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Inverse blebs operate as hydraulic pumps during mouse blastocyst formation

Nature Cell Biology volume 26, pages 1669–1677 (2024)Cite this article

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Abstract

During preimplantation development, mouse embryos form a fluid-filled lumen. Pressurized fluid fractures cell–cell contacts and accumulates into pockets, which coarsen into a single lumen. How the embryo controls intercellular fluid movement during coarsening is unknown. Here we report inverse blebs growing into cells at adhesive contacts. Throughout the embryo we observed hundreds of inverse blebs, each filling with intercellular fluid and retracting within a minute. Inverse blebs grow due to pressure build-up resulting from fluid accumulation and cell–cell adhesion, which locally confines fluid. Inverse blebs retract due to actomyosin contraction, practically pushing fluid within the intercellular space. Importantly, inverse blebs occur infrequently at contacts formed by multiple cells, which effectively serve as fluid sinks. Manipulation of the embryo topology reveals that without sinks inverse blebs pump fluid into one another in futile cycles. We propose that inverse blebs operate as hydraulic pumps to promote luminal coarsening, thereby constituting an instrument used by cells to control fluid movement.

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Fig. 1: Inverse blebs at the onset of blastocoel formation.
Fig. 2: Spatiotemporal dynamics of inverse blebs throughout blastocoel formation.
Fig. 3: Inverse bleb formation requires cell–cell adhesion and fluid accumulation.
Fig. 4: Inverse blebs retract with actomyosin contractility.
Fig. 5: Inverse blebs pump fluid into topological sinks.

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Data availability

The raw microscopy data, regions of interests and analyses are available at the BioImage Archive (accession number S-BIAD1193) under a CC BY-NC-SA license (https://doi.org/10.6019/S-BIAD1193, https://www.ebi.ac.uk/biostudies/bioimages/studies/S-BIAD1193). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

The MATLAB analysis codes are available at the BioImage Archive (accession number S-BIAD1193) under a CC BY- NC-SA license (https://doi.org/10.6019/S-BIAD1193, https://www.ebi.ac.uk/biostudies/bioimages/studies/S-BIAD1193).

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Acknowledgements

We thank the imaging platform of the Genetics and Developmental Biology Unit at the Institut Curie (PICT-IBiSA@BDD, member of the French National Research Infrastructure France-BioImaging ANR-10-INBS-04) for their outstanding support, in particular O. Leroy for his help writing Metamorph journals, and the animal facility of the Institut Curie for their invaluable help. We thank Viventis Microscopy for custom Python scripts for the LS1 Live. We thank Y. Bellaïche, C. Blanch-Mercader and the members of the Maître laboratory for critical reading of the manuscript. Research in the laboratory of J.-L.M. is supported by the Institut Curie, the Centre National de la Recherche Scientifique (CNRS) and the Institut National de la Santé Et de la Recherche Médicale (INSERM), and is funded by grants from the Fondation Schlumberger pour l’Éducation et la Recherche via the Fondation pour la Recherche Médicale, the European Research Council Starting Grant ERC-2017-StG 757557, the Agence Nationale de la Recherche (ANR-21-CE13-0027-01), the European Molecular Biology Organization Young Investigator programme (EMBO YIP), the INSERM transversal programme Human Development Cell Atlas (HuDeCA), Paris Sciences Lettres (PSL) QLife (17-CONV-0005) grant and Labex DEEP (ANR-11-LABX-0044), which are part of the IDEX PSL (ANR-10-IDEX-0001-02). M.F.S. is funded by a Convention Industrielle de Formation pour la Recherche (grant number 1113 2019/0253) between the Agence Nationale de la Recherche and Carl Zeiss SAS as well as La Ligue contre le cancer. M.F.S. thanks the support from La Fondation des Treilles. A.M. acknowledges funding from the QBio Junior Research Chair programme of the QBio initiative of ENS-PSL and the Parisante Campus.

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Authors and Affiliations

  1. Institut Curie, CNRS UMR3215, INSERM U934, PSL Research University and Sorbonne Université, Paris, France

    Markus F. Schliffka, Julien G. Dumortier, Diane Pelzer & Jean-Léon Maître

  2. Carl Zeiss SAS, Marly-le-Roy, France

    Markus F. Schliffka

  3. Laboratoire de physique de l’École Normale Supérieure, CNRS UMR 8023, PSL Research University, Sorbonne Université and Université Paris Cité, Paris, France

    Arghyadip Mukherjee

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Contributions

M.F.S., J.G.D. and D.P. performed experiments and prepared data for analyses. M.F.S., J.G.D. and J.-L.M. designed the project and analysed the data. A.M. developed the theoretical analysis. M.F.S., A.M. and J.-L.M. wrote the manuscript. M.F.S. and J.-L.M. acquired funding.

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Correspondence to Arghyadip Mukherjee or Jean-Léon Maître.

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Competing interests

During this project M.F.S. was partly employed by Carl Zeiss SAS via a public PhD programme (Conventions Industrielles de Formation par la Recherche, CIFRE) cofunded by the Association Nationale de la Recherche et de la Technologie (ANRT).

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Nature Cell Biology thanks Marino Arroyo, Carien Niessen, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Dynamics of individual inverse blebs.

a) Initial recruitment time of LA–GFP (green, n = 100 blebs from 13 embryos from 5 experiments) and MYH9–GFP (red, n = 21 blebs from 5 embryos from 4 experiments) to inverse blebs. Mean and SD are shown in black bracket. p value results from Mann–Whitney U-test. b) Maximal recruitment time of LA–GFP (green, n = 100 blebs from 13 embryos from 5 experiments) and MYH9–GFP (red, n = 21 blebs from 5 embryos from 4 experiments) to inverse blebs. Mean and SD are shown in black bracket. p value results from Mann–Whitney U-test. c) Still images following the lifetime of an inverse blebs labelled with LA–GFP in green and mTmG in magenta for a TE (top lane) and an ICM (bottom lane) cell. Images taken every 6.9 s with time indicated relative to the maximal inverse bleb size. Scale bars, 2 µm.

Extended Data Fig. 2 Inverse bleb growth directionality.

a) Schematic diagram of 32-cell stage embryo enclosed within the zona pellucida (grey) showing inverse blebs (asymmetric intrusions coated with green actomyosin and filled with blue luminal fluid) and microlumens (symmetric pockets of blue luminal fluid) between surface trophectoderm (pink, T) and inner cell mass cells (peach, I). b) Proportion of contact length relative to the cell–cell contact network within the imaging plane for TE-TE (TT, pink), ICM-ICM (II, peach) or TE-ICM (TI, brown-purple) from 12 embryos from 5 experiments. c) Proportion of inverse blebs growing at TE-TE (pink), ICM-ICM (peach) and TE-ICM interfaces with the direction of growth into TE (brown) or ICM (purple) indicated for TE-ICM interfaces from 12 embryos from 5 experiments. d) Number of inverse blebs observed per minute normalized by the length of cell–cell contacts at given tissue interfaces in the optical slice of embryos imaged using spinning disk microscopy. Inverse blebs are captured growing between trophectoderm cells (TT, n = 51), inner cell mass cells (II, n = 132), into inner cell mass cells (TI->I, n = 67) or into trophectoderm cells (TI->T, n = 52) at the interface between trophectoderm and inner cell mass cells of 12 embryos from 5 experiments. Mean and SD are shown in black bracket. p value results from Kruskal–Wallis test.

Source data

Extended Data Fig. 3 Dynamics of adhesion molecules in individual inverse blebs.

a) Still images showing cell–cell contacts before and after the formation of microlumens with CDH1-GFP in green and mTmG in magenta. Arrowheads point at foci of CDH1 around microlumens. Scale bar, 5 µm. b) Equatorial plane of a 32-cell stage embryo with CDH1-GFP in green and mTmG in magenta (Supplementary Movie 6). Scale bar, 20 µm. Dashed rectangle shows a cell–cell contact with an inverse bleb magnified on the left. Scale bar, 5 µm. c) Still images following the lifetime of an inverse bleb shown with mTmG in magenta and CDH1-GFP in green. Images taken every 5 s with time indicated relative to the maximal inverse bleb size. Scale bar, 2 µm. d) Mean contour length (grey), normalized CDH1-GFP (green) and mTmG (magenta) intensities of 31 inverse blebs from 7 embryos synchronized to their maximal extension from 3 experiments. Error bars show SEM.

Source data

Extended Data Fig. 4 Global and local effects of adhesion loss on microlumens and inverse blebs.

a) Still images of mTmG in WT (top) and mCdh1+/− (bottom) embryos during lumen formation (Supplementary Movies 7, 8). Arrows point at microlumens. Scale bars, 20 µm. b) Duration of microlumens in WT (n = 28 embryos from 3 experiments) and mCdh1+/− (n = 29 embryos from 3 experiments) embryos. Mean and SD are shown in black bracket. p value results from Student’s t test. c) Proportion of WT (n = 18 embryos from 3 experiments) and mCdh1+/− (n = 15 from embryos 3 experiments) embryos showing inverse blebs. p value results from Chi-square test.

Source data

Extended Data Fig. 5 Inverse blebs require accumulation of luminal fluid.

Proportion of embryos showing inverse blebs in DMSO (1:100 or 1:200, 13 embryos from 6 experiments), EIPA (15 µM, n = 5 embryos from 2 experiments) or 500 µM (n = 9 embryos from 3 experiments) or 1 mM (n = 9 embryos from 3 experiments) Ouabain treatments during the 32-cell stage. p values result from Chi-square test.

Source data

Extended Data Fig. 6 Dynamics of individual inverse blebs with impaired actomyosin contractility.

a) Still images following the lifetime of inverse blebs labelled with LA–GFP in green and mTmG in magenta in 1:1200 DMSO (top) or 25 µM para-Nitroblebbistatin (p-Nbb, bottom) media (Supplementary Movie 11). Images taken every 5 s with time indicated relative to the maximal inverse bleb size. Scale bars, 2 µm. b) Duration of retraction of inverse blebs for embryos treated with 1:1200 DMSO (purple, n = 55 from 6 embryos from 4 experiments) or 25 µM para-Nitroblebbistatin (violet, n = 26 from 6 embryos from 4 experiments) media. Mean and SD are shown in black bracket. p value results from Mann–Whitney U-test. c) Mean normalized LA–GFP intensity as a function of mean contour length of inverse blebs synchronized to their maximal extension for embryos treated with 1:1200 DMSO (purple, n = 55 from 6 embryos from 4 experiments) or 25 µM para-Nitroblebbistatin (violet, n = 26 from 6 embryos from 4 experiments) media. Error bars show SEM. Arrows indicate the extension and retraction dynamics of inverse blebs. d–f) Maximal contour length, initial and maximal recruitment time of LA–GFP to inverse blebs of embryos treated with 1:1,200 DMSO (purple, n = 55 blebs from 6 embryos from 4 experiments) or with 25 µm para-Nitroblebbistatin (violet, n = 26 blebs from 6 embryos from 4 experiments). Mean and SD are shown in black bracket. p values result from Mann–Whitney U-test. g–i) Maximal contour length, initial and maximal recruitment time of LA–GFP to inverse blebs of WT (grey, n = 100 blebs from 13 embryos from 5 experiments), mMyh9+/− (red, n = 67 blebs from 10 embryos from 4 experiments) or mMyh10+/− (blue, n = 41 blebs from 6 embryos from 3 experiments) embryos. Mean and SD are shown in black bracket. p values result from Kruskal–Wallis and Dunn's multiple comparisons tests. j) Left: immunostaining of 32-cell stage embryos showing phalloidin (green) and the phosphorylated form of non-muscle myosin heavy chain IIA (pMYH9, magenta). Arrowheads point at fixed inverse blebs. Scale bar, 20 µm. Right: greyscale images on the right show separate signals of the inverse bleb pointed by the red arrowhead. Scale bar, 2 µm. k) Duration of microlumens in WT (n = 28 embryos from 3 experiments) and mMyh9+/− (n = 20 embryos from 3 experiments) embryos. Mean and SD are shown in black bracket. p value results from Student’s t test.

Source data

Extended Data Fig. 7 Inverse bleb growth in WT/WT and mMyh9+/−/WT chimaeric embryos.

a) Left: Schematic diagrams of chimeric embryos with contacts of homo- or heterotypic contractility. A WT 8-cell stage blastomere is grafted onto a WT host morula to create homotypic contacts or onto a mMyh9+/− host embryo to create heterotypic contacts. Hosts and grafts are distinguished by the intensity of their membrane signal resulting from either paternal (host) or maternal (graft) expression of mTmG. Alternatively, grafts express NLS–mKate2 (ref. 45) in addition to paternal mTmG (not shown; see Methods). All cells express LifeAct–GFP to detect inverse blebs. Right: Examples of inverse blebs at homotypic (top; Supplementary Movie 13) and heterotypic contacts (bottom; Supplementary Movie 14). White boxes are magnified on the right. White arrowheads indicate inverse blebs expanded into a grafted blastomere. Scale bars, 20 (left) and 5 µm (right). b) Proportions of inverse blebs forming into graft or host blastomeres in WT/WT or mMyh9+/−/WT chimeras. The number of inverse blebs counted at host-graft interfaces is indicated. WT/WT: n = 7 chimeric embryos from 3 experiments. mMyh9+/−/WT: n = 7 chimeric embryos from 3 experiments. p value results from Chi-square test.

Source data

Extended Data Fig. 8 Geometry and topology of reduced embryos.

a) Schematic diagrams of the dissociation process used to produce quartets and doublets of 32-cell stage blastomeres. A 16-cell stage embryo is dissociated into singlets and doublets which then divide into doublets and quartets respectively. Quartets can adopt 3 different configurations: from left to right, 4 outer cells, 3 outer and 1 inner cell or 2 outer and 2 inner cells, which have at least 2 multicellular microlumens. Doublets can consist of 2 outer cells or 1 outer and 1 inner cell, which do not have multicellular microlumen. b) Length of the network of cell–cell contact measured at the equatorial plane of whole embryos (n = 18 embryos from 3 experiments), quartets (n = 32 quartets from 7 experiments) or doublets (n = 27 doublets from 6 experiments) shown with a logarithmic scale. Mean and SD are shown in black bracket. p values result from ANOVA and Dunnett’s multiple comparisons tests.

Supplementary information

Supplementary Information

Supplementary text.

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Supplementary Video 1

Dynamic recruitment of actin during inverse blebs retraction. Time-lapse imaging at the equatorial plane of a 32-cell-stage embryo expressing LA-GFP (green) and mTmG (magenta) imaged every 6.9 s using spinning disk microscopy. Scale bar, 20 µm.

Supplementary Video 2

Dynamic recruitment of non-muscle myosin II during inverse blebs retraction. Time-lapse imaging at the equatorial plane of a 32-cell-stage embryo expressing MYH9-GFP (green) and mTmG (magenta) imaged every 6.9 s using spinning disk microscopy. Scale bar, 20 µm.

Supplementary Video 3

Inverse blebs fill with intercellular fluid before flushing it back into the microlumen network. Time-lapse imaging at the equatorial plane of a 32-cell-stage embryo expressing mTmG (grey) incubated with dextran–Alexa Fluor 488 (cyan) at the 16-cell stage before sealing of tight junctions imaged every 5 s using spinning disk microscopy. Scale bar, 20 µm.

Supplementary Video 4

Inverse bleb activity peaks before lumen expansion. Nested time-lapse imaging of a LA–GFP embryo imaged every 10 s for 5 min, repeated every 30 min, using light-sheet microscopy. The equatorial plane (left) and maximum projection (right) are shown. Developmental time relative to peak blebbing activity measured throughout the entire embryo volume is indicated. Scale bar, 20 µm.

Supplementary Video 5

Inverse blebs coexist with microlumens throughout lumen formation. Time-lapse imaging at the equatorial plane of a 32-cell-stage embryo expressing LA-GFP (green) and mTmG (magenta) imaged every minute using spinning disk microscopy. Scale bar, 20 µm.

Supplementary Video 6

Dynamics of adhesion molecules during inverse blebbing. Time-lapse imaging at the equatorial plane of a 32-cell-stage embryo expressing CDH1-GFP (green) and mTmG (magenta) imaged every 5 s using spinning disk microscopy. Scale bar, 20 µm.

Supplementary Video 7

Microlumen duration in WT embryos. Time-lapse imaging of a 32-cell-stage WT embryo expressing mTmG imaged every 5 min using spinning disk microscopy to capture the time of microlumen appearance and disappearance. The equatorial plane is shown. Scale bar, 20 µm.

Supplementary Video 8

Microlumen duration in mCdh1+/− embryos. Time-lapse imaging of a 32-cell-stage mCdh1+/−embryo expressing mTmG imaged every 5 min using spinning disk microscopy to capture the time of microlumen appearance and disappearance. The equatorial plane is shown. Scale bar, 20 µm.

Supplementary Video 9

Unlike WT embryos, mCdh1+/− embryos rarely form inverse blebs. Time-lapse imaging at the equatorial plane of 32-cell-stage WT and mCdh1+/− embryos expressing LA-GFP (green) and mTmG (magenta) imaged every minute using spinning disk microscopy. Embryos are synchronized to the time of appearance of microlumens at the equatorial plane. Scale bar, 20 µm.

Supplementary Video 10

EIPA treatment acutely stops inverse blebs. Time-lapse imaging at the equatorial plane of a 32-cell-stage embryo expressing LA-GFP (green) and mTmG (magenta) imaged every 5 s using spinning disk microscopy. The embryo was first treated with 1:2,500 DMSO containing medium (left) and then with 20 µM EIPA for 15 min before the start of the time lapse (right). Scale bar, 20 µm.

Supplementary Video 11

para-Nitroblebbistatin treatment acutely slows inverse bleb retraction. Time-lapse imaging at the equatorial plane of a 32-cell-stage embryo expressing LA-GFP (green) and mTmG (magenta) imaged every 5 s using spinning disk microscopy. Treatment with 25 µM para-Nitroblebbistatin (p-Nbb) for 15 min before the start of the time lapse causes inverse bleb retraction to slow down compared with DMSO treatment. Scale bar, 20 µm.

Supplementary Video 12

mMyh9+/− embryos retract inverse blebs at a slower rate compared with WT and mMyh10+/− embryos. Time-lapse imaging at the equatorial plane of WT, mMyh9+/− and mMyh10+/− embryos expressing LA-GFP (green) and mTmG (magenta) imaged every 6.9 s using spinning disk microscopy. Scale bar, 20 µm.

Supplementary Video 13

Inverse bleb formation in a WT/WT chimaeric embryo. Time-lapse imaging at the equatorial plane of a chimaeric embryo with a WT host expressing LA–GFP (green) and paternal mTmG (magenta), and a WT graft expressing LA–GFP and maternal mTmG appearing brighter, imaged every minute at the equatorial plane using spinning disk microscopy. Scale bar, 20 μm.

Supplementary Video 14

Inverse bleb formation in a mMyh9+/−/WT chimaeric embryo. Time-lapse imaging at the equatorial plane of a chimaeric embryo with a mMyh9+/− host expressing LA–GFP (green) and paternal mTmG (magenta), and a WT graft expressing LA–GFP and maternal mTmG appearing brighter, imaged every minute at the equatorial plane using spinning disk microscopy. Scale bar, 20 μm.

Supplementary Video 15

Microlumen duration in mMyh9+/− embryos. Time-lapse imaging of a 32-cell-stage mMyh9+/− embryo expressing mTmG imaged every 5 min using spinning disk microscopy to capture the time of microlumen appearance and disappearance. The equatorial plane is shown. Scale bar, 20 µm.

Supplementary Video 16

Quartets of 32-cell-stage blastomeres form a lumen rapidly. Time-lapse imaging at the equatorial plane of a quartet of 32-cell-stage blastomeres expressing LA–GFP (green) and mTmG (magenta) imaged every 1 min using spinning disk microscopy. Time relative to the appearance of microlumens at the equatorial plane is indicated. Scale bar, 20 µm.

Supplementary Video 17

Doublets of 32-cell-stage blastomeres do not form a lumen rapidly. Time-lapse imaging at the equatorial plane of a doublet of 32-cell-stage blastomeres expressing LA–GFP (green) and mTmG (magenta) imaged every 1 min using spinning disk microscopy. Time relative to the appearance of microlumens at the equatorial plane is indicated. Scale bar, 20 µm.

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Schliffka, M.F., Dumortier, J.G., Pelzer, D. et al. Inverse blebs operate as hydraulic pumps during mouse blastocyst formation. Nat Cell Biol 26, 1669–1677 (2024). https://doi.org/10.1038/s41556-024-01501-z

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