‘Polonaise movements’ persist under suppression of Wnt/PCP pathway.

(A) Testing model of relationships between the polonaise movements, primitive streak (PS) morphogenesis, and the Wnt/PCP pathway. A-P and L-R; anterior-posterior and left-right body axes, respectively. (B) Diagram of experimental set up. (C, D) In situ hybridizations for Brachyury (Bra) in control- and ΔDEP-GFP-misexpressing embryos. (E) Overlay of embryos hybridized for Bra (n=4 for each). (F) Cell number in PS [control 8.5±0.4x103 cells vs ΔDEP 8.8±1.2x103 cells, p=0.68, n=3 for each (two-tailed Student’s t test)]. (G-H’’’) Trace of cell flow path of electroplated epiblast cells with Flowtrace. The initiation of the polonaise movements was set as Time 0 (t=0). See also Movie S1. (I-J’’’) Vector fields overlaid with fluorescent images, generated from the images of the control (G-G’’’) and aphidicolin-treated embryos (H-H’’’). (K-L’’’) Streamlines, visualizing averaged cell flows during each time period. (M-N’’’) Vorticity plots, displaying an averaged measure of the local rotation during each time period. Blue, clockwise; red, counter-clockwise rotation.

Early phase of ‘polonaise movements’ remains under global mitotic arrest.

(A) Testing model of relationships between the polonaise movements, primitive streak (PS) morphogenesis, and mitosis. Axes as in Fig. 1. (B) Diagram of experimental set up. (C, D) BrdU incorporation in control-sham operated and aphidicolin treated in embryos. White boxes show enlarged areas. (C’, C”) High magnification of boxed areas in PS and non-PS, respectively. (E) In situ hybridizations for Brachyury (Bra), Vg1, and Sox3 in control and aphidicolin-treated embryos. Arrow heads indicate diminished PSs. Black boxes, enlarged area including the diminished PS. (F-G’’’) Trace of cell flow path with Flowtrace analysis (green is trace, yellow indicates endpoint) of fluorescently-tagged epiblast cells. The initiation of the polonaise movements was set as Time 0 (t=0). See also Movie S2. (H-I’’’) Vector fields, generated from the images of control (F-F’’’) and aphidicolin-treated embryos (G-G’’’). (J-K’’’) Streamlines, visualizing averaged cell flows during each time period. Interpolated vectors are displayed in orange. (L-M’’’) Vorticity plots, displaying averaged measure of the local rotation during each time period. Blue, clockwise; red, counter-clockwise rotation. Note: Since vorticity is calculated for all deviation from set point, slight curves and full rotations receive the same color indication, as seen in M’’’ which is not maintaining a bilateral vortex-like-rotating cell flows. See Materials and Methods section for a full discussion.

Authentic PS extends under disruption of authentic ‘polonaise movements’.

(A) Testing model of relationships between the polonaise movements, primitive streak (PS) morphogenesis, using ectopic Vg1-inducing system. Axes as in Fig. 1. (B) Diagram of experimental set up. (C, D) In situ hybridizations for Brachyury (Bra) in control and ectopic Vg1-induced PS, respectively. (E-F’’’) Trace of cell flow path of electroplated epiblast cells by using Imaris tracking analysis. See also Movie S3. White circles, COS cell implanted site. The initiation of the polonaise movements was set as Time 0 (t=0). (G-H’’’) Vector fields, generated from the images of control-GFP/COS-(E-E’’’) and Vg1/COS-implanted embryos (F-F’’’). (I-J’’’) Streamlines, visualizing averaged cell flows during each time period of the vector fields. (K-L’’’) Vorticity plots, displaying an averaged measure of the local rotation during each time period in the vector fields. Blue, clockwise; red, counter-clockwise rotation. Note: vorticity is identified in both a full-rotation and curve, particularly in (L-L’’’). The white asterisks indicate the polonaise movements at the induced PS which are opposite direction to the authentic PS. (M) Summary of the cell flow patterns in control-GFP/COS- and Vg1/COS-implanted embryos.

Induced ‘polonaise movements’ persist despite defective PS morphogenesis.

(A) Testing model of relationships between the polonaise movements, primitive streak (PS) morphogenesis, and Vg1. Axes as in Fig. 1. (B) Diagram of experimental set up. (C, D) BrdU incorporation in control-GFP/COS- and Vg1/COS-implanted embryos under aphidicolin-treatment. White boxes showed enlarged areas. (E, F) In situ hybridizations for Brachyury (Bra, n=3 for each). Black arrows indicate diminished PSs. (G-H’’’) Trace of cell flow path of electroplated epiblast cells by using Imaris tracking analysis. See also Movie S5. White circles, COS cell implanted site. The initiation of the polonaise movements was set as Time 0 (t=0). (I-J’’’) Vector fields, generated from the images of control-GFP/COS-(G-G”’) and Vg1/COS-implanted embryos (H-H’”) under aphidicolin-treatment. (K-L’’’). Streamlines, visualizing averaged cell flows during each time period of the vector fields. (M-N’’’) Vorticity plots, displaying an averaged measure of the local rotation during each time period in the vector fields. Note: vorticity is identified in both a full-rotation and curve, particularly in (L-L’’’). Blue, clockwise; red, counter-clockwise rotation. White asterisks, the polonaise movements at the induced axis which are opposite direction to the authentic midline axis. Note: vorticity is identified in both a full-rotation and curve in (M’’’) and (N’’’). See description in the material and method section.

Summary of the patterns of the cell flow and PS morphogenesis.

(A) Cell flow pattern under experimental manipulation of PS morphogenesis.

(B) Authentic PS morphogenesis under disrupting authentic polonaise movements.

(C) Vg1-induced cell flow pattern under defective PS morphogenesis.

(D) Summary of the relationships between polonaise movements, PS morphogenesis, mitosis, and Wnt/PCP pathway.

Distance between the LR rotations increases under suppression of the Wnt/PCP pathway.

(A, C) Immunofluorescent staining images of control- and ΔDEP-GFP-misexpressing PSs at HH3 with GFP (green) and DAPI (cyan). (B, D) Immunostaining images of epiblast and PS cells in control- and ΔDEP-GFP-misexpressing embryos at HH3 with GFP (green), Prickle1 (Pk1; magenta), and DAPI (blue). (E, G) Averaged bilateral vortex-like-rotating cell flows (6 hours), visualized by vorticity plot, in control- and ΔDEP-GFP-misexpressing embryos. Black lines, measurement of the distance between the left-right (LR) rotations. Blue, clockwise; red, counter-clockwise rotation. (F, H) Averaged bilateral vortex-like-rotating cell flows (6 hours), visualized by streamline plot, in control- and ΔDEP-GFP-misexpressing embryos. (I) Distance between the LR rotations in control- and ΔDEP-GFP-misexpressing embryos [control-GFP 860.2±86.2 μm vs ΔDEP-GFP 1008.4±183.8μm, p=0.026, n=4 for each (two-tailed Student’s t test)].

PS extension dose-dependently responds to aphidicolin.

(A) Dose-dependency of aphidicolin-induced mitotic arrest (PS, control 98.8±1.3% vs 10uM aphidicolin 10.3±4.3% vs 100uM aphidicolin 0±0%, p=0.002e-4 in one-way ANOVA; embryo, control 87.9±9.0% vs 10uM aphidicolin 9.0±4.4% vs 100uM aphidicolin 0.02±0.01%; n=4 for each; p=0.003e-5 in one-way ANOVA). (B-C) In situ hybridization for Brachyury (Bra) to different concentrations of aphidicolin treatment. (D) Length of PS at HH3 for different concentrations of aphidicolin treatment (control 1.3±0.2mm vs 0.1μM aphidicolin 1.3±0.3mm vs 1μM aphidicolin 0.8±0.4mm vs 10μM aphidicolin 0.3±0.1mm vs 100μM aphidicolin 0.1±0.1mm; n=6 for each, p=6.2e-9 in one way ANOVA).

Aphidicolin-treatment maintains apoptotic index and pMyosin cables.

(A-B) TUNEL staining at stage HH 3 in control (A) and aphidicolin-treated embryos (B). (C) Apoptotic index in control and aphidicolin-treated embryos (control 10.6±3.5% vs aphidicolin 12.1±3.7%, p=0.20, n=4 for each). (D-F) Immunofluorescent staining images of control- and aphidicolin-treated embryos at HH3 with ZO-1 (magenta) and pMyosin (cyan). White boxes show enlarged areas.

Time evolution of cell flow in control-GFP/COS-implanted embryo.

(A) Trace of cell flow path of electroplated epiblast cells by using Imaris tracking analysis. See also Movie S3. A-P and L-R; anterior-posterior and left-right body axes, respectively. White circles, COS cell implanted site. (B) Vector fields, generated from the images of control-GFP/COS-implanted embryos shown in (A). (C) Streamlines, visualizing averaged cell flows during each time period of the vector fields shown in (B). (D) Vorticity plots, displaying averaged measure of the local rotation during each time period in the vector fields shown in (B). Blue, clockwise; red, counter-clockwise rotation.

Time evolution of cell flow in Vg1/COS-implanted embryo.

(A) Trace of cell flow path of electroplated epiblast cells by using Imaris tracking analysis. See also Movie S3. A-P and L-R; anterior-posterior and left-right body axes, respectively. White circles, COS cell implanted site. (B) Vector fields, generated from the images of control-GFP/COS-implanted embryos shown in (A). (C) Streamlines, visualizing averaged cell flows during each time period of the vector fields. (D) Vorticity plots, displaying averaged measure of the local rotation during each time period. Blue, clockwise; red, counter-clockwise rotation. White asterisks, the polonaise movements at the induced axis which are opposite direction to the authentic axis.

Time evolution of cell flow in control-GFP/COS-implanted embryo under aphidicolin-treatment.

(A) Trace of cell flow path of electroplated epiblast cells by using Imaris tracking analysis. See also Movie S5. A-P and L-R; anterior-posterior and left-right body axes, respectively. White circles, COS cell implanted site. White circles, COS cell implanted site.

(B) Vector fields, generated from the images of control-GFP/COS-implanted embryos shown in (A).

(C) Streamlines, visualizing averaged cell flows during each time period of the vector fields.

(D) Vorticity plots, displaying averaged measure of the local rotation during each time period. Blue, clockwise; red, counter-clockwise rotation.

Time evolution of cell flow in Vg1/COS-implanted embryo under aphidicolin-treatment.

(A) Trace of cell flow path of electroplated epiblast cells by using Imaris tracking analysis. See also Movie S5. Axes are shown in Fig S4. White circles, COS cell implanted site. (B) Vector fields, generated from the images of control-GFP/COS-implanted embryos shown in (A). (C) Streamlines, visualizing averaged cell flows during each time period of the vector fields. (D) Vorticity plots, displaying averaged measure of the local rotation during each time period. Blue, clockwise; red, counter-clockwise rotation. White asterisks, the polonaise movements at the induced axis which are opposite direction to the authentic axis.