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Zygotic contractility awakening during mouse preimplantation development

Özgüç Ö1, de Plater L1, Kapoor1, Tortorelli A1, Jean-Léon Maître1 
11 Jul 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this article, the authors used periodic cortical waves of contraction (PeCoWaCo) to study the awakening of actomyosin contractility during preimplantation development.
Abstract: Actomyosin contractility is a major engine of preimplantation morphogenesis, which starts at the 8-cell stage during mouse embryonic development. Contractility becomes first visible with the appearance of periodic cortical waves of contraction (PeCoWaCo), which travel around blastomeres in an oscillatory fashion. How contractility of the mouse embryo becomes active remains unknown. We have taken advantage of PeCoWaCo to study the awakening of contractility during preimplantation development. We find that PeCoWaCo become detectable in most embryos only after the 2nd cleavage and gradually increase their oscillation frequency with each successive cleavage. To test the influence of cell size reduction during cleavage divisions, we use cell fusion and fragmentation to manipulate cell size across a 20-60 μm range. We find that the stepwise reduction in cell size caused by cleavage divisions does not explain the presence of PeCoWaCo or their accelerating rhythm. Instead, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. Therefore, during cleavage stages, cortical softening awakens zygotic contractility before preimplantation morphogenesis.

Summary (3 min read)

Introduction

  • During embryonic development, the shape of animal cells and tissues largely relies on the contractility of the actomyosin cortex (Coravos et al., 2017; Murrell et al., 2015; Özgüç and Maître, 2020).
  • Contractile stresses of the actomyosin cortex mediate crucial cellular processes such as the ingression of the cleavage furrow during cytokinesis (Fujiwara and Pollard, 1976; Straight et al., 2003; Yamamoto et al., 2021), the advance of cells’ back during migration (Eddy et al., 2000; Tsai et al., 2019) or the retraction of blebs (Charras et al., 2006; Taneja and Burnette, 2019).
  • During mouse preimplantation development, PeCoWaCo become visible before compaction (Maître et al., 2015), the first morphogenetic movements leading to the formation of the blastocyst (Maître, 2017; Özgüç and Maître, 2020; White et al., 2018).
  • Interestingly, although removing cellcell contacts free-up a large surface for the contractile waves to propagate, the oscillation period seems robust to the manipulation (Maître et al., 2015).

Results

  • PeCoWaCo during cleavage stages PeCoWaCo have been observed at the 8-, 16-cell, and blastocyst stages.
  • This is further supported by the fact that PeCoWaCo are detected at the same rate and with the same oscillation period during the early or late halves of the 2-, 4- and 8-cell stages (Fig S1).
  • Proportion of cells showing detectable oscillations and their detected period.

Control

  • Furthermore, the period of oscillation is identical to 4-cell stage embryos in both control and drug-treated conditions (Fig 2D).
  • First, the authors analyzed for the presence of PeCoWaCo in dissociated 2-cell stage blastomeres and then proceeded to reduce their size (Fig 2EF, Movie 3).
  • To test whether cell size determines PeCoWaCo oscillation period, the authors set out to manipulate cell size over a broad range.
  • CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Discussion

  • During cleavage stages, blastomeres halve their size with successive divisions.
  • Therefore, during cleavage stages, cortical softening awakens zygotic contractility before preimplantation morphogenesis.
  • Keratin intermediate filaments appear at the onset of blastocyst morphogenesis (Schwarz et al., 2015) and become preferentially inherited by prospective TE cells (Lim et al., 2020).
  • As cell-cell contacts grow during compaction and apical domains expand (Korotkevich et al., 2017; Zenker et al., 2018), the available excitable cortical area for PeCoWaCo eventually vanishes.
  • Together, their study uncovers the maturation of the actomyosin cortex, which softens and speeds up the rhythm of contractions during the cleavage stages of the mouse embryo.

Author contributions

  • V. K. wrote the curvature analysis code following Ö. Ö. and J.-L.M. initial plans.
  • Ö. Ö. and J.-L.M. acquired funding, analyzed the data, designed the project and wrote the manuscript.

Recovery and culture

  • All animal work is performed in the animal facility at the Institut Curie, with permission by the institutional veterinarian overseeing the operation (APAFIS #11054- 2017082914226001).
  • The animal facilities are operated according to international animal welfare rules.
  • Embryos are isolated from superovulated female mice mated with male mice.
  • Only embryos surviving the experiments were analyzed.

Isolation of Blastomeres

  • ZP-free 2-cell or 4-cell stages embryos are aspirated multiple times (typically between 3– 5 times) through a smoothened glass pipette (narrower than the embryo but broader than individual cells) until dissociation of cells.
  • For 16-cell-stage embryos, they are placed into EDTA containing Ca2+ free KSOM (Biggers et al., 2000) for 8–10 min before dissociation.
  • Cells are then washed with KSOM for 1 h before experiment.

Chemical reagents and treatments

  • To prevent mitosis, 2-cell-stage embryos are cultured in 2.5 µM Vx-680 for 3 h shortly prior to the 2nd cleavage and then washed in KSOM.
  • 2-3 repeated aspirations are typically sufficient to clip cells into to 2 large fragments, one containing the nucleus and one without.
  • HVJ envelope is resuspended following manufacturer’s instructions and diluted in FHM for use.
  • To fuse blastomeres of embryos at the 16-cell stage, embryos are incubated in 1:50 HVJ envelope for 15 min at 37°C followed by washes in KSOM.
  • To soften cells, 2-cell stage embryos are imaged in medium containing Latrunculin A covered with mineral oil for 2 h.

Microscopy

  • MTmG embryos are imaged at the 2- and 16- cell stage using an inverted Zeiss Observer Z1 microscope with a CSU-X1 spinning disc unit .
  • Excitation is achieved using a 561 nm laser through a 63x/1.2 C Apo Korr water immersion objective.
  • The microscope is equipped with an incubation chamber to keep the sample at 37°C and supply the atmosphere with 5% CO2.

Image analysis

  • Manual shape measurements FIJI (Schindelin et al., 2012) is used to measure cell, embryo, pipette sizes, and wave velocity.
  • Spectra of individual embryos are checked for the presence of a distinct amplitude peak to extract the oscillation period.
  • The peak value between 50 s and 200 s was taken as the amplitude, as this oscillation period range is detectable by their imaging method.
  • The strip is then moved by 1 pixel along the segmented cell and a new circle is fitted.
  • Kymograph of local curvature values around the perimeter over time is produced by plotting the perimeter of the strip over time.

Statistics

  • Data are plotted using Excel and R-based SuperPlotsOfData tool (Goedhart, 2021).
  • Mean, standard deviation, median, one-tailed Student’s t-test, Fisher exact test, and Chi2 p values are calculated using Excel or R (R Foundation for Statistical Computing).
  • Statistical significance is considered when p < 10-2. The sample size was not predetermined and simply results from the repetition of experiments.
  • No sample that survived the experiment, as assessed by the continuation of cell divisions, was excluded.
  • The investigators were not blinded during experiments.

Code availability

  • The code used to analyze the oscillation frequencies from PIV and local curvature analyses can be found at github.com/MechaBlasto/PeCoWaCo.git.
  • The Fiji plugin for local curvature analysis WizardofOz can be found under the MTrack repository.

Movie legends

  • Particle image velocimetry (PIV) analysis during cleavage stages, also known as Movie 1.
  • Pictures are taken every 5 s and PIV analysis is performed between two consecutive images.
  • PIV vectors are overlaid on top of the images with vectors pointing upward in magenta and downward in green.
  • Time-lapse imaging of 4-cell stage embryos showing PeCoWaCo after treatment with DMSO or 2.5 µM Vx680 at the time of the 2nd cleavage division.
  • Surface deformation tracking of fragmented 16-cell-stage blastomeres, also known as Movie 5.

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Zygotic contractility awakening during mouse preimplantation development
Özge Özgüç
1
, Ludmilla de Plater
1
, Varun Kapoor
1
, Anna Francesca Tortorelli
1
and
Jean-Léon Maître
1
*
1
Institut Curie, PSL Research University, Sorbonne Universite!, CNRS UMR3215, INSERM U934, Paris,
France.
*Correspondence to jean-leon.maitre@curie.fr
Abstract
Actomyosin contractility is a major engine of preimplantation morphogenesis, which starts at
the 8-cell stage during mouse embryonic development. Contractility becomes first visible with
the appearance of periodic cortical waves of contraction (PeCoWaCo), which travel around
blastomeres in an oscillatory fashion. How contractility of the mouse embryo becomes active
remains unknown. We have taken advantage of PeCoWaCo to study the awakening of
contractility during preimplantation development. We find that PeCoWaCo become detectable
in most embryos only after the 2
nd
cleavage and gradually increase their oscillation frequency
with each successive cleavage. To test the influence of cell size reduction during cleavage
divisions, we use cell fusion and fragmentation to manipulate cell size across a 20-60 µm
range. We find that the stepwise reduction in cell size caused by cleavage divisions does not
explain the presence of PeCoWaCo or their accelerating rhythm. Instead, we discover that
blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially
softening cells enhances PeCoWaCo prematurely. Therefore, during cleavage stages, cortical
softening awakens zygotic contractility before preimplantation morphogenesis.
Introduction
During embryonic development, the
shape of animal cells and tissues largely
relies on the contractility of the actomyosin
cortex (Coravos et al., 2017; Murrell et al.,
2015; Özgüç and Maître, 2020). The
actomyosin cortex is a sub-micron-thin layer
of crosslinked actin filaments, which are put
under tension by non-muscle myosin II
motors (Kelkar et al., 2020). Tethered to the
plasma membrane, the actomyosin cortex is
a prime determinant of the stresses at the
surface of animal cells (Clark et al., 2014;
Kelkar et al., 2020). Contractile stresses of
the actomyosin cortex mediate crucial cellular
processes such as the ingression of the
cleavage furrow during cytokinesis (Fujiwara
and Pollard, 1976; Straight et al., 2003;
Yamamoto et al., 2021), the advance of cells’
back during migration (Eddy et al., 2000; Tsai
et al., 2019) or the retraction of blebs
(Charras et al., 2006; Taneja and Burnette,
2019). At the tissue scale, spatiotemporal
changes in actomyosin contractility drive
apical constriction (Martin et al., 2009; Solon
et al., 2009) or the remodeling of cell-cell
contacts (Bertet et al., 2004; Maitre et al.,
2012). Although tissue remodeling takes
place on timescales from tens of minutes to
hours or days, the action of the actomyosin
cortex is manifest on shorter timescales of
tens of seconds (Coravos et al., 2017;
Michaud et al., 2021; Özgüç and Maître,
2020). In fact, actomyosin is often found to act
via pulses of contraction during
morphogenetic processes among different
animal species from nematodes to human
cells (Baird et al., 2017; Bement, 2015;
Blanchard et al., 2010; Kim and Davidson,
2011; Maître et al., 2015; Martin et al., 2009;
Munro et al., 2004; Roh-Johnson et al., 2012;
Solon et al., 2009). A pulse of actomyosin
begins with the polymerization of actin
filaments and the sliding of myosin mini-
filaments until maximal contraction of the
local network within about 30 s (Dehapiot et
al., 2020; Ebrahim et al., 2013; Martin et al.,
2009). Then, the actin cytoskeleton
disassembles, and myosin is inactivated,
which relaxes the local network for another 30
s (Mason et al., 2016; Munjal et al., 2015;
Vasquez et al., 2014). These cycles of
contractions and relaxations are governed by
the turnover of the Rho GTPase and its
effectors, which are well-characterized
regulators of actomyosin contractility
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451745doi: bioRxiv preprint
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451745doi: bioRxiv preprint
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451745doi: bioRxiv preprint
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(Bement, 2015; Graessl et al., 2017; Munjal
et al., 2015). In instances where a sufficient
number of pulses occur, pulses of contraction
display a clear periodicity. The oscillation
period of pulsed contractions ranges from 60
to 200 s (Baird et al., 2017; Bement, 2015;
Maître et al., 2015; Solon et al., 2009). The
period appears fairly defined for cells of a
given tissue but can vary between tissues of
the same species. What determines the
oscillation period of contraction is poorly
understood, although the Rho pathway may
be expected to influence it (Bement, 2015;
Munjal et al., 2015; Vasquez et al., 2014).
Finally, periodic contractions can propagate
into travelling waves. Such periodic cortical
waves of contraction (PeCoWaCo) were
observed in cell culture, starfish, and frog
oocytes as well as in mouse preimplantation
embryos (Bement, 2015; Driscoll et al., 2015;
Kapustina et al., 2013; Maître et al., 2015). In
starfish and frog oocytes, mesmerizing Turing
patterns of Rho activation with a period of 80
s and a wavelength of 20 µm appear in a cell
cycle dependent manner (Bement, 2015;
Wigbers et al., 2021). Interestingly,
experimental deformation of starfish oocytes
revealed that Rho activation wave front may
be coupled to the local curvature of the cell
surface (Bischof et al., 2017), which was
proposed to serve as a mechanism for cells
to sense their shape (Wigbers et al., 2021). In
mouse embryos, PeCoWaCo with a period of
80 s were observed at the onset of blastocyst
morphogenesis (Maître et al., 2015; Maître et
al., 2016). What controls the propagation
velocity, amplitude, and period of these
waves is unclear and the potential role of
such evolutionarily conserved phenomenon
remains a mystery.
During mouse preimplantation
development, PeCoWaCo become visible
before compaction (Maître et al., 2015), the
first morphogenetic movements leading to the
formation of the blastocyst (Maître, 2017;
Özgüç and Maître, 2020; White et al., 2018).
During the second morphogenetic
movement, prominent PeCoWaCo are
displayed in prospective inner cells before
their internalization (Maître et al., 2016). In
contrast, cells remaining at the surface of the
embryo display PeCoWaCo of lower
amplitude due to the presence of a domain of
apical material that inhibits the activity of
myosin (Maître et al., 2016). Then, during the
formation of the blastocoel, high temporal
resolution time-lapse hint at the presence of
PeCoWaCo as microlumens coarsen into a
single lumen (Dumortier et al., 2019).
Therefore, PeCoWaCo appear throughout
the entire process of blastocyst formation
(Özgüç and Maître, 2020). However, little is
known about what initiates and regulates
PeCoWaCo. The analysis of maternal zygotic
mutants suggests that PeCoWaCo in mouse
blastomeres result primarily from the action of
the non-muscle myosin heavy chain IIA
(encoded by Myh9) rather than IIB (encoded
by Myh10) (Schliffka et al., 2021).
Dissociation of mouse blastomeres indicates
that PeCoWaCo are cell-autonomous since
they persist in single cells (Maître et al.,
2015). Interestingly, although removing cell-
cell contacts free-up a large surface for the
contractile waves to propagate, the oscillation
period seems robust to the manipulation
(Maître et al., 2015). Similarly, when cells
form an apical domain taking up a large
portion of the cell surface, the oscillation
period does not seem to be different from
cells in which the wave can propagate on the
entire cell surface (Maître et al., 2016). This
raises the question of how robust PeCoWaCo
are to geometrical parameters, especially in
light of recent observations in starfish oocytes
(Bischof et al., 2017; Wigbers et al., 2021).
This question becomes particularly relevant
when considering that, during preimplantation
development, cleavage divisions halve cell
volume with each round of cytokinesis (Aiken
et al., 2004; Niwayama et al., 2019).
In this study, we investigate how the
contractility of the cleavage stages emerges
before initiating blastocyst morphogenesis.
We take advantage of the slow development
of the mouse embryo to study thousands of
pulsed contractions and of the robustness of
the mouse embryo to size manipulation to
explore the geometrical regulation of
PeCoWaCo. We discover that the initiation,
maintenance, or oscillatory properties of
PeCoWaCo do not depend on cell size.
Instead, we discover a gradual softening of
blastomeres with each successive cleavage,
which conceals PeCoWaCo. Together, this
study reveals how preimplantation
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contractility is robust to the geometrical
changes of the cleavage stages during which
the zygotic contractility awakens.
Results
PeCoWaCo during cleavage stages
PeCoWaCo have been observed at
the 8-, 16-cell, and blastocyst stages. To
know when PeCoWaCo first appear, we
imaged embryos during the cleavage stages
and performed Particle Image Velocimetry
(PIV) and Fourier analyses (Fig 1A-C, Movie
1). This reveals that PeCoWaCo are
detectable in fewer than half of zygote and 2-
cell stage embryos and become visible in
most embryos from the 4-cell stage onwards
(Fig 1D). Furthermore, PeCoWaCo only
display large amplitude from the 4-cell stage
onwards (Fig 1B-C). Interestingly, the period
of oscillations of the detected PeCoWaCo
Figure 1: Analysis of PeCoWaCo during cleavage stages. A) Representative images of
a short-term time-lapse overlaid with a subset of velocity vectors from Particle Image
Velocimetry (PIV) analysis during cleavage stages (Movie 1). Magenta for positive and
green for negative Y directed movement. Scale bars, 20 µm. B) Velocity over time for a
representative velocity vector of each embryo shown in A. C) Mean
power spectrum resulting from Fourier transform of PIV analysis of Zygote (grey, n = 13),
2-cell (blue, n = 22), 4-cell (orange, n = 33) and 8-cell (green, n = 22) stages embryos
showing detectable oscillations. Data show as mean ± SEM. D) Proportion of Zygote (grey,
n = 27), 2-cell (blue, n = 52), 4-cell (orange, n = 39), 8-cell stage (green, n =
34) embryos showing detectable oscillations after Fourier transform of PIV analysis. Light
grey shows non-oscillating embryos. Chi
2
p values comparing different stages are indicated.
E) Oscillation period of Zygote (grey, n = 13), 2-cell (blue, n = 22), 4-cell (orange, n = 33),
8-cell (green, n = 22) stages embryos. Larger circles show median values. Student’s t test
p values are indicated.
Analysis of PeCoWaCo during cleavage stages
A) Representative images of a shortterm timelapse overlaid with a subset of velocity vectors from Particle Image
Velocimetry (PIV) analysis during cleavage stages. Magenta for positive and green for negative Y directed
movement. Scale bars, 20 µm. B) Velocity over time for a representative velocity vector of 4-cell stage embryo
shown in A. C) Proportion of Zygote (grey, n = 27), 2-cell stage (blue, n = 52), 4-cell stage (orange, n = 39), 8-cell
stage (green, n = 34) embryos showing detectable oscillations after Fourier transform of PIV analysis. Chi
2
p value
comparing to xx is indicated D) Mean power spectrum resulting from Fourier transform of PIV analysis of Zygote
(grey, n = 13), 2-cell stage (blue, n = 22), 4-cell stage (orange, n = 33), 8-cell stage (green, n = 22) embryos
showing detectable oscillations. Data show mean ± SEM. E) Oscillation period of Zygote (grey, n = 13), 2-cell stage
(blue, n = 22), 4-cell stage (orange, n = 33), 8-cell stage (green, n = 22) embryos. Larger circles show median
values. Following One-way ANOVA with f-ratio = 32.741 and p-value < .00001, Post Hoc Tukey HSD result is
indicated on the graph.
10 10 0 10 00
Amplitude (µm/s)
Period (s)
0.6
0.3
0.0
Zygote
2-cell
4-cell
8-cell
p < 10
-2
p < 10
-7
p > 10
-1
250
200
150
50
100
0
Period (s)
Zygote 2-cell 4-cell 8-cell
A
B
C D E
0 20 0 40 0 60 0
Time (s)
-1
Velocity (µm/s)
1
-0.5
0
0.5
0 20 0 40 0 60 0
Time (s)
-1
Velocity (µm/s)
1
-0.5
0
0.5
0 200 40 0 60 0
Time (s)
-1
Velocity (µm/s)
1
-0.5
0
0.5
0 20 0 40 0 60 0
Time (s)
-1
Velocity (µm/s)
1
-0.5
0
0.5
Zygote 2-cell stage 4-cell stage 8-cell stage
0
50
10 0
Early
La te
Early
La te
Early
La te
2-cell 4-cell 8-cell
% of embryos with detected oscillation
0
50
10 0
Zygote 2-cell 4-cell 8-cell
13 22 33 22
14 30 6
12
% of embryos with
detected oscillation
p > 10
-2
p < 10
-2
D
0
50
10 0
Zygote 2-cell 4-cell 8-cell
13 22 33 22
14 30 6
12
% of embryos with
detected oscillation
p > 10
-1
p < 10
-2
p < 10
-4
p > 10
-2
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451745doi: bioRxiv preprint

shows a gradual decrease from 200 s to 80 s
between the zygote and 8-cell stages (Fig
1E). The acceleration of PeCoWaCo rhythm
could simply result from the stepwise
changes of cell size after cleavage divisions.
Indeed, we reasoned that if the contractile
waves travel at constant velocity, the period
will scale with cell size and shape. This is
further supported by the fact that PeCoWaCo
are detected at the same rate and with the
same oscillation period during the early or late
halves of the 2-, 4- and 8-cell stages (Fig S1).
Therefore, we set to investigate the
relationship between cell size and periodic
contractions.
Cell size is not critical for the initiation or
maintenance of PeCoWaCo
First, to test whether the initiation of
PeCoWaCo in most 4-cell stage embryos
depends on the transition from the 2- to 4-cell
stage blastomere size, we prevented
cytokinesis. Using transient exposure to Vx-
680 to inhibit the activity of Aurora kinases
Initiation of PeCoWaCo is independent of cell size.
Graphical description of Blocking division with Vx680. Proportion of embryos showing
detectable oscillations and their detected period. Control (n= 19) Vx-680 treated (n = 12)
Graphical description of cellular fragmentation at 2-cell stage. Proportion of cells showing
detectable oscillations and their detected period. before (n=17 ), Control (n = 8) , fragmented
cell (n= 8) fragment (n=9)
Isolated
blastomere
Isolated
blastomere
Mechanical
control
Fragmented
cell
Enucleated
fragment
Radius (µm)
40
20
0
30
10
.285871.
Control
Fragmented cell
Fragment
17
9
2 3
DMSO
Vx-680
% of embryos with detected oscillations
Period (s)
250
200
100
0
DMSO Vx-680
150
50
A
B
C D
E F
0
50
10 0
% of embryos with detected oscillations
Mechanical
control
Fragmented
cell
Enucleated
fragment
G
8 8 7
00 1
p > 10
-1
p > 10
-1
p > 10
-1
Figure 2: Initiation of PeCoWaCo is independent of cell size. A) Schematic diagram of
PeCoWaCo analysis after blocking the 2
nd
cleavage division with 2.5 µM Vx680. B)
Representative images of DMSO and Vx-680 treated embryos overlaid with a subset of
velocity vectors from Particle Image Velocimetry (PIV) analysis (Movie 2). C-D) Proportion
(C) of embryos showing detectable oscillations and their detected period (D, DMSO n = 19
and Vx-680 n = 12). Chi
2
(C) and Student’s t test (D) p values comparing two conditions
are indicated. Larger circles show median values. E) Schematic diagram of PeCoWaCo
analysis after fragmentation of 2-cell stage blastomeres. F) Representative images of
mechanical control, fragmented cell and enucleated fragments overlaid with a subset of
velocity vectors from PIV analysis (Movie 3). G) Proportion of cells showing detectable
oscillations in mechanical controls (n = 8), fragmented cells (n= 8) and enucleated
fragments (n=9). Fisher exact test p values comparing different conditions are indicated.
Light grey shows non-oscillating embryos.
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451745doi: bioRxiv preprint

triggering chromosome separation, we
specifically blocked the 2- to 4-cell stage
cytokinesis without compromising the next
cleavage to the 8-cell stage (Fig 2A-B, Movie
2). This causes embryos to reach the 4-cell
stage with tetraploid blastomeres the size of
2-cell stage blastomeres. At the 4-cell stage,
we detect PeCoWaCo in most embryos
whether they have 4- or 2-cell stage size
blastomeres (Fig 2C). Furthermore, the
period of oscillation is identical to 4-cell stage
embryos in both control and drug-treated
conditions (Fig 2D). This suggests that 4-cell
stage blastomere size is not required to
initiate PeCoWaCo in the majority of
embryos.
Then, we tested whether PeCoWaCo
could be triggered prematurely by artificially
reducing 2-cell stage blastomeres to the size
of a 4-cell stage blastomere. First, we
analyzed for the presence of PeCoWaCo in
dissociated 2-cell stage blastomeres and
then proceeded to reduce their size (Fig 2E-
F, Movie 3). To reduce cell size, we treated
dissociated 2-cell stage blastomeres with the
actin cytoskeleton inhibitor Cytochalasin D
before deforming repeatedly them into a
narrow pipette (Fig 2E, Fig S2). By adapting
the number of aspirations of softened
blastomeres, we could carefully fragment
blastomeres while keeping their sister cell
mechanically stressed but intact. While the
fragmented cell was reduced to the size of a
4-cell stage blastomere, both fragmented and
manipulated cells eventually succeeded in
dividing to the 4-cell stage. After waiting 1 h
for cells to recover from this procedure, we
examined for the presence of PeCoWaCo. At
the 2-cell stage, PeCoWaCo were rarely
detected in either control or fragmented cells
(Fig 2G). This suggests that 4-cell stage
blastomere size is not sufficient to trigger
PeCoWaCo in the majority of embryos.
Cell size does not influence the properties of
PeCoWaCo
The transition from 2- to 4-cell stage
blastomere size is neither required nor
sufficient to initiate PeCoWaCo.
Nevertheless, the decrease in period of
PeCoWaCo remarkably scales with the
stepwise decrease in blastomere size (Fig
1E). Given a constant propagation velocity,
PeCoWaCo may reduce their period
according to the reduced distance to travel
around smaller cells. To test whether cell size
determines PeCoWaCo oscillation period, we
set out to manipulate cell size over a broad
range. By fusing varying numbers of 16-cell
stage blastomeres, we built cells equivalent in
size to 8-, 4- and 2-cell stage blastomeres
(Fig 3A-D, Movie 4). In addition, by
fragmenting 16-cell stage blastomeres, we
made smaller cells equivalent to 32-cell stage
blastomeres (Fig 3E-G, Movie 5)(Niwayama
et al., 2019). Together, we could image 16-
cell stage blastomeres with sizes ranging
from 10 to 30 µm in radius (Fig 3H-I). Finally,
to identify how the period may scale with cell
size by adjusting the velocity of the contractile
wave, we segmented the outline of cells to
compute the local curvature, which, unlike
PIV analysis, allows us to track contractile
waves and determine their velocity in addition
to their period (Fig 3A) (Maître et al., 2015;
Maître et al., 2016). We find that fused and
fragmented 16-cell stage blastomeres show
the same period, regardless of their size (Fig
3H). This could be explained if the wave
velocity would scale with cell size. However,
we find that the wave velocity remains
constant regardless of cell size (Fig 3I).
Therefore, both the oscillation period and
wave velocity are properties of PeCoWaCo
that are robust to changes in cell size and
associated curvature.
Fusion of cells causes blastomeres to
contain multiple nuclei, while cell
fragmentation creates enucleated fragments.
Interestingly, enucleated fragments
continued oscillating with the same period
and showing identical propagation velocities
as the nucleus-containing fragments (Fig 3F).
These measurements indicate that
PeCoWaCo are robust to the absence or
presence of single or multiple nuclei and their
associated functions.
Together, using fusion and
fragmentation of cells, we find that
PeCoWaCo oscillation properties are robust
to a large range of size perturbations.
Therefore, other mechanisms must be at play
to regulate periodic contractions during
preimplantation development.
.CC-BY 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451745doi: bioRxiv preprint

References
More filters
Journal ArticleDOI
TL;DR: Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis that facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system.
Abstract: Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.

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Journal ArticleDOI
01 Sep 2007-Genesis
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Abstract: The Cre/loxP system has been used extensively for conditional mutagenesis in mice. Reporters of Cre activity are important for defining the spatial and temporal extent of Cre-mediated recombination. Here we describe mT/mG, a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision. We show that reporter expression is nearly ubiquitous, allowing visualization of fluorescent markers in live and fixed samples of all tissues examined. We further demonstrate that mG labeling is Cre-dependent, complementary to mT at single cell resolution, and distinguishable by fluorescence-activated cell sorting. Both membrane-targeted markers outline cell morphology, highlight membrane structures, and permit visualization of fine cellular processes. In addition to serving as a global Cre reporter, the mT/mG mouse may also be used as a tool for lineage tracing, transplantation studies, and analysis of cell morphology in vivo.

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Journal ArticleDOI
14 Mar 2003-Science
TL;DR: It is shown that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis and continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.
Abstract: Completion of cell division during cytokinesis requires temporally and spatially regulated communication from the microtubule cytoskeleton to the actin cytoskeleton and the cell membrane. We identified a specific inhibitor of nonmuscle myosin II, blebbistatin, that inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly. Using blebbistatin and other drugs, we showed that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis. Continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.

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Proceedings ArticleDOI
09 Jun 2011
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Abstract: Segmentation is the process of partitioning digital images into meaningful regions. The analysis of biological high content images often requires segmentation as a first step. We propose ilastik as an easy-to-use tool which allows the user without expertise in image processing to perform segmentation and classification in a unified way. ilastik learns from labels provided by the user through a convenient mouse interface. Based on these labels, ilastik infers a problem specific segmentation. A random forest classifier is used in the learning step, in which each pixel's neighborhood is characterized by a set of generic (nonlinear) features. ilastik supports up to three spatial plus one spectral dimension and makes use of all dimensions in the feature calculation. ilastik provides realtime feedback that enables the user to interactively refine the segmentation result and hence further fine-tune the classifier. An uncertainty measure guides the user to ambiguous regions in the images. Real time performance is achieved by multi-threading which fully exploits the capabilities of modern multi-core machines. Once a classifier has been trained on a set of representative images, it can be exported and used to automatically process a very large number of images (e.g. using the CellProfiler pipeline). ilastik is an open source project and released under the BSD license at www.ilastik.org.

1,158 citations

Journal ArticleDOI
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Abstract: Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. Here we show, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. Our results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally.

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Frequently Asked Questions (1)
Q1. What are the contributions mentioned in the paper "Zygotic contractility awakening during mouse preimplantation development" ?

The authors have taken advantage of PeCoWaCo to study the awakening of contractility during preimplantation development. To test the influence of cell size reduction during cleavage divisions, the authors use cell fusion and fragmentation to manipulate cell size across a 20-60 μm range. The authors find that the stepwise reduction in cell size caused by cleavage divisions does not explain the presence of PeCoWaCo or their accelerating rhythm. CC-BY 4. 0 International license made available under a ( which was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.