.
Videos
Video showing the 2-day-old sample without Chiron from Figure 2.
Video showing the 3-day-old sample without Chiron from Figure 2.
Video showing the 3-day-old sample with Chiron from Figure 2.
Video showing the 4-day-old sample without Chiron from Figure 2.
Video showing the 4-day-old sample with Chiron from Figure 1.
References:
@article{anlacs2024early,
title={Early autonomous patterning of the anteroposterior axis in gastruloids},
author={Anla{\c{s}}, Kerim and Gritti, Nicola and Nakaki, Fumio and Salam{\'o} Palau, Laura and Tlili, Sham Leilah and Oriola, David and Arat{\'o}, Krisztina and Le Lim, Jia and Sharpe, James and Trivedi, Vikas},
journal={Development},
volume={151},
number={22},
year={2024},
publisher={The Company of Biologists}
}