Figure 2.
Nuclear deformation and dispersion during tissue extension. (A) Still images from a time-lapse movie of embryo expressing nuclear (NLS: RFP) and cell outline marker (Spider:GFP) showing nuclear deformation as GBE proceeds (related to Video 2). Nuclei are color-coded according to their major-to-minor axis ratio in the XY plane. Image slice from ∼10 µm below the apical surface. (B) Nuclear shape changes represented by ratio of major to minor axis at the nuclear midplane from pre- to mid-GBE (−10 to +20 min); n = 605 nuclei from k = 5 embryos. (C) Still frames showing nuclear dispersion indicated by disappearance of nuclei from the given imaging plane (marked by arrows) during a T1 cell intercalation event (related to Video 3). Image slice from ∼8 µm below the apical surface. (D) Cells with their nuclei in apical–basal axis at −5 min (top), 10 min (center), and 20 min (bottom) of GBE onset showing nuclei undergo dispersion relative to cells as GBE proceeds. (E) Heatmap showing wide distribution of nuclei in apical–basal axis (absolute midplane position) as GBE progresses. Color bar indicates the probability of nuclear midplane position; n = 606 nuclei from k = 3 embryos. (F) Probability distribution of nuclei along apical–basal axis relative to their neighboring nuclei in pre- (−5 to 0 min, blue), early (0–10 min, yellow), and mid- (15–20 min, red) GBE; n = 16225 pairs of nuclei from k = 3 embryos. (G) Mean nuclear velocities at pre- (−10 to 0 min) and mid- (15–20 min) GBE, n = 213 nuclei for pre- and n = 512 for mid-GBE from k = 3. (H) MSD (pre = −5 to 0 min, early = 0–10 min, and mid = 15–20 min); n = 16,225 pairs of nuclei from k = 3 embryos. (I) Maximum packing ratio achieved by clusters of nuclei as measured by the dispersion metric (see Materials and methods) at pre- (−5 min) and mid- (+20 min) GBE; n = 152 clusters of cells from k = 3 embryos. Schematics on the top right exemplifies ratio of 1.0 when nuclei (blue) are in the same apical–basal plane (red dotted line) and the ratio decreases as nuclei spread across different planes (bottom right). (J) Average cytoplasmic area (ACA) ratio available in cell clusters (schematics depict cytoplasmic areas in blue and nuclei areas in black) at the level of the nuclear midplane of a given central cell during GBE (left) and box and whisker plot showing significance if ACA metric at 5 min before (pre) and 20 min (mid) after the onset of GBE (right); n = 206 clusters of cells from k = 3 embryos. For B and J, light blue background indicates GBE. Error envelopes indicate SD. For A and C, scale bar = 5 µm and for D, scale bar = 10 μm. The measured n values are regardless of timepoints where not indicated. For I, statistical significance was calculated using Student’s t test. For G and J, statistical significance was calculated using the Mann–Whitney U-test. **P < 0.01, ****P < 0.0001.

Nuclear deformation and dispersion during tissue extension. (A) Still images from a time-lapse movie of embryo expressing nuclear (NLS: RFP) and cell outline marker (Spider:GFP) showing nuclear deformation as GBE proceeds (related to Video 2). Nuclei are color-coded according to their major-to-minor axis ratio in the XY plane. Image slice from ∼10 µm below the apical surface. (B) Nuclear shape changes represented by ratio of major to minor axis at the nuclear midplane from pre- to mid-GBE (−10 to +20 min); n = 605 nuclei from k = 5 embryos. (C) Still frames showing nuclear dispersion indicated by disappearance of nuclei from the given imaging plane (marked by arrows) during a T1 cell intercalation event (related to Video 3). Image slice from ∼8 µm below the apical surface. (D) Cells with their nuclei in apical–basal axis at −5 min (top), 10 min (center), and 20 min (bottom) of GBE onset showing nuclei undergo dispersion relative to cells as GBE proceeds. (E) Heatmap showing wide distribution of nuclei in apical–basal axis (absolute midplane position) as GBE progresses. Color bar indicates the probability of nuclear midplane position; n = 606 nuclei from k = 3 embryos. (F) Probability distribution of nuclei along apical–basal axis relative to their neighboring nuclei in pre- (−5 to 0 min, blue), early (0–10 min, yellow), and mid- (15–20 min, red) GBE; n = 16225 pairs of nuclei from k = 3 embryos. (G) Mean nuclear velocities at pre- (−10 to 0 min) and mid- (15–20 min) GBE, n = 213 nuclei for pre- and n = 512 for mid-GBE from k = 3. (H) MSD (pre = −5 to 0 min, early = 0–10 min, and mid = 15–20 min); n = 16,225 pairs of nuclei from k = 3 embryos. (I) Maximum packing ratio achieved by clusters of nuclei as measured by the dispersion metric (see Materials and methods) at pre- (−5 min) and mid- (+20 min) GBE; n = 152 clusters of cells from k = 3 embryos. Schematics on the top right exemplifies ratio of 1.0 when nuclei (blue) are in the same apical–basal plane (red dotted line) and the ratio decreases as nuclei spread across different planes (bottom right). (J) Average cytoplasmic area (ACA) ratio available in cell clusters (schematics depict cytoplasmic areas in blue and nuclei areas in black) at the level of the nuclear midplane of a given central cell during GBE (left) and box and whisker plot showing significance if ACA metric at 5 min before (pre) and 20 min (mid) after the onset of GBE (right); n = 206 clusters of cells from k = 3 embryos. For B and J, light blue background indicates GBE. Error envelopes indicate SD. For A and C, scale bar = 5 µm and for D, scale bar = 10 μm. The measured n values are regardless of timepoints where not indicated. For I, statistical significance was calculated using Student’s t test. For G and J, statistical significance was calculated using the Mann–Whitney U-test. **P < 0.01, ****P < 0.0001.

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