Pulsatile cell-autonomous contractility drives compaction in the mouse embryo

Journal name:
Nature Cell Biology
Volume:
17,
Pages:
849–855
Year published:
DOI:
doi:10.1038/ncb3185
Received
Accepted
Published online
Corrected online

Abstract

Mammalian embryos initiate morphogenesis with compaction, which is essential for specifying the first lineages of the blastocyst. The 8-cell-stage mouse embryo compacts by enlarging its cell–cell contacts in a Cdh1-dependent manner. It was therefore proposed that Cdh1 adhesion molecules generate the forces driving compaction. Using micropipette aspiration to map all tensions in a developing embryo, we show that compaction is primarily driven by a twofold increase in tension at the cell–medium interface. We show that the principal force generator of compaction is the actomyosin cortex, which gives rise to pulsed contractions starting at the 8-cell stage. Remarkably, contractions emerge as periodic cortical waves when cells are disengaged from adhesive contacts. In line with this, tension mapping of mzCdh1/ embryos suggests that Cdh1 acts by redirecting contractility away from cell–cell contacts. Our study provides a framework to understand early mammalian embryogenesis and original perspectives on evolutionary conserved pulsed contractions.

At a glance

Figures

  1. Spatiotemporal map of tensions during compaction.
    Figure 1: Spatiotemporal map of tensions during compaction.

    (a) Diagram of the tension mapping method. Using a micropipette of radius Rp, a pressure Pc is applied to the surface of blastomeres of curvature 1/Rc. The surface tension γcm is calculated using the Young–Laplace equation. From γcm, the external and internal contact angles (θe and θi, respectively) of adjacent cells, we use the Young–Dupré equation to calculate the interfacial tension γcc and the compaction parameter α. (b) Representative images of tension mapping experiments. Time post-division (hh:mm); scale bar, 10 μm (Supplementary Video 2). (c) Surface tension γcm as a function of contact angle θi measured on n = 466 blastomeres pooled from 28 embryos representative of 11 independent experiments. Pearson correlation R = −0.517 (P < 0.001). (df) Time course of contact angles θi and θe (d), tensions γcm and γcc/2 (e) and compaction parameter α (f). Mean ± s.d. over 2 h of n = 466 contacting blastomeres pooled from 28 embryos. Statistics source data are shown in Supplementary Table 1. Images in b are representative of 11 independent experiments.

  2. Role of cell-cell contacts in regulating tensions during compaction.
    Figure 2: Role of cell–cell contacts in regulating tensions during compaction.

    (ac) Representative images of tension mapping experiment on a whole embryo (a), 2/8th (b) or 1/8th blastomeres (c). Time post-division (hh:mm); scale bars, 10 μm. (d) Surface tension γcm at 0–6 and 6–12 h post-division (hpd). Mean ± s.d. surface tension of blastomeres from whole 8-cell-stage embryos (n = 40 and 46 cells pooled from 4 embryos), 2/8th (n = 32 and 28 cells pooled from 4 embryos) or 1/8th (n = 23 and 24 cells pooled from 4 embryos) representative of 3 independent experiments. P values result from Students t-test; NS: not significant (P = 0.7 when comparing means between 8-cell and 1/8th or 2/8th and 1/8th at 0–6 hpd, and P = 0.1 and P = 0.5 at 6–12 hpd). Statistics source data are shown in Supplementary Table 1. e, Surface tension γcm as a function of contact angle θi. Contacting blastomeres from whole 8-cell embryos (n = 86 cells pooled from 4 embryos) and 2/8th doublets (n = 60 cells pooled from 4 embryos). WT from Fig. 1c in grey. Pearson correlation R = −0.645 for 8-cell and −0.478 for 2/8th (P < 0.001). Images in ac are each representative of 3 independent experiments.

  3. Role of Cdh1 in regulating tensions during compaction.
    Figure 3: Role of Cdh1 in regulating tensions during compaction.

    (a,b) Representative images of tension mapping experiment on mCdh1+/ (a) or mzCdh1/ embryos (b). Time post-division (hh:mm); scale bars, 10 μm. (c) Surface tension γcm at 0–6 and 6–12 h post-division (hpd). Mean ± s.d. of contacting blastomeres from mCdh1+/ (n = 54 and 78 cells pooled from 7 embryos) and mzCdh1/ (n = 41 and 54 cells pooled from 5 embryos) embryos representative of 5 independent experiments. P value results from Students t-test; NS: not significant (P = 0.8 for 0–6 hpd and 0.2 for 6–12 hpd). Statistics source data are shown in Supplementary Table 1. (d) Surface tension γcm as a function of contact angle θi. Contacting blastomeres from mCdh1+/ (n = 264 cells pooled from 7 embryos) and mzCdh1/ (n = 190 cells pooled from 5 embryos) embryos. WT from Fig. 1c in grey. Pearson correlation R = −0.377 for mCdh1+/ and −0.230 for mzCdh1/ embryos (P < 0.001 and 0.01 respectively). (e) Interfacial tension γcc/2 at 0–6 and 6–12 hpd. Mean ± s.d. of contacting blastomeres from mCdh1+/ (n = 54 and 78 cells pooled from 7 embryos) and mzCdh1/ (n = 41 and 54 cells pooled from 5 embryos) embryos. P values result from Students t-test; NS: not significant (P = 0.5). Images in a and b are each representative of 3 independent experiments.

  4. Role of contractility in generating tensions during compaction.
    Figure 4: Role of contractility in generating tensions during compaction.

    (a) Time-lapse of LifeAct–GFP-expressing embryo. Time post-division (hh:mm); scale bar, 10 μm (Supplementary Video 3). (b) Immunostaining of double-phosphorylated myosin regulatory light chain (ppMRLC) early and late during the 8-cell stage. Scale bar, 10 μm. (c) Contact/cortex ratio of LifeAct–GFP over time. Mean ± s.d. of n = 23 contacts pooled from 8 embryos followed every 30 min for 5–8 h. Top-right inset describes how the contact/cortex ratio is calculated from the intensities measured at the cell–medium Icm1/2 and cell–cell Icc interface. (d) Contact/cortex ratio as a function of contact angle θe. Mean ± s.d. of n = 120 contacts pooled from 29 embryos stained for ppMRLC and n = 23 contacts pooled from 8 LifeAct–GFP-expressing embryos representative of 3 independent experiments each. Pearson correlation R = −0.825 and −0.599 (P < 0.001) for LifeAct–GFP and ppMRLC respectively. (e,f) Representative images of embryos treated with 25 μM blebbistatin (Bb) (+) (e) or Bb (−) (f). Scale bars, 10 μm. (g) Surface tension γcm as a function of contact angle θi. Contacting blastomeres of Bb (+)- (n = 164 cells pooled from 5 embryos) or Bb (−)- (n = 242 cells pooled from 7 embryos) treated embryos representative of 4 independent experiments. WT from Fig. 1c in grey. Pearson correlation R = −0.656 for Bb (+) and −0.172 for Bb (−) (P < 0.01 and 0.05 respectively). (h,i) Tension γcm (h) and γcc/2 (i) of Bb (+)- or Bb (−)-treated embryos at 0–6 and 6–12 h post-division. Mean ± s.d. of contacting blastomeres from Bb (+)-treated embryos (n = 62 and 102 cells pooled from 5 embryos) and Bb (−)-treated embryos (n = 76 and 166 pooled from 7 embryos). P values result from Students t-test. Statistics source data are shown in Supplementary Table 1. Images in a, b, e and f are representative of 3, 3, 4 and 4 independent experiments, respectively.

  5. Non-cell-autonomous control of contractility by Cdh1.
    Figure 5: Non-cell-autonomous control of contractility by Cdh1.

    (a) Immunostaining of ppMRLC of mCdh1+/ and mzCdh1/ embryos. Red arrowheads point at cell–medium interfaces, green ones at cell–cell contacts. Scale bars, 10 μm. (b) Contact/cortex ratio as a function of contact angle θe. Mean ± s.d. of n = 46, 40 and 120 contacts pooled from, respectively, 11 mCdh1+/ (blue), 14 mzCdh1/ (green) and 29 WT (grey, from Fig. 4d) embryos stained for ppMRLC. Pearson correlation R = −0.610 (P < 0.005) for mCdh1+/ and mzCdh1/ embryos representative of 3 independent experiments. (c) Tension γcm and γcc/2 of mzCdh1/ embryos treated with Bb (+) or Bb (−). Mean ± s.d. of n = 27 and 33 contacting blastomeres pooled from 4 and 5 embryos for Bb (+) and (−) respectively from 2 representative experiments. P values result from Students t-test. Images in a are representative of 3 independent experiments.

  6. Phase diagram of compaction.
    Figure 6: Phase diagram of compaction.

    Phase diagram showing the compaction parameter α as a function of γcc/2 and γcm. Means ± s.d. of WT, mzCdh1/ and Bb (−) embryos are shown as arrows starting at 0–2 and ending at 8–10 h post-division. The compaction parameter α = γcc/2γcm is colour-coded on the right, with diagrams of the corresponding doublet shapes to the side.

  7. Periodic contractions.
    Figure 7: Periodic contractions.

    (a) Particle image velocimetry (PIV)-tracking of an 8-cell embryo (Supplementary Video 4). Velocity is colour-coded, green for positive y-velocity or red for negative. Scale bars, 10 μm. (b) PIV tracking y-velocity over time of a vector from Supplementary Video 4 . (c) PIV tracking amplitude as a function of the oscillation period for control (n = 23) and Bb (−) (n = 19) embryos representative of 6 and 3 independent experiments, respectively. Mean ± s.d. (d) Time-lapse of an en face optical section through the cortex of a LifeAct–GFP-expressing embryo (Supplementary Video 6). The red circle indicates the measured region of interest. Scale bar, 5 μm. (e) LifeAct–GFP intensity over time for 4 blastomeres from 4 embryos representative of 2 independent experiments (embryo shown in d and Supplementary Video 6 in red). Images in a are representative of 6 independent experiments, images in d are representative of 2.

  8. Periodic cortical waves.
    Figure 8: Periodic cortical waves.

    (ad) Time-lapse of a mTmG- (b) and LifeAct–GFP- (c) expressing 1/8th blastomere with bright-field shown in a and the local curvature overlaid on mTmG images in d. Scale bars, 10 μm. Supplementary Video 11 . (e) Kymographs of curvature (top) and cortical LifeAct–GFP intensity (bottom) from Supplementary Video 11 . Images in ad are each representative of 3 independent experiments.

  9. Blastomeres volume during the 8-cell stage.
    Supplementary Fig. 1: Blastomeres volume during the 8-cell stage.

    a, Images of cell volume reconstruction showing 6 cells of an 8-cell stage embryo expressing mTmG (grey) artificially labelled in distinct colours using Bitplane Imaris. Scale bar, 10 μm. b, Volume of blastomeres at the beginning (30 min after the last 4- to 8-cell stage division) and end (30 min before rounding up of the first 8- to 16-cell stage division) of the 8-cell stage. Individual examples from (a) are shown in colour while the whisker plot in black shows the minimum and maximum values, and upper and lower quartiles of n = 32 blastomeres pooled from 4 embryos from 1 representative experiment. p value indicates the result of Students t-test.

  10. Theoretical modelling of surface energy minimization during compaction of 2/8th doublets.
    Supplementary Fig. 2: Theoretical modelling of surface energy minimization during compaction of 2/8th doublets.

    a, Diagram of two blastomeres from 8-cell stage mouse embryo forming a 2/8th doublet shaped by surface tension γcm at the cell–medium interface and interfacial tension γcc at the cell–cell interface. The shape of each blastomere is characterized by a radius of curvature Rc, an area of cell–medium interface A and a volume V0. The shape of the cell–cell contact is characterized by its radius r and contact angle θe. The 2/8th doublet is symmetric with respect to a rotational axis Φ and to the cell–cell contact. b, Rescaled energy E/E0 as a function of the contact angle θe/2 for different values of the dimensionless parameter 0 ≤ α = γcc/2γcm ≤ 1. The minimum of the surface energy, overlaid as a diagram, corresponds to the mechanical equilibrium α = cos(θe/2) for the 2/8th doublet.

  11. Natural variation in shape and tensions during compaction.
    Supplementary Fig. 3: Natural variation in shape and tensions during compaction.

    ad, Mean ± s.d. are shown for the blastomeres and contacts of 3 individual embryos. Embryo shown in Fig. 1b is shown in red. a, Radius of curvature Rc over time. b, Contact angle θe over time. c, Surface tension γcm over time. d, Interfacial tension γcc/2 over time. e, Plot of each surface tension γcm of each contacting blastomeres against one another. N = 233 contacting blastomeres pooled from 28 embryos from 11 representative experiments, Pearson correlation R = 0.778 (p < 0.001). f, Plot of each contact angle θi of each contacting blastomeres against one another. N = 233 contacting blastomeres pooled from 28 embryos, Pearson correlation R = 0.603 (p < 0.001).

  12. Surface tension at the 4-cell stage.
    Supplementary Fig. 4: Surface tension at the 4-cell stage.

    a, Surface tension measurements of an early (top) and late (bottom) 4-cell stage embryo. bc, Contact angles θi and θe (b) and tensions γcm and γcc/2 (c) of n = 42 and 46 contacting blastomeres pooled from 6 embryos early and late in the 4-cell stage from 2 representative experiments. d, Surface tension measurements (n = 88)γcm of a 4-cell stage embryo as a function of the contact angle θi in blue, together with 8-cell stage measurements shown in Fig. 1c in grey. p value indicates the result of Students t-test.

  13. Role of contractility in generating tensions during compaction.
    Supplementary Fig. 5: Role of contractility in generating tensions during compaction.

    a, Immunostaining of Myh9 early and late in the 8-cell stage. Red arrowheads point at cell–medium interfaces, green ones at cell–cell contacts. Scale bar, 10 μm. b, Mean ± s.d. contact/cortex ratio, n = 194 contacting blastomeres pooled from 22 embryos from 2 representative experiments, Pearson correlation R = −0.538 (p < 0.001). c, Phalloidin staining of F-actin early and late in the 8-cell stage. Red arrowheads point at cell–medium interfaces, green ones at cell–cell contacts. d, Mean ± s.d. contact/cortex ratio, 113 embryos from 9 representative experiments, Pearson correlation R = −0.752 (p < 0.001). e, Time-lapse of mTmG expressing embryo. Time post-division (hh:mm), Supplementary Video 3 . f, Contact/cortex ratio of mTmG and LifeAct-GFP over time. Mean ± s.d. of n = 23 contacts pooled from 8 embryos from 3 representative experiments followed every 30 min for 5–8 h. g, Surface tension measurement of DMSO or 1 μM CCD treated embryos. Scale bar, 10 μm. h, Surface tension γcm as a function of the contact angle θi of n = 10 and 42 contacting blastomeres pooled from 2 and 3 embryos treated with DMSO (black) or CCD (yellow) respectively from 1 representative experiment. Pearson correlation R = −0.028 for DMSO and −0.005 for CCD with (p > 0.1). WT (grey) shown in Fig. 1c.

  14. Examples of PIV and Fourier analysis of periodic contractions in different conditions.
    Supplementary Fig. 6: Examples of PIV and Fourier analysis of periodic contractions in different conditions.

    af, Four examples of PIV and Fourier analysis are shown for 8-cell stage embryos (a, black), 4-cell stage embryos (b, blue), 8-cell stage embryos without zona pellucida (c, no ZP, purple), 8-cell stage embryos treated with 25 μM Bb (−) (d, orange) or 1 μM CCD (e, yellow), and mzCdh1/ 8-cell stage embryos (f, green) are shown. The left column shows the velocity of one vector in one direction over time. The middle column shows the corresponding power spectrum. The right column shows the amplitude as a function of the period.

  15. Periodic contractions in different conditions.
    Supplementary Fig. 7: Periodic contractions in different conditions.

    ad, Mean ± s.d. amplitude of periodic contractions as a function of the oscillation period for control 8-cell stage embryos (ad, in black, n = 23 from 6 representative experiments), 1 μM CCD-treated 8-cell stage embryos (a, in yellow, n = 14 from 2 representative experiments), 4-cell stage embryos (b, in blue, n = 8 from 2 representative experiments), 8-cell stage embryos without zona pellucida (c, No ZP, in purple, n = 6 from 2 representative experiments). mzCdh1/ 8-cell stage embryos (d, in green, n = 7 from 3 representative experiments). e, After Fourier analysis, the mean power spectra are analysed for the presence of a distinct peak of amplitude at a specific frequency. Distinct peaks could be observed in 16 out of n = 23 embryos at 8-cell stage, 1 out of n = 8 embryos at 4-cell stage, 0 out of n = 17 embryos at 8-cell stage with 25 μM Bb (−), 0 out of n = 14 embryos at 8-cell stage with 1 μM CCD, 6 out of n = 6 embryos at 8-cell stage without zona pellucida (ZP), 7 out of n = 7 mzCdh1/ embryos at 8-cell stage, 7 out of n = 7 blastomeres isolated at the 8-cell stage. f, Periods that could be extracted are plotted as mean ± s.d. for embryos at 8-cell stage after PIV analysis (n = 16) and LifeAct-GFP intensity changes (n = 4), n = 6 embryos at 8-cell stage without zona pellucida (ZP) after PIV analysis, n = 7 mzCdh1/ embryos at 8-cell stage after PIV analysis, n = 7 blastomeres isolated at the 8-cell stage after curvature analysis and LifeAct-GFP intensity changes.

  16. Examples of periodic cortical waves.
    Supplementary Fig. 8: Examples of periodic cortical waves.

    Three examples of 1/8th blastomeres showing periodic cortical waves. Left column shows the curvature computed from the segmented membrane signal. 2nd column shows LifeAct-GFP. 3rd column shows a kymograph of the curvature along the cell perimeter (y-axis, arbitrary unit) as a function of time (x-axis over 15 min). 4th column shows a kymograph of LifeAct-GFP signal along the cell perimeter (y-axis, arbitrary unit) as a function of time (x-axis over 15 min).

Tables

  1. Supplementary Table 1:

    a, mean ± s.d. of contact angles and surface tensions over time shown in Fig. 1c. b, mean ± s.d. of contact angles and surface tensions over time for whole 8-cell embryos, 2/8th doublets and 1/8th single blastomeres shown in Fig. 2e. c, mean ± s.d. of contact angles and surface tensions over time for mCdh1+/ and mzCdh1/ embryos shown in Fig. 3d. d, mean ± s.d. of contact angles and surface tensions over time for 25 μM Bb (+) or Bb (−) treated embryos shown in Fig. 4g.

Videos

  1. Pre-implantation development.
    Video 1: Pre-implantation development.
    Brightfield (BF) images of a developing zygote with mechanically removed ZP until blastocyst stage. At the 8-cell stage, compaction starts at 68:00 and ends at 76:00. Images are taken every 30 min and shown at 10 frames per second (fps). Time post-division (hh:mm), scale bar, 10 μm.
  2. Mapping of surface tension in space and time.
    Video 2: Mapping of surface tension in space and time.
    BF images of surface tension mapping of an embryo. Each blastomeres is measured 5 times throughout the 8-cell stage (from ~3:00 to ~10:30 post-division). A picture is taken for each pressure step when the deformation reached a stable shape and shown at 10 fps. Time post-division (hh:mm), scale bar, 10 μm.
  3. LifeAct-GFP and mTmG compaction.
    Video 3: LifeAct-GFP and mTmG compaction.
    Fluorescent confocal imaging of an mTmG (right) and LifeAct-GFP (left) expressing transgenic embryo throughout the 8-cell stage. Timelapse imaging consists of a picture taken every 30 min and is displayed at 5 fps. Time post-division (hh:mm), scale bar, 10 μm.
  4. PIV analysis of control 8-cell stage embryo.
    Video 4: PIV analysis of control 8-cell stage embryo.
    BF images of an 8-cell stage embryo undergoing pulsed contractions. Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  5. PIV analysis of 4-cell stage embryo.
    Video 5: PIV analysis of 4-cell stage embryo.
    BF images of a 4-cell stage embryo. Pictures are acquired every 2 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red and half of the images are shown to match Supplementary Videos 4, 710 display. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  6. LifeAct-GFP pulses.
    Video 6: LifeAct-GFP pulses.
    En face confocal slice of the cortex of an 8-cell stage embryo undergoing pulsed contractions. On the left, LifeAct-GFP shows patchy accumulation of signal at the cortex of cells. In the middle, mT shows membrane movements while the cell remains in focus. On the right, LifeAct-GFP (green) and mT (red) signals are merged. Timelapse imaging consists of a picture taken every 5 s and is displayed at 20 fps. Scale bar, 5 μm.
  7. PIV analysis of 8-cell stage embryo treated with 25 M Bb (-).
    Video 7: PIV analysis of 8-cell stage embryo treated with 25 μM Bb (-).
    BF images of an 8-cell stage embryo treated with 25 μM Bb (-). Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  8. PIV analysis of 8-cell stage embryo treated with 1 M CCD.
    Video 8: PIV analysis of 8-cell stage embryo treated with 1 μM CCD.
    BF images of an 8-cell stage embryo treated with 1 μM CCD. Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  9. PIV analysis of 8-cell stage embryo without zona pellucida
    Video 9: PIV analysis of 8-cell stage embryo without zona pellucida
    BF images of an 8-cell stage embryo without ZP. Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  10. PIV analysis of mzCdh1 embryo.
    Video 10: PIV analysis of mzCdh1/ embryo.
    BF images of an 8-cell stage mzCdh1/ embryo. Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  11. Periodic cortical waves.
    Video 11: Periodic cortical waves.
    Montage of BF (top left), mTmG (top right), LifeAct-GFP (bottom left) and local curvature measurement (bottom right) of a 1/8th blastomeres undergoing periodic cortical waves. Timelapse imaging consists of a picture taken every 5 s and is displayed at 10 fps. Scale bar, 10 μm.

Change history

Corrected online 05 July 2017
In the version of this Article originally published, the wave velocity reported in the sentence beginning 'In the absence of cell–cell contacts...' was incorrectly calculated and should have read '0.8 ± 0.1 μm s–1'. The wave velocity is calculated by dividing the cell perimeter by the oscillation period, and further dividing this by two as there are two waves travelling around the cell. The number of waves was not taken into account in the originally published version of the Article. This mistake does not affect the main conclusions of the paper, as this sentence only describes a property of the phenomenon identified. This error has been corrected in the online version of the Article.

References

  1. Wolpert, L. Principles of Development (Oxford Univ. Press, 2011).
  2. Stephenson, R. O., Yamanaka, Y. & Rossant, J. Disorganized epithelial polarity and excess trophectoderm cell fate in preimplantation embryos lacking E-cadherin. Development 137, 33833391 (2010).
  3. Johnson, M. H., Chakraborty, J., Handyside, A. H., Willison, K. & Stern, P. The effect of prolonged decompaction on the development of the preimplantation mouse embryo. J. Embryol. Exp. Morphol. 54, 241261 (1979).
  4. Larue, L., Ohsugi, M., Hirchenhain, J. & Kemler, R. E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc. Natl Acad. Sci. USA 91, 82638267 (1994).
  5. Ducibella, T., Ukena, T., Karnovsky, M. & Anderson, E. Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo. J. Cell Biol. 74, 153167 (1977).
  6. Goel, N. S., Doggenweiler, C. F. & Thompson, R. L. Simulation of cellular compaction and internalization in mammalian embryo development as driven by minimization of surface energy. Bull. Math. Biol. 48, 167187 (1986).
  7. Kemler, R., Babinet, C., Eisen, H. & Jacob, F. Surface antigen in early differentiation. Proc. Natl Acad. Sci. USA 74, 44494452 (1977).
  8. Shirayoshi, Y., Okada, T. S. & Takeichi, M. The calcium-dependent cell–cell adhesion system regulates inner cell mass formation and cell surface polarization in early mouse development. Cell 35, 631638 (1983).
  9. Hyafil, F., Morello, D., Babinet, C. & Jacob, F. A cell surface glycoprotein involved in the compaction of embryonal carcinoma cells and cleavage stage embryos. Cell 21, 927934 (1980).
  10. Foty, R. & Steinberg, M. The differential adhesion hypothesis: a direct evaluation. Dev. Biol. 278, 255263 (2005).
  11. Steinberg, M. S. & Takeichi, M. Experimental specification of cell sorting, tissue spreading, and specific spatial patterning by quantitative differences in cadherin expression. Proc. Natl Acad. Sci. USA 91, 206209 (1994).
  12. Maître, J-L. & Heisenberg, C-P. Three functions of cadherins in cell adhesion. Curr. Biol. 23, R62633 (2013).
  13. Krieg, M. et al. Tensile forces govern germ-layer organization in zebrafish. Nat. Cell Biol. 10, 429436 (2008).
  14. Maître, J-L. et al. Adhesion functions in cell sorting by mechanically coupling the cortices of adhering cells. Science 338, 253256 (2012).
  15. Stirbat, T. V. et al. Fine tuning of tissues viscosity and surface tension through contractility suggests a new role for α-catenin. PLoS ONE 8, e52554 (2013).
  16. Yamada, S. & Nelson, W. J. Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell–cell adhesion. J. Cell Biol. 178, 517527 (2007).
  17. Tinevez, J-Y. et al. Role of cortical tension in bleb growth. Proc. Natl Acad. Sci. USA 106, 1858118586 (2009).
  18. Yeung, A. & Evans, E. Cortical shell-liquid core model for passive flow of liquid-like spherical cells into micropipets. Biophys. J. 56, 139149 (1989).
  19. Mitchison, J. M. & Swann, M. M. The mechanical properties of the cell surface I. The cell elastimeter. J. Exp. Biol. 31, 443460 (1954).
  20. Fierro-González, J. C., White, M. D., Silva, J. C. & Plachta, N. Cadherin-dependent filopodia control preimplantation embryo compaction. Nat. Cell Biol. 15, 110 (2013).
  21. Clark, A. G., Wartlick, O., Salbreux, G. & Paluch, E. K. Stresses at the cell surface during animal cell review morphogenesis. Curr. Biol. 24, R484R494 (2014).
  22. Dai, J., Ting-Beall, H., Hochmuth, R., Sheetz, M. & Titus, M. Myosin I contributes to the generation of resting cortical tension. Biophys. J. 77, 11681176 (1999).
  23. Sobel, J. S. Localization of myosin in the preimplantation mouse embryo. Dev. Biol. 95, 227231 (1983).
  24. Martin, A., Kaschube, M. & Wieschaus, E. Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457, 495499 (2009).
  25. Roh-Johnson, M. et al. Triggering a cell shape change by exploiting preexisting actomyosin contractions. Science 335, 12321235 (2012).
  26. Solon, J., Kaya-Copur, A., Colombelli, J. & Brunner, D. Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure. Cell 137, 13311342 (2009).
  27. Bertet, C., Sulak, L. & Lecuit, T. Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation. Nature 429, 667671 (2004).
  28. Winkel, G. K., Ferguson, J. E., Takeichi, M. & Nuccitelli, R. Activation of protein kinase C triggers premature compaction in the four-cell stage mouse embryo. Dev. Biol. 138, 115 (1990).
  29. Kawai, Y., Yamaguchi, T., Yoden, T., Hanada, M. & Miyake, M. Effect of protein phosphatase inhibitors on the development of mouse embryos: protein phosphorylation is involved in the E-cadherin distribution in mouse two-cell embryos. Biol. Pharm. Bull. 25, 179183 (2002).
  30. Ohsugi, M., Butz, S. & Kemler, R. β-catenin is a major tyrosine-phosphorylated protein during mouse oocyte maturation and preimplantation development. Dev. Dyn. 216, 168176 (1999).
  31. Riedl, J. et al. Lifeact mice for studying F-actin dynamics. Nat. Methods 7, 168169 (2010).
  32. Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593605 (2007).
  33. Boussadia, O., Kutsch, S., Hierholzer, A., Delmas, V. & Kemler, R. E-cadherin is a survival factor for the lactating mouse mammary gland. Mech. Dev. 115, 5362 (2002).
  34. de Vries, W. N. et al. Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis 26, 110112 (2000).
  35. Tsunoda, Y., Yasui, T., Nakamura, K., Uchida, T. & Sugie, T. Effect of cutting the zona pellucida on the pronuclear transplantation in the mouse. J. Exp. Zool. 240, 119125 (1986).
  36. Biggers, J. D., McGinnis, L. K. & Raffin, M. Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium. Biol. Reprod. 63, 281293 (2000).
  37. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676682 (2012).
  38. Thielicke, W. & Stamhuis, E. J. PIV Lab—Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB (version: 1.35) (2010).
  39. Driscoll, M. K. et al. Cell shape dynamics: from waves to migration. PLoS Comput. Biol. 8, e1002392 (2012).
  40. Sommer, C., Strähle, C., Köthe, U. & Hamprecht, F. A. Proc. 8th IEEE Int. Symp. Biomed. Imaging 230233 (2011).
  41. Turlier, H., Audoly, B., Prost, J. & Joanny, J-F. Furrow constriction in animal cell cytokinesis. Biophys. J. 106, 114123 (2014).

Download references

Author information

Affiliations

  1. European Molecular Biology Laboratory, Meyerhofstrasse 1 69117 Heidelberg, Germany

    • Jean-Léon Maître,
    • Ritsuya Niwayama,
    • Hervé Turlier,
    • François Nédélec &
    • Takashi Hiiragi

Contributions

J-L.M. and T.H. designed the project. J-L.M. designed, performed and analysed the experiments. R.N. implemented the image analysis. H.T. conceived the supporting theory. All authors discussed the significance of the results and helped write the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Blastomeres volume during the 8-cell stage. (426 KB)

    a, Images of cell volume reconstruction showing 6 cells of an 8-cell stage embryo expressing mTmG (grey) artificially labelled in distinct colours using Bitplane Imaris. Scale bar, 10 μm. b, Volume of blastomeres at the beginning (30 min after the last 4- to 8-cell stage division) and end (30 min before rounding up of the first 8- to 16-cell stage division) of the 8-cell stage. Individual examples from (a) are shown in colour while the whisker plot in black shows the minimum and maximum values, and upper and lower quartiles of n = 32 blastomeres pooled from 4 embryos from 1 representative experiment. p value indicates the result of Students t-test.

  2. Supplementary Figure 2: Theoretical modelling of surface energy minimization during compaction of 2/8th doublets. (286 KB)

    a, Diagram of two blastomeres from 8-cell stage mouse embryo forming a 2/8th doublet shaped by surface tension γcm at the cell–medium interface and interfacial tension γcc at the cell–cell interface. The shape of each blastomere is characterized by a radius of curvature Rc, an area of cell–medium interface A and a volume V0. The shape of the cell–cell contact is characterized by its radius r and contact angle θe. The 2/8th doublet is symmetric with respect to a rotational axis Φ and to the cell–cell contact. b, Rescaled energy E/E0 as a function of the contact angle θe/2 for different values of the dimensionless parameter 0 ≤ α = γcc/2γcm ≤ 1. The minimum of the surface energy, overlaid as a diagram, corresponds to the mechanical equilibrium α = cos(θe/2) for the 2/8th doublet.

  3. Supplementary Figure 3: Natural variation in shape and tensions during compaction. (373 KB)

    ad, Mean ± s.d. are shown for the blastomeres and contacts of 3 individual embryos. Embryo shown in Fig. 1b is shown in red. a, Radius of curvature Rc over time. b, Contact angle θe over time. c, Surface tension γcm over time. d, Interfacial tension γcc/2 over time. e, Plot of each surface tension γcm of each contacting blastomeres against one another. N = 233 contacting blastomeres pooled from 28 embryos from 11 representative experiments, Pearson correlation R = 0.778 (p < 0.001). f, Plot of each contact angle θi of each contacting blastomeres against one another. N = 233 contacting blastomeres pooled from 28 embryos, Pearson correlation R = 0.603 (p < 0.001).

  4. Supplementary Figure 4: Surface tension at the 4-cell stage. (228 KB)

    a, Surface tension measurements of an early (top) and late (bottom) 4-cell stage embryo. bc, Contact angles θi and θe (b) and tensions γcm and γcc/2 (c) of n = 42 and 46 contacting blastomeres pooled from 6 embryos early and late in the 4-cell stage from 2 representative experiments. d, Surface tension measurements (n = 88)γcm of a 4-cell stage embryo as a function of the contact angle θi in blue, together with 8-cell stage measurements shown in Fig. 1c in grey. p value indicates the result of Students t-test.

  5. Supplementary Figure 5: Role of contractility in generating tensions during compaction. (552 KB)

    a, Immunostaining of Myh9 early and late in the 8-cell stage. Red arrowheads point at cell–medium interfaces, green ones at cell–cell contacts. Scale bar, 10 μm. b, Mean ± s.d. contact/cortex ratio, n = 194 contacting blastomeres pooled from 22 embryos from 2 representative experiments, Pearson correlation R = −0.538 (p < 0.001). c, Phalloidin staining of F-actin early and late in the 8-cell stage. Red arrowheads point at cell–medium interfaces, green ones at cell–cell contacts. d, Mean ± s.d. contact/cortex ratio, 113 embryos from 9 representative experiments, Pearson correlation R = −0.752 (p < 0.001). e, Time-lapse of mTmG expressing embryo. Time post-division (hh:mm), Supplementary Video 3 . f, Contact/cortex ratio of mTmG and LifeAct-GFP over time. Mean ± s.d. of n = 23 contacts pooled from 8 embryos from 3 representative experiments followed every 30 min for 5–8 h. g, Surface tension measurement of DMSO or 1 μM CCD treated embryos. Scale bar, 10 μm. h, Surface tension γcm as a function of the contact angle θi of n = 10 and 42 contacting blastomeres pooled from 2 and 3 embryos treated with DMSO (black) or CCD (yellow) respectively from 1 representative experiment. Pearson correlation R = −0.028 for DMSO and −0.005 for CCD with (p > 0.1). WT (grey) shown in Fig. 1c.

  6. Supplementary Figure 6: Examples of PIV and Fourier analysis of periodic contractions in different conditions. (353 KB)

    af, Four examples of PIV and Fourier analysis are shown for 8-cell stage embryos (a, black), 4-cell stage embryos (b, blue), 8-cell stage embryos without zona pellucida (c, no ZP, purple), 8-cell stage embryos treated with 25 μM Bb (−) (d, orange) or 1 μM CCD (e, yellow), and mzCdh1/ 8-cell stage embryos (f, green) are shown. The left column shows the velocity of one vector in one direction over time. The middle column shows the corresponding power spectrum. The right column shows the amplitude as a function of the period.

  7. Supplementary Figure 7: Periodic contractions in different conditions. (395 KB)

    ad, Mean ± s.d. amplitude of periodic contractions as a function of the oscillation period for control 8-cell stage embryos (ad, in black, n = 23 from 6 representative experiments), 1 μM CCD-treated 8-cell stage embryos (a, in yellow, n = 14 from 2 representative experiments), 4-cell stage embryos (b, in blue, n = 8 from 2 representative experiments), 8-cell stage embryos without zona pellucida (c, No ZP, in purple, n = 6 from 2 representative experiments). mzCdh1/ 8-cell stage embryos (d, in green, n = 7 from 3 representative experiments). e, After Fourier analysis, the mean power spectra are analysed for the presence of a distinct peak of amplitude at a specific frequency. Distinct peaks could be observed in 16 out of n = 23 embryos at 8-cell stage, 1 out of n = 8 embryos at 4-cell stage, 0 out of n = 17 embryos at 8-cell stage with 25 μM Bb (−), 0 out of n = 14 embryos at 8-cell stage with 1 μM CCD, 6 out of n = 6 embryos at 8-cell stage without zona pellucida (ZP), 7 out of n = 7 mzCdh1/ embryos at 8-cell stage, 7 out of n = 7 blastomeres isolated at the 8-cell stage. f, Periods that could be extracted are plotted as mean ± s.d. for embryos at 8-cell stage after PIV analysis (n = 16) and LifeAct-GFP intensity changes (n = 4), n = 6 embryos at 8-cell stage without zona pellucida (ZP) after PIV analysis, n = 7 mzCdh1/ embryos at 8-cell stage after PIV analysis, n = 7 blastomeres isolated at the 8-cell stage after curvature analysis and LifeAct-GFP intensity changes.

  8. Supplementary Figure 8: Examples of periodic cortical waves. (687 KB)

    Three examples of 1/8th blastomeres showing periodic cortical waves. Left column shows the curvature computed from the segmented membrane signal. 2nd column shows LifeAct-GFP. 3rd column shows a kymograph of the curvature along the cell perimeter (y-axis, arbitrary unit) as a function of time (x-axis over 15 min). 4th column shows a kymograph of LifeAct-GFP signal along the cell perimeter (y-axis, arbitrary unit) as a function of time (x-axis over 15 min).

Video

  1. Video 1: Pre-implantation development. (7.05 MB, Download)
    Brightfield (BF) images of a developing zygote with mechanically removed ZP until blastocyst stage. At the 8-cell stage, compaction starts at 68:00 and ends at 76:00. Images are taken every 30 min and shown at 10 frames per second (fps). Time post-division (hh:mm), scale bar, 10 μm.
  2. Video 2: Mapping of surface tension in space and time. (6.05 MB, Download)
    BF images of surface tension mapping of an embryo. Each blastomeres is measured 5 times throughout the 8-cell stage (from ~3:00 to ~10:30 post-division). A picture is taken for each pressure step when the deformation reached a stable shape and shown at 10 fps. Time post-division (hh:mm), scale bar, 10 μm.
  3. Video 3: LifeAct-GFP and mTmG compaction. (1.6 MB, Download)
    Fluorescent confocal imaging of an mTmG (right) and LifeAct-GFP (left) expressing transgenic embryo throughout the 8-cell stage. Timelapse imaging consists of a picture taken every 30 min and is displayed at 5 fps. Time post-division (hh:mm), scale bar, 10 μm.
  4. Video 4: PIV analysis of control 8-cell stage embryo. (1.5 MB, Download)
    BF images of an 8-cell stage embryo undergoing pulsed contractions. Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  5. Video 5: PIV analysis of 4-cell stage embryo. (1.9 MB, Download)
    BF images of a 4-cell stage embryo. Pictures are acquired every 2 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red and half of the images are shown to match Supplementary Videos 4, 710 display. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  6. Video 6: LifeAct-GFP pulses. (4.36 MB, Download)
    En face confocal slice of the cortex of an 8-cell stage embryo undergoing pulsed contractions. On the left, LifeAct-GFP shows patchy accumulation of signal at the cortex of cells. In the middle, mT shows membrane movements while the cell remains in focus. On the right, LifeAct-GFP (green) and mT (red) signals are merged. Timelapse imaging consists of a picture taken every 5 s and is displayed at 20 fps. Scale bar, 5 μm.
  7. Video 7: PIV analysis of 8-cell stage embryo treated with 25 μM Bb (-). (1.38 MB, Download)
    BF images of an 8-cell stage embryo treated with 25 μM Bb (-). Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  8. Video 8: PIV analysis of 8-cell stage embryo treated with 1 μM CCD. (1.39 MB, Download)
    BF images of an 8-cell stage embryo treated with 1 μM CCD. Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  9. Video 9: PIV analysis of 8-cell stage embryo without zona pellucida (1.25 MB, Download)
    BF images of an 8-cell stage embryo without ZP. Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  10. Video 10: PIV analysis of mzCdh1/ embryo. (1.77 MB, Download)
    BF images of an 8-cell stage mzCdh1/ embryo. Pictures are acquired every 5 s and PIV analysis is performed between two successive images. Resulting PIV vectors are overlaid on top of the first of the two images analysed with vectors pointing up in green and vectors pointing down in red. Pictures are displayed at 10 fps. Scale bar, 10 μm.
  11. Video 11: Periodic cortical waves. (14.43 MB, Download)
    Montage of BF (top left), mTmG (top right), LifeAct-GFP (bottom left) and local curvature measurement (bottom right) of a 1/8th blastomeres undergoing periodic cortical waves. Timelapse imaging consists of a picture taken every 5 s and is displayed at 10 fps. Scale bar, 10 μm.

Supplementary Tables

  1. Supplementary Table 1: (417 KB)

    a, mean ± s.d. of contact angles and surface tensions over time shown in Fig. 1c. b, mean ± s.d. of contact angles and surface tensions over time for whole 8-cell embryos, 2/8th doublets and 1/8th single blastomeres shown in Fig. 2e. c, mean ± s.d. of contact angles and surface tensions over time for mCdh1+/ and mzCdh1/ embryos shown in Fig. 3d. d, mean ± s.d. of contact angles and surface tensions over time for 25 μM Bb (+) or Bb (−) treated embryos shown in Fig. 4g.

PDF files

  1. Supplementary Information (2 MB)

    Supplementary Information

Additional data