Cell fragmentation in mouse preimplantation embryos induced by ectopic activation of the polar body extrusion pathway

Abstract

Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

Keywords: cytoskeleton; meiosis; mitosis; morphogenesis; preimplantation development.

Figure 1. Myo1cKO embryos form fragments

1. A

   Representative image of a Control (left) and Myo1cKO (right) embryo. Top: max projections of embryos stained with Phalloidin (green) and DAPI (gray). Bottom: magnifications at dashed rectangles of Phalloidin (gray) with white arrows pointing at fragments.

2. B, C

   Number of fragments without DNA (B) per embryo and mean radius (C) in Control (gray) and Myo1cKO (salmon) embryos (left, Control n = 20, Myo1cKO n = 20). Boxplot shows median, upper and lower quartiles, min and max values. Whisker plot shows median, upper and lower quartiles.

3. D

   Representative images of a time‐lapse of a Myo1cKO embryo undergoing fragmentation outside of division (top) and during division (bottom). Max projections of embryos expressing mTmG (gray) and mCherry‐EB3 (gray) imaged with light sheet microscopy are shown. White arrows point at the forming fragments.

4. E

   Ratio of fragmentation events during and outside of division (n = 13 cells from 7 embryos).

Data information: Mann–Whitney test P‐values are indicated. Scale bars, 10 μm.

Figure EV1. Characterization of Myo1cKO embryos development

1. A

   Mouse single cell RNA sequencing analysis adapted from Deng et al (2014), showing the expression levels of members for the Myosin 1 family at the zygote, 2‐, 4‐, 8‐, 16‐cell, early and late blastocyst stages. Data show mean ± SD of cells at the zygote (n = 4), 2‐ (n = 8), 4‐ (n = 13), 8‐ (n = 19), 16‐cell (n = 57), early (n = 43), and late (n = 28) blastocyst stages.

2. B–E

   Number of fragments without DNA as a function of the number of cells (B), percentage of fragments containing DNA per embryo (C), percentage of fragments located at the embryo apical surface (D), cell number per embryo (E) in Control (gray) and Myo1cKO (salmon) embryos at E3.5 (Control n = 20, Myo1cKO n = 20) and E4.5 (Control n = 13, Myo1cKO n = 28).

3. F

   Mean contact angle at maximal compaction (Control n = 11, Myo1cKO n = 39).

4. G

   Representative image of surface tension measurement.

5. H

   Surface tension of control (gray, n = 6 embryos, 27 contacts) and Myo1cKO (salmon, n = 6 embryos, 53 contacts) blastomeres as a function of their contact angle. Representative image of tension measurement on a control 8‐cell stage embryo.

6. I

   Representative images of Control (left) and Myo1cKO (right) embryos at E5.5 stained for Pard6b (yellow), Aqp3 (magenta) and DAPI (gray).

7. J

   Representative images of Control (left) and Myo1cKO (right) embryos at E5.5 stained for Cdx2 (cyan) Sox2 (red) and Phalloidin (gray).

8. K

   Sox2 nuclear to cytoplasmic (N/C) ratio as a function of Cdx2 N/C ratio of individual cells in Control (gray, n = 7) and Myo1cKO (salmon, n = 11) embryos.

9. L

   Cdx2 N/C ratio of cells located at the surface (left) or inside (right) the embryo in Control (n = 8) and Myo1cKO (n = 21) embryos.

10. M

    Sox2 N/C ratio of cells located at the surface (left) or inside (right) the embryo in Control (n = 11) and Myo1cKO (n = 16) embryos.

Data information: the Mann–Whitney test (F) or the Kruskal–Wallis test (C, D, E, L, M) P‐values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 μm.

Figure 2. Defective spindle anchoring leads to fragmentation

1. A

   Representative images of Control (left) and Myo1cKO (right) embryos expressing mTmG and mCherry–EB3 (gray) shown as max projections. White arrows indicate the mitotic spindle.

2. B, C

   Spindle persistence in Control (n = 6) and Myo1cKO (n = 9) embryos (B) and in non‐fragmenting and fragmenting cells of Myo1cKO embryos (C, n = 42 and 12 cells from 7 Myo1cKO embryos). Spindle persistence measures the time from appearance to disassembly of the spindle.

3. D, E

   Spindle straightness (D) and rotation (E) in Control (n = 6) and Myo1cKO (n = 9) embryos. Straightness reports the pole to pole distance of the spindle divided by its continuous length. Rotation describes the angle between the initial orientation of the spindle pole and its maximal deviation in 2D.

4. F

   Representative images of embryos treated with DMSO (left) and 37.5 μM Ciliobrevin D (right), stained with Phalloidin. Top: Maximum projection. Bottom: magnifications of a single plane at dashed rectangles of Phalloidin with white arrows pointing at fragments.

5. G

   Number of fragments without DNA per embryo in DMSO (gray) and Ciliobrevin D (honeydew) embryos at E2.5 (left, Control n = 9, Myo1cKO n = 7).

Data information: the Mann–Whitney test P‐values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 μm.

Figure EV2. Mitotic spindle movement in Ciliobrevin D treated embryos

1. A

   Representative images of DMSO (left) and Ciliobrevin D (right) treated embryos expressing mTmG and mCherry–EB3 (gray) shown as max projections. White arrows indicate the mitotic spindle. Scale bar, 10 μm.

2. B–D

   Spindle persistence (B), straightness (C) and rotation (D) in DMSO (n = 11) and Ciliobrevin D (n = 11) treated embryos. Persistence measures the time from appearance to disassembly of the spindle. Straightness reports the pole to pole distance of the spindle divided by its continuous length. Rotation describes the angle between the initial orientation of the spindle pole and its maximal deviation in 2D.

Data information: the Mann–Whitney test P‐values are indicated. Boxplots show median, upper and lower quartiles, min and max values.

Figure 3. Actomyosin cap formation in proximity of DNA

1. A

   Top: Representative images of a time‐lapse of a Myo1cKO embryo labeled with LifeAct–GFP (green) and SiRDNA (gray) with one blastomere undergoing mitosis shown as max projections. Bottom: magnifications of dashed rectangles.

2. B

   In Myo1cKO embryo shown in (A) LifeAct–GFP intensities at the cortex region of eventual contact with DNA (I_c) are normalized to the intensity at a reference region (I_ref). LifeAct–GFP I_c/I_ref values (green) are plotted over time. The DNA‐cortex distance (gray) between the region of eventual contact and the center of the DNA signal is also measured. A threshold distance of 20 μm is indicated with a dashed line.

3. C

   LifeAct‐GFP I_c/I_ref values at DNA‐cortex distances < 20 and > 20 μm (Myo1cKO embryos n = 13, cells n = 21).

4. D

   Top: Representative images of a time‐lapse of a Myo1cKO embryo expressing Myh9–GFP (green) and mTmG (magenta) while labeled with SiRDNA (gray) undergoing fragmentation during mitosis shown as max projections. Bottom: magnifications of Myh9‐GFP (gray) at dashed rectangles showing cortical accumulation (middle) followed by fragmentation (right).

5. E

   Control (gray) and Myo1cKO (salmon) embryos Myh9–GFP I_c/I_ref values at DNA‐cortex distances < 20 and > 20 μm (Control embryos n = 10, cells n = 35; Myo1cKO embryos n = 9, cells n = 26).

6. F

   Comparison of the increase in Myh9–GFP intensity in Control and Myo1cKO embryos when DNA is in close proximity to the cortex. The ratio I_c0‐20/I_c > 20 is calculated from Myh9–GFP intensities at the contact region when DNA‐cortex distances are < 20 μm (I_c0‐20) divided by intensities when distances are > 20 μm (I_c > 20; Control embryos n = 10, cells n = 33; Myo1cKO embryos n = 9, cells n = 26).

Data information: Mann–Whitney test (C, F) or Kruskal–Wallis and pairwise the Mann–Whitney tests (E) P‐values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 μm.

Figure EV3. Characterization of chromosome movements and nuclear envelope dynamics during mitosis of Myo1cKO embryos

1. A, B

   Measured distance between the DNA visualized with SiRDNA or H2B‐mCherry and the cell cortex (A) and standard deviation of this distance throughout division (B) for Control (n = 10) and Myo1cKO embryos (n = 9).

2. C

   Representative images of Control (left) and Myo1cKO (right) embryos expressing mTmG (magenta) and Lap2b–GFP (cyan).

3. D

   Comparison of the time from nuclear envelope breakdown to its reassembly between Control (n = 8) and Myo1cKO embryos (n = 15).

Data information: Mann–Whitney test (A) or Student's t‐test (B and D) P values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bar, 10 μm.

Figure 4. The polar body extrusion (PBE) pathway is necessary and sufficient to induce fragmentation during cleavage stages

1. Representative images of Control (left) and Myo1cKO (right) embryos labeled with SiRDNA (gray) and expressing a reporter of Cdc42 activity (yellow) in one blastomere, which is undergoing mitosis, shown as max projections. The second blastomere is outlined with a dashed line. White arrows point at the accumulation of active Cdc42 at the cortex in close proximity to DNA.

2. The ratio of active Cdc42 I_c/I_ref was compared between < 20 and 20 μm DNA‐cortex distances for Control (dark yellow) and Myo1cKO (yellow) embryos (Control embryos n = 10; Myo1cKO embryos n = 10).

3. Representative images of Myo1cKO embryos expressing LifeAct–GFP (green) alone (left) or together with dominant negative Cdc42 (DNCdc42, right) labeled with SiRDNA (gray) shown as max projections. White arrows point at the cortex in close proximity to DNA.

4. LifeAct–GFP I_c/I_ref values at DNA‐cortex distances < 20 and > 20 μm for Myo1cKO (salmon) and Myo1cKO + DNCdc42 (yellow) embryos (Myo1cKO embryos n = 8, cells n = 19; Myo1cKO + DNCdc42 embryos n = 12, cells n = 30).

5. Representative images of a GFP (left) and Ect2–GFP (right) expressing embryo stained with Phalloidin (green) and DAPI (gray) shown as max projections. White arrows indicate fragments.

6. Number of fragments without DNA per embryo in GFP (gray) and Ect2–GFP (blue) embryos (GFP n = 14; Ect2–GFP n = 18).

Data information: The Kruskal–Wallis and pairwise Mann–Whitney tests (B, D) or Mann–Whitney test (F) P‐values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 μm.

Figure EV4. Characterization of Ect2–GFP expressing embryos

1. Representative images of GFP (top, blue) and Ect2–GFP (bottom, blue) expressing embryos during mitosis labeled with LifeAct‐RFP (green) and SiRDNA (white) shown as max projections. Scale bar, 10 μm.

2. LifeAct–RFP I_c/I_ref ratio compared between DNA‐cortex distances < 20 and > 20 μm for GFP (gray) and Ect2–GFP (blue) embryos (GFP embryos n = 7, cells n = 26; Ect2‐GFP embryos n = 9, cells n = 26).

3. Surface tension of 4‐cell stage embryos in Control (gray) and Ect2–GFP (blue) embryos (Control n = 8; Ect2–GFP n = 12).

Data information: the Kruskal–Wallis and pairwise Mann–Whitney tests (B) or unpaired Student's t‐test (C) P‐values are indicated. Boxplots show median, upper and lower quartiles, min and max values.

Figure 5. Schematic diagram of the steps leading to cell fragmentation in preimplantation embryos

Spindle in red, DNA in white, signals from the DNA in yellow and actomyosin in green.