The EMBO Journal Peer Review Process File - EMBO-2016-94264
Manuscript EMBO-2016-94264
Polarized Cortical Tension drives Zebrafish Epiboly
Movements
Amayra Hernandez-Vega, Maria Marsal, Philippe-Alexandre Pouille, Sebastien Tosi, Julien
Colombelli, Tomas Luque, Daniel Navajas, Ignacio Pagonabarraga and Enrique Martin-Blanco
Corresponding author: Enrique Martin-Blanco, Instituto de Biologia Molecular de Barcelona CSIC
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Editorial Decision:
Revision received:
Accepted:
17 March 2016
13 May 2016
31 July 2016
02 September 2016
18 September 2016
26 September 2016
Editor: David del Alamo
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
1st Editorial Decision
13 May 2016
Thank you for the submission of your manuscript entitled "Polarized cortical tension drives
zebrafish epiboly movements" and for your patience during the review process. We have now
received and analyzed the reports from two of the referees that accepted to evaluate your work,
which I copy below. The third referee has not sent his/her report, but we will forward it to you in
case we receive it.
As you can see from their comments, both referees are supportive of your work, but point out to a
number of significant concerns that will require your attention before your manuscript can be
published in The EMBO Journal. I will not repeat here the referee concerns, but in summary, both
referees agree that the manuscript is only poorly organized and unnecessarily complex.
Additionally, questions regarding the comparison of the method proposed with other existing
methods are being raised, and further experimental validation might be required. I believe the
concerns of the referees are reasonable and addressable, but please contact me if you have any
questions, need further input on the referee comments or if you anticipate any problems in
addressing any of their points.
-----------------------------------------------REFEREE REPORTS
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2016-94264
Referee #1:
In this manuscript, Hernandez-Vega and colleagues develop a novel, non-invasive method to
quantify mechanical properties in living animals at an intermediate scale between cells and tissues.
Using this approach, the authors investigate the mechanical contribution of different structures (the
Enveloping Layer - EVL, the External Yolk Syncytial Layer - E-YSL and the yolk) to epiboly in
zebrafish. First, the authors characterize the increasing speed of the EVL and the decreasing width
of the E-YSL as epiboly progresses. They use particle image velocimetry to demonstrate the
presence of vortices in the yolk, beneath the cortex. They introduce hydrodynamic regression (HR)
to estimate cortical stress based on the flows immediately above and below the cortex. Using HR,
the authors make predictions about local surface stress patterns, and they confirm those predictions
using laser microsurgery and atomic force microscopy. Based on the patterns identified by HR, the
authors propose that epiboly, at least during its second half, is driven by a combination of contractile
force at the E-YSL and differential stiffness along the animal-vegetal pole of the animal, with
greater stiffness at the yolk that at the EVL. Simulations using the HR framework confirm that the
proposed model could explain the dynamics of epiboly.
The manuscript is very interesting from the methodological and biological points of view. However,
there are substantial problems with both aspects of the work. From the method-development
perspective, the establishment of non-invasive methods to measure tissue mechanics in vivo is
critical for our understanding of animal development. However, some such methods already exist
(see below) and no comparison is provided to discuss the advantages and disadvantages of the
proposed technique. Furthermore, the methodological section is long, with lots of redundancies
between the supplement and the main text, and, more concerning, is written in an obscure way that
makes it difficult to follow. The organization of the figures and the absence of details in some cases
do not help. From the biological perspective, the model put forward by the authors is interesting, but
the validation is limited, and without further experimental evidence disrupting the yolk or EVL
stiffness, the manuscript may be more appropriate for a specialized journal in the field of biophysics.
I propose to address the following points before publication:
MAJOR
1. Image-based methods have been previously developed to estimate cell and tissue mechanics. See
for instance (Ishihara and Sugimura, J Theor Biol, 2012; Brodland et al., PLoS One, 2014). The
authors should at least discuss the advantages and disadvantages of their method with respect to
some of the existing ones.
2. Several values appear with no obvious calculation or references. The sources for these values
should be explicitly provided. For instance, yolk density (pg. 8), cortical elastic modulus (pg. 9) and
cortical viscosity at long time scales (pg. 9). What cortical width are the authors considering in pg.
9?
3. The authors should show images of their AV and CC laser ablation experiments, either in Figure
5 or in Supp. Fig. 7. Otherwise, it is difficult to judge the experiments simply based on the plots.
4. The authors should validate (using laser cuts or AFM) the HR prediction that at 60-80% epiboly
the difference between CC and AV tension is positive at the EVL side of the E-YSL and negative in
the yolk (Figure 4e); and that the CC tension switches from negative to positive (Figure 4e) at the
interface between EVL and yolk, particularly when the authors are arguing mechanical differences
between EVL and yolk are critical for epiboly to progress.
5. In Figure 7, the authors should show that msn1 morpholinos affect actomyosin recruitment to the
E-YSL, and try to control for the specificity of the treatment. Are actomyosin dynamics normal in
the EVL and the yolk cortex in msn1 morphants? If not, could that explain part of the epiboly
phenotype?
6. The authors propose that increased stiffness at the yolk cortex with respect to the EVL promotes
epiboly. How is the stiffness of the yolk regulated? Why is the yolk cortex stiffer than the EVL?
Why does its stiffness increase as epiboly progresses?
7. According to their model, after 50% epiboly the EVL does not actively contribute to epiboly. Can
© European Molecular Biology Organization
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the authors disrupt the EVL and show that epiboly still happens (maybe even faster if they can
decrease its stiffness)? Alternatively, can the authors reduce the stiffness of the yolk (maybe by
making repeated incisions with their laser) and demonstrate that that is sufficient to locally stop
epiboly?
MINOR
1. The description of hydrodynamic regression should move to the Methods (right now it is in the
middle of the Results section). In the Results, perhaps the authors can provide an explanation that is
easier to follow by a biological readership.
2. If I understand HR correctly, flow should only be measured within a relatively thin layer around
the cortex; otherwise, friction may affect interpretation of the results. The authors should explicitly
indicate the "thickness" of tissue used to quantify flows for HR analysis.
3. In Figure 1a, the authors should indicate the three different axes (r, theta and phi) to make things
easier for non-zebrafish readers.
4. In Figure 2, tau refers to time; in hydrodynamic regression (Figure 3), tau refers to cortical
tension. The authors should use different symbols/greek letters to represent each of these
parameters.
5. In plots, authors should display the units next to the axis label (e.g. Fig. 2b-e). What are the units
of the y axis in 2e? They are not indicated in the figure legend either.
6. The panel names in Figure 3 do not match the legend (e.g. the legend for Figure 3c corresponds to
panel 3b in the figure).
7. In Figure 4c, what is the difference between the three images shown in the top row? If they are in
different stages of epiboly, that should be indicated.
8. In Figures 4d and 4e, displaying both the progress into epiboly and the RMSE as a percentage and
side by side is confusing. Maybe the RMSE can be shown to the right of the text "Pressure (or
Power) RMSE:", and the progress through epiboly can be shown on top of the images as "40%
epiboly", "60% epiboly", etc.
9. In Figure 4e, is phi the horizontal axis? The authors should label the horizontal axes of the three
plots in this panel, and add the range of values that this angle can take, as in Supp. Fig. 4.
10. What are the units in the Y-axis in Figure 5a for differential tension and stress?
11. What is the normalized recoil velocity shown in Figure 5b normalized to?
12. The authors should indicate the marker shown in Figure 6a.
TYPOS
1. Page 3, paragraph 2, line 5: "Syncitial" should be "Syncytial".
2. Pg. 4, p. 1, l. 8: "19 20" should be "19, 20".
3. Pg. 6, p. 2, l.1: "although appears to be clear" should be "although it appears to be clear".
4. Pg. 6, p. 2, l. 3: please revise.
5. Pg. 6, last paragraph, l. 2: "HR let to retrieve" should be "HR allows retrieval of".
6. Pg. 7, p. 2, l. 13: "REFS" should be fixed.
7. Pg. 7, p. 4: "the rate of mechanical energy produced by the unbalanced cortical tension per unit of
time" is redundant, and should be "the rate of mechanical energy produced by the unbalanced
cortical tension" or "the mechanical energy produced by the unbalanced cortical tension per unit of
time".
8. Pg. 11, p. 4, l. 11: "mesoscopicaly" should be mesoscopically.
9. Pg. 14, p. 2, l. 3: "As stands today" should be "As it stands".
Referee #2:
© European Molecular Biology Organization
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This manuscript describes and models the epiboly movement in the zebrafish embryo. Epiboly is a
conserved morphogenetic movement, during which cell layers dramatically reorganize. The current
description of this movement posits that spreading of the enveloping layer (EVL) over the yolk cell
is driven by a contractile actomyosin ring at the margin of the EVL. According to this model, the
contractile ring would produce a flow-friction force, enabling the spreading of the EVL . Here the
authors propose an alternative mechanism, based on polarized cortical tension.
The novelty of the present manuscript resides in the method used to infer stresses and the
combination of theoretical description and mechanical measurements. The method, called
Hydrodynamic Regression (HR), infer stresses from velocity measurements. Using such inference
and rheological measurements, the authors are able to predict the epiboly movement with rather
good accuracy. The results shown in this article are interesting but the authors fail to present them in
a comprehensive way.
The organization of the manuscript is such that I have had a lot of difficulties to follow the
argumentation, and one needs to navigate back and forth in the manuscript and in the SI to
understand the findings. I think the manuscript should be rewritten and the dialogue between
experiments and analysis/model should be clarified. What is part of verification of the method ?
What are the hypotheses and what are experiments/analyses to test them ?
I see important possible improvements by :
avoiding jargon (e.g. dynamical stresses);
checking consistency of notation (e.g. time is tau in Fig 2, but tau refers to cortical tension
elsewhere);
checking the figures: some units are missing (Fig 2), or incorrect (g' and g', in pN/um in Fig 4b) and
labelling (Fig 2 b and c)
I understand that this manuscript encompasses a broad spectrum of approaches and disciplines ; this
requires a very careful writing, especially if the aim of the authors is to reach a broad audience.
1st Revision - authors' response
31 July 2016
Answers to Reviewer's comments (italics)
Referee #1:
In this manuscript, Hernandez-Vega and colleagues develop a novel, non-invasive method to
quantify mechanical properties in living animals at an intermediate scale between cells and tissues.
Using this approach, the authors investigate the mechanical contribution of different structures (the
Enveloping Layer - EVL, the External Yolk Syncytial Layer - E-YSL and the yolk) to epiboly in
zebrafish. First, the authors characterize the increasing speed of the EVL and the decreasing width
of the E-YSL as epiboly progresses. They use particle image velocimetry to demonstrate the
presence of vortices in the yolk, beneath the cortex. They introduce hydrodynamic regression (HR)
to estimate cortical stress based on the flows immediately above and below the cortex. Using HR,
the authors make predictions about local surface stress patterns, and they confirm those predictions
using laser microsurgery and atomic force microscopy. Based on the patterns identified by HR, the
authors propose that epiboly, at least during its second half, is driven by a combination of contractile
force at the E-YSL and differential stiffness along the animal-vegetal pole of the animal, with
greater stiffness at the yolk that at the EVL. Simulations using the HR framework confirm that the
proposed model could explain the dynamics of epiboly.
The manuscript is very interesting from the methodological and biological points of view. However,
there are substantial problems with both aspects of the work. From the method-development
perspective, the establishment of non-invasive methods to measure tissue mechanics in vivo is
critical for our understanding of animal development. However, some such methods already exist
(see below) and no comparison is provided to discuss the advantages and disadvantages of the
proposed technique. Furthermore, the methodological section is long, with lots of redundancies
between the supplement and the main text, and, more concerning, is written in an obscure way that
makes it difficult to follow. The organization of the figures and the absence of details in some cases
do not help. From the biological perspective, the model put forward by the authors is interesting, but
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2016-94264
the validation is limited, and without further experimental evidence disrupting the yolk or EVL
stiffness, the manuscript may be more appropriate for a specialized journal in the field of biophysics.
I propose to address the following points before publication:
MAJOR
1. Image-based methods have been previously developed to estimate cell and tissue mechanics. See
for instance (Ishihara and Sugimura, J Theor Biol, 2012; Brodland et al., PLoS One, 2014). The
authors should at least discuss the advantages and disadvantages of their method with respect to
some of the existing ones.
A thorough discussion comparing HR with those methods from Ishihara and Sugimura and
Brodland et al was already presented in Supplementary Note 8 (Pages 25-26).
The Bayesian method of Ishihara and Sugimura was developed for an epithelia at rest where all
elastic forces are equilibrated. It could not be applied in dynamic morphogenetic movements with
contributions of materials with different properties (elastic and viscous). Brodland et al take into
account edge tensions and internal pressures in cells but do not consider viscosity. They only
consider in plane forces and not the third dimension. This method could not be applied to the
process of epiboly where we know that the movements in the yolk are important for the overall
equilibrium of forces.
2. Several values appear with no obvious calculation or references. The sources for these values
should be explicitly provided. For instance, yolk density (pg. 8), cortical elastic modulus (pg. 9) and
cortical viscosity at long time scales (pg. 9). What cortical width are the authors considering in pg.
9?
The yolk density had been previously measured and is very similar to that of water. The reference
was missing in the previous version and it has been added in the current one: Fujimura, Y., et al
(2007) (Page 8).
The cortical elastic modulus and the cortical viscosity were inferred from the analysis of the AFM
data. The appropriate calculations were performed via Equations 43, 47 and 49 in Supplementary
Methods and Supplementary Note 5 (we computed the cortex shear modulus assuming the contact
model of an equivalent elastic half-space indented by a spherical tip by AFM). The texts in Page 8 of
the main manuscript and Pages 17-18 and 30-33 of the Supplementary Text have been slightly
modified to make these points more clear.
Precise measurements provide values between 1 and 4 mm for the yolk cortex thickness (see Figure
S2 and Rawson et al., 2000). In any case, the cortex depth will always be at least two orders of
magnitude smaller than the embryo diameter. These data and reference are now discussed in the text
(Page 8).
3. The authors should show images of their AV and CC laser ablation experiments, either in Figure
5 or in Supp. Fig. 7. Otherwise, it is difficult to judge the experiments simply based on the plots.
Images of perpendicular and parallel cuts have now been added to the Supplementary Figure 7 and
refereed in the text (Page 10).
4. The authors should validate (using laser cuts or AFM) the HR prediction that at 60-80% epiboly
the difference between CC and AV tension is positive at the EVL side of the E-YSL and negative in
the yolk (Figure 4e); and that the CC tension switches from negative to positive (Figure 4e) at the
interface between EVL and yolk, particularly when the authors are arguing mechanical differences
between EVL and yolk are critical for epiboly to progress.
Our own laser cuts in the yolk (Figure 5), together with those described in the literature for the yolk
and EVL marginal cells (Campinho et al., 2013) fully validate our claims. Campinho et al found no
differences in the recoiled velocity between parallel (AV) and perpendicular cuts (CC) in the
marginal EVL cells at the beginning of epiboly. However, as epiboly progressed, AV tension
increased (parallel cuts) while CC tension decreased. Thus, a difference between AV and CC
tension, being AV higher than CC, was generated over time. In the yolk the AV vs. CC differences,
which develop throughout epiboly are opposite to those of the marginal cells, being AV lower than
CC.
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In the kymograph shown in Figure 6A (New added Panel) can also be clearly observed that, upon
laser microsurgery close to the EVL margin at the yolk, the wound edges are pulled towards the
vegetal pole. This, indirectly, also points to a lower tensional contribution at the EVL when
compared to the yolk.
We now discuss these issues in more detail in the text (Discussion - Page 14).
5. In Figure 7, the authors should show that msn1 morpholinos affect actomyosin recruitment to the
E-YSL, and try to control for the specificity of the treatment. Are actomyosin dynamics normal in
the EVL and the yolk cortex in msn1 morphants? If not, could that explain part of the epiboly
phenotype?
It has been shown that Msn1 MOs (msn1MO-splice) (Koppen et al, 2006), despite not leading to
complete protein loss, show a reproducible delay in epiboly. We fully reproduced this observation.
This effect was yolk specific as it was suppressed by injection of wild type msn1 mRNA in the YSL
(Koppen et al, 2006). Indeed, epiboly delay was induced by late injection of Msn1 MO in the yolk
after compartmentalization (our own data - Figure 7).
Actin and phospho-myosin 2 failed to accumulate at the YSL in morphants and the EVL cells at the
margin do not stretch longitudinally (Koppen et al, 2006). ZO-1 accumulation (septate junctions)
was not affected. Altogether, these data provide strong support for a specific role of Msn1 in the
recruitment of actin and myosin 2 to the YSL. This information is now discussed in the text (Page
11).
6. The authors propose that increased stiffness at the yolk cortex with respect to the EVL promotes
epiboly. How is the stiffness of the yolk regulated? Why is the yolk cortex stiffer than the EVL?
Why does its stiffness increase as epiboly progresses?
These questions are extremely relevant but, in our opinion, they go well beyond the scope of the
present report. We do not know how the stiffness of the yolk is regulated or why increases over time
and becomes much stiffer than the EVL although we are currently studying these issues in depth.
We could, however, speculate. The EVL cells could easily respond to nearby pulling forces from the
YSL increasing their apical surface area at the expense of their height, flattening passively. The
yolk, on the contrary, may have no way to increase its surface area in response to the YSL
contraction. As a result of these differences, tension would accumulate at the yolk membrane as
epiboly progresses, which would lead to an overall progressive reduction of stiffness of the EVL.
Indeed, when we artificially increased myosin contraction in the YSL by microsurgery, we observed
that nearby EVL cells deformed more than faraway cells, while the vegetal part of the myosin ring
did not deform (see updated Figure 6A panel). We introduced this discussion in the manuscript
(Page 16).
7. According to their model, after 50% epiboly the EVL does not actively contribute to epiboly. Can
the authors disrupt the EVL and show that epiboly still happens (maybe even faster if they can
decrease its stiffness)? Alternatively, can the authors reduce the stiffness of the yolk (maybe by
making repeated incisions with their laser) and demonstrate that that is sufficient to locally stop
epiboly?
Trinkaus and colleagues performed different types of surgical interventions in Fundulus at the
contact edge between the EVL and the yolk (Betchaku and Trinkaus, 1978). These authors observed
that when the EVL/yolk contact margin was locally disrupted (1/8 of the circumferential perimeter),
the E-YSL progressed faster towards the vegetal pole (as no resistance by the EVL was placed to its
forward movement) and the yolk closure occurred excentrically, biased towards the nonmanipulated areas of the EVL perimeter. Further, when the contact between the EVL and the yolk
was disrupted the nearby EVL cells retracted indicating that the EVL/yolk contact was under
tension. This last outcome has also been observed for zebrafish (Behrndt et al., 2012). Thus, the
EVL, after 50% epiboly appears to limit the speed of epiboly progression.
We performed repeated laser incisions as suggested by the reviewer but they resulted uninformative
as a quick healing response was induced, leading to a great accumulation of actin and myosin. High
contractile responses masked any meaningful analysis.
We now discuss these data in the text (Page 15).
MINOR
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1. The description of hydrodynamic regression should move to the Methods (right now it is in the
middle of the Results section). In the Results, perhaps the authors can provide an explanation that is
easier to follow by a biological readership.
We consider essential to keep a section on the description and implementation of the hydrodynamic
regression (HR) method. However, we agreed with the reviewer that it was too complex in its
current format and we moved a substantial amount of information to the Methods section
simplifying the main text as much as we could.
2. If I understand HR correctly, flow should only be measured within a relatively thin layer around
the cortex; otherwise, friction may affect interpretation of the results. The authors should explicitly
indicate the "thickness" of tissue used to quantify flows for HR analysis.
The assessment of the reviewer is fully correct. The measurement of velocity gradients at the
proximity of the cortex can be employed to infer the values of the cortical stresses. However, the
determination of the velocity gradients adjacent to the cortex is in most cases not feasible
experimentally. We then relied on the evaluation of the complete velocity profile of surrounding
flows. Due to the force balance between the cortex and adjacent fluids, the cortical stresses can be
determined from the adjacent 3D fluid velocity fields. Further, as mentioned, fitting an analytical
solution of the 3D velocity field increases significantly the precision in the spatial derivations and
enables to compute the analytical dynamic pressure distribution. Stokeslet pairs model local cortical
contractions or expansions and are used to fit a simulated velocity field to the complete
experimental 3D velocity fields estimated by PIV.
We have simplified and clarified the text to avoid any confusion (see Supplementary Note 2).
3. In Figure 1a, the authors should indicate the three different axes (r, theta and phi) to make things
easier for non-zebrafish readers.
Animal (A) and vegetal (V) poles and φ (embryo surface - red) and r (internal to external axis green) are now indicated in Fig 1a and its legend.
4. In Figure 2, tau refers to time; in hydrodynamic regression (Figure 3), tau refers to cortical
tension. The authors should use different symbols/greek letters to represent each of these
parameters.
When referring to time, tau has been substituted by t in all figures (and in the main text, legends and
supplementary materials).
5. In plots, authors should display the units next to the axis label (e.g. Fig. 2b-e). What are the units
of the y axis in 2e? They are not indicated in the figure legend either.
Axis have been labeled in the figures and introduced in the legend when missing.
6. The panel names in Figure 3 do not match the legend (e.g. the legend for Figure 3c corresponds to
panel 3b in the figure).
The figure legend has been modified accordingly.
7. In Figure 4c, what is the difference between the three images shown in the top row? If they are in
different stages of epiboly, that should be indicated.
They indeed correspond to different epiboly stages equivalent to those showed in (d) and (e). They
have been labeled accordingly.
8. In Figures 4d and 4e, displaying both the progress into epiboly and the RMSE as a percentage and
side by side is confusing. Maybe the RMSE can be shown to the right of the text "Pressure (or
Power) RMSE:", and the progress through epiboly can be shown on top of the images as "40%
epiboly", "60% epiboly", etc.
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RMSE values for 4d and 4e have been moved to the bottom left of each panel and the labels
rearranged accordingly. Now, percentage of epiboly and RMSE labels are uncoupled.
9. In Figure 4e, is phi the horizontal axis? The authors should label the horizontal axes of the three
plots in this panel, and add the range of values that this angle can take, as in Supp. Fig. 4.
The horizontal axis in 4e is phi, which ranges from 0 to 1. This is indicated in the figure panel and
described in the legend.
10. What are the units in the Y-axis in Figure 5a for differential tension and stress?
In Figure 5a, as explained in the legend, are shown the differential tension represented by the recoil
velocities differences between CC and AV laser cuts (cyan) and their difference calculated by
regression analysis (blue) in normalized relative units fitted by applying a constant adjustment
ratio.
11. What is the normalized recoil velocity shown in Figure 5b normalized to?
We apologize for mistakenly labeling normalized velocities. The graph represents averaged recoiled
velocities (mm/s). The labeling has been corrected and the figure legend has been modified.
12. The authors should indicate the marker shown in Figure 6a.
Myosin-GFP transgenic embryo [Tg (β-actin:myl 12.1-e-GFP)]. This information has been
introduced in the figure legend.
TYPOS
1. Page 3, paragraph 2, line 5: "Syncitial" should be "Syncytial".
2. Pg. 4, p. 1, l. 8: "19 20" should be "19, 20".
3. Pg. 6, p. 2, l.1: "although appears to be clear" should be "although it appears to be clear".
4. Pg. 6, p. 2, l. 3: please revise.
5. Pg. 6, last paragraph, l. 2: "HR let to retrieve" should be "HR allows retrieval of".
6. Pg. 7, p. 2, l. 13: "REFS" should be fixed.
7. Pg. 7, p. 4: "the rate of mechanical energy produced by the unbalanced cortical tension per unit of
time" is redundant, and should be "the rate of mechanical energy produced by the unbalanced
cortical tension" or "the mechanical energy produced by the unbalanced cortical tension per unit of
time".
8. Pg. 11, p. 4, l. 11: "mesoscopicaly" should be mesoscopically.
9. Pg. 14, p. 2, l. 3: "As stands today" should be "As it stands".
All typos have been corrected.
Referee #2:
This manuscript describes and models the epiboly movement in the zebrafish embryo. Epiboly is a
conserved morphogenetic movement, during which cell layers dramatically reorganize. The current
description of this movement posits that spreading of the enveloping layer (EVL) over the yolk cell
is driven by a contractile actomyosin ring at the margin of the EVL. According to this model, the
contractile ring would produce a flow-friction force, enabling the spreading of the EVL . Here the
authors propose an alternative mechanism, based on polarized cortical tension.
The novelty of the present manuscript resides in the method used to infer stresses and the
combination of theoretical description and mechanical measurements. The method, called
Hydrodynamic Regression (HR), infer stresses from velocity measurements. Using such inference
and rheological measurements, the authors are able to predict the epiboly movement with rather
good accuracy. The results shown in this article are interesting but the authors fail to present them in
a comprehensive way.
The organization of the manuscript is such that I have had a lot of difficulties to follow the
argumentation, and one needs to navigate back and forth in the manuscript and in the SI to
© European Molecular Biology Organization
8
The EMBO Journal Peer Review Process File - EMBO-2016-94264
understand the findings. I think the manuscript should be rewritten and the dialogue between
experiments and analysis/model should be clarified. What is part of verification of the method ?
What are the hypotheses and what are experiments/analyses to test them ?
To reach a broader audience, the manuscript has been extensively edited and simplified without
losing scientific soundness. Hypothesis, models, methodologies, experiments, analyses and
conclusions are presented in logical order in each section.
I see important possible improvements by :
avoiding jargon (e.g. dynamical stresses);
Some expressions such as dynamic stress or power density have been simplified to mechanical stress
and power
checking consistency of notation (e.g. time is tau in Fig 2, but tau refers to cortical tension
elsewhere);
This issue has already been tackled (see answers to Reviewer 1).
checking the figures: some units are missing (Fig 2), or incorrect (g' and g', in pN/um in Fig 4b) and
labelling (Fig 2 b and c)
These issues have been already corrected (see answers to Reviewer 1 and amendments to Figures).
However, since the yolk behaves mechanically as an elastic cortex enclosing a viscous fluid and not
as a homogenous material we have computed an effective complex modulus (g*) as the complex
force-indentation ratio (equation 48, Supplementary Methods). This effective modulus plotted in
Figure 4b has units’ pN/mm. To compare the stiffness of the embryo with that of other biological
materials we also computed the complex shear modulus (G*) assuming the contact model of an
equivalent homogeneous half-space. We have clarified the difference definition of these two moduli
en Supplementary Page 33.
I understand that this manuscript encompasses a broad spectrum of approaches and disciplines ; this
requires a very careful writing, especially if the aim of the authors is to reach a broad audience.
See above.
Referee #3:
This is an excellent paper on the mechanics of epiboly in zebrafish. The authors have combined an
impressive array of biophysical measurement techniques (imaging, PIV, particle tracking, etc.)
along with a novel hydrodynamic method for interpreting flow patterns to arrive at a comprehensive
picture of the mechanics of this fascinating progress. I suspect this paper will be seen as a landmark
in the field.
There is, however, one clear weakness in the paper, which I trust the authors can fix. While the HR
method allows them to deduce force distributions throughout the organism, the body of the paper
does not provide any clear interpretation of the magnitude of those forces (and dipoles).
Applying AFM and microrheology we were able to extract diverse absolute values for several
parameters. The Reynold's number of the yolk fluid is 3.2 10-7, the viscosity of the yolk is 129 mPa·s,
the elastic modulus of the yolk cortex G’ ∼ 100 Pa, the effective cortex viscosity at long-time scales
(fluid-like response) is in the order of hc ~ 10 Pa.s, and the absolute values of cortical tension (Tc)
for the yolk membrane in the order of 100 pN / µm. However, HR cannot infer absolute power or
stress values and just provides relative spatio-temporal differences that could be normalized to the
overall power or stress of the embryo. Laser microsurgery does not help either as it, again, provides
only relative information (relative recoiled speed).
Yet, relative mechanical information is extremely relevant in our case as for morphogenetic events
the dynamic balance between the different tissues (dynamic equilibrium) and the relative stress
distribution are major instrumental elements. The absolute stress values will not help to understand
directional movements.
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2016-94264
On the other hand, we cannot directly interpret the magnitudes we measure. In our opinion this goes
beyond the focus of the paper. It goes into the question of the physical mechanisms that generate
these tensions. A hydrodynamic model cannot answer this issue.
In the text we present a full discussion about these issues in the Discussion (Page 13) and
Supplementary Note 8.
Moreover, there are places (such as Figure 6c&d and Figure 7d) in which no units are given for
surface stress. If we are to understand these forces to arise from, e.g. molecular motors and
biofilaments, I would expect there to be an interpretation in terms of numbers of motors, elastic
properties of filaments, etc. Can the authors provide this?
The graphs in Figures 4e, 6c, 6d and 7d correspond to normalized values in arbitrary units for AV
and CC stresses and for their differences plotted with respect to the surface angle (f) (from animal to
vegetal) at different time points. We have clarified these issues in the figure legends.
Our data (employing HR) infers the spatio-temporal pattern of power and stress during epiboly,
while our simulations, laser microsurgery and interference in yolk contractility point to a model in
which actomyosin contraction at the E-YSL together with a tensional gradient at the yolk surface
are instrumental for epiboly progression.
Beyond this scenario, we have not explored yet in quantitative terms the motors involved or the roles
that microtubules or actomyosin flows may have. We consider that this type of analysis would
require to develop specific analytical methods as the hydrodynamic method, as such, does not
provide any information about the physical mechanism that generates the stresses. Modeling making
use of the measured stresses may be employed as input to define the roles for microtubule bundles
or biofilaments and to predict their functional roles. These analyses are currently being unfold in
the laboratory and in our opinion go beyond the focus of the present manuscript. This issue is
discussed in Page 16 of the Discussion and the Supplementary Note 8.
Dimensional analysis would suffice. Moreover, is there a toy problem that can be worked out
analytically so the reader can understand the overall mechanics?
The dimensional analyses are not sensitive to the details of the geometry, which are those precisely
tackled by HR.
As a toy model we could consider the liquid droplet paradigm that we employed as a test for HR,
whose geometry is similar to that of the zebrafish embryo. In this model we go beyond simple
dimensional analyses applying HR to infer relative profiles of tension adapted to its geometry with
spatial resolution.
However, the complexity of the epiboly of the EVL and E-YSL goes well beyond this simple model
and we are reluctant to place it as an example that could mislead readers' interpretations.
I am reminded of the situation in cytoplasmic streaming in large plant cells [e.g. Chara; PNAS 105,
3663 (2008)] in which the flow inside the vacuole arises from molecular motors walking along actin
filaments. Measuring the flow [JFM 642, 5 (2010)] one finds consistency with a certain velocity
distribution at the cell periphery, which can ultimately be interpreted in terms of motor density and
forces [Plant Cell Physiol. 47, 1427 (2006)]. Perhaps there is a useful parallel here.
The first mentioned articles analyze the dynamics of the cytoplasmic flow in Chara and its relation
to the cytoskeleton. Velocity fields were determined by magnetic resonance and compared to a
kinematic model. The last paper deals with the potential role that myosin level may have in the
implementation of Chara's cytoplasm helicoidal streaming. In these works, the distributions of
power and/or stress were not inferred. Indeed, HR could be a very useful method to approach these
problems in this system. These articles are now mentioned in the Discussion. We also describe now
as related models the cytoplasmic streaming in C. elegans and in the mice oocyte (Nimayawa et al.,
2011, Yi et al., 2011), where it could be generated by stress anisotropies at the surface.
Minor issues:
In a few places the English is a bit awkward. I suggest a proofreading by a native speaker.
We have made a big effort to simplify the manuscript text and expect to have gain substantial
accessibility to its content.
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The EMBO Journal Peer Review Process File - EMBO-2016-94264
2nd Editorial Decision
02 September 2016
Thank you for the submission of your revised manuscript to The EMBO Journal. As you will see
below, your article was sent back to former referee #1, who now considers that you have properly
dealt with the main concerns originally raised in the review process, and therefore I am writing with
an 'accept in principle' decision. This means that I will be happy to formally accept your manuscript
for publication once a few more minor issues have been addressed.
As you can see from the referee report, a couple of minor suggestions have been made and we agree
with the referee that they would add some more clarity to the paper. Browsing through the
manuscript myself I have noticed another cosmetic issue that will need to be addressed in the final
version of the paper: error bars shown in several figures need to be defined in the figure legend.
-----------------------------------------------REFEREE REPORTS
Referee #1:
The authors have addressed most of my concerns. I just have two minor, additional suggestions:
1. I suggest moving the discussion of the advantages of HR with respect to other image-based
methods to the main text. I missed it as Supplementary Note 8, and other readers may also miss it.
Also, it would be important to discuss weaknesses (e.g. with regards to the resolution of the
measurements).
2. In Figure S7, the authors should include images of the cortex before ablation, and an overlay of
the flow field for a CC cut indicating, both for the AV and CC examples, the epiboly stage. It would
help if flow field vectors were colored consistent with panel S7E.
2nd Revision - authors' response
18 September 2016
We have added the reviewer and editor recommendations to our manscript.
3rd Editorial Decision
26 September 2016
I am pleased to inform you that your manuscript has been accepted for publication in the EMBO
Journal.
© European Molecular Biology Organization
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Manuscript Number: 2016-­‐94264R
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