Fluid extraction from the left-right organizer uncovers mechanical properties needed for symmetry breaking
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eLife assessment:
Sampaio and colleagues utilize an elegant approach to manipulate fluid dynamics in zebrafish Kupffer's vesicle to ask if fluid movement or something in the fluid governs the break in symmetry. These valuable results support a role for fluid movement and detection as important in breaking symmetry in a ciliated left-right organizer and help set a time window when fluid flow is critical for this process. However, as the fluid extraction experiments affect both chemical and physical features, the authors need to provide further convincing evidence to support their mechanosensory hypothesis or temper the claims.
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Abstract
Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development. The mechanism behind left-right establishment is not fully understood. Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a physiological developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Next, aiming to test the presence of any secreted determinant, we replaced the extracted LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered, demonstrating that symmetry breaking does not depend on the nature of the fluid content but is rather sensitive to fluid mechanics. Altogether, we conclude that the zebrafish LRO is more sensitive to fluid dynamics for symmetry breaking.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, Sampaio et al. tackle the role of fluid flow during left-right axis symmetry breaking. The left-right axis is broken in the left-right organiser (LRO) where cilia motility generates a directional flow that permit to dictate the left from the right embryonic side. By manipulating the fluid moved by cilia in zebrafish, the authors conclude that key symmetry breaking event occurs within 1 hour through a mechanosensory process.
Overall, while the study undeniably represents a huge amount of work, the conclusions are not sufficiently backed up by the experiments. Furthermore, the results provided present a limited advance to the field: the transient activity of the LRO is well established, and narrowing down this activity to 1 hour (even though unclear from the presented …
Author Response
Reviewer #1 (Public Review):
In this manuscript, Sampaio et al. tackle the role of fluid flow during left-right axis symmetry breaking. The left-right axis is broken in the left-right organiser (LRO) where cilia motility generates a directional flow that permit to dictate the left from the right embryonic side. By manipulating the fluid moved by cilia in zebrafish, the authors conclude that key symmetry breaking event occurs within 1 hour through a mechanosensory process.
Overall, while the study undeniably represents a huge amount of work, the conclusions are not sufficiently backed up by the experiments. Furthermore, the results provided present a limited advance to the field: the transient activity of the LRO is well established, and narrowing down this activity to 1 hour (even though unclear from the presented data that it is a valid conclusion) does not help to understand better the mechanism of symmetry breaking.
We thank the reviewer1 for acknowledging the hard experimental set up. However, we must argue that knowing the exact timing that is more sensitive to fluid flow manipulations is a very important advance we provide here. The reason is because this type of experiment is giving us the physiological timing in a WT embryo. It is one thing to know the system can respond to optical tweezers earlier than 5 ss and later than 5 ss, as Yuan lab did recently, but quite another to constrain the physiological timing at which the process occurs in an unperturbed manner (as much as possible). Our aim was the latter. Our rationale is that knowing the physiological time is important to provide clues, for example we had these types of questions at the time: is the physiological time before or after cell rearrangements occur? is it falling in a directional or non-directional flow regime? Is it governed by a mild flow or stronger one? Is it before or after dand5 becomes asymmetric? Some of these questions that we think we all know the answers for, could be challenged by our experiments… so it is indeed very important to not assume we know the answer, and ask the question again in an unbiased way with every new technique available! We wanted to be unbiased, and we think that is the beauty of our time-window experiment. Indeed, it shows the physiological time-window peaks at 5 ss which is later than Yuan’s lab calcium transient recording and before dand5 asymmetric expression. In our opinion this is compatible and makes perfect sense because although the system already shows calcium transients before and can respond to lack of Pkd2 or optical tweezer cilia manipulations at 1 ss – 3 ss, it is from 4 to 6 ss, peaking at 5 ss, that it is most responsive physiologically to the fluid extraction and therefore both mechanical and chemical perturbations.
We have made additional experiments and used smFISH on WT embryos for detecting dand5 expression with cellular resolution, and we have quantified asymmetries in dand5 number of transcripts as early as 6 ss (new Figure 7 and new author: Catarina Bota) that further support our time-window claim. Degradation of dand5 mRNA has been the mechanism suggested to be at the base of the asymmetric dand5 expression, which is usually a very fast mechanism. This new piece of evidence supports that the physiological breaking of symmetry is stronger around 5 ss. (see new discussion on this subject on page 27).
Regarding the symmetry breaking. The fact that anterior angular velocity was the major difference between embryos that recovered without LR defects versus those that did not, reveals that angular velocity must be tightly regulated by cilia motility and CFTR activity to bring back fluid and flow directionality, which together confer the robustness of flow. This is now better explained in the manuscript. We agree that the novelty regarding angular velocity may seem incremental compared to our work from 2014, where we only analyzed speed (Sampaio et al, 2014). However, here we provided more resolution and detailed parameters of angular velocity per sections of the LRO as well as tangential and radial velocities, the components of angular velocity. The Radial component shows a trend towards left anterior that is now discussed in the text as evidence for a left difference. The present work shows that anterior angular velocity has a major role in the successful recovery of the symmetry breaking process, which was not claimed before. Here we challenged the embryo to bring to light the most important parameters.
Importantly, the authors do not provide any convincing experiments to back up the mechanosensory hypothesis because the fluid extraction experiments affect both the chemical and physical features of the LRO, so it is impossible to disentangle the two with this approach.
We agree the first extraction experiment (Figures 1-3 and Table 1) affects both mechanisms and does not disentangle them, and that was, in fact, our goal for the first experiment - the finding of the exact time-window for symmetry breaking. However, in the second part of the work (Figures 4-5 and Table 2) we provide a 20,000 times dilution experiment, this dilution experiment is very different than the extraction one. We apologize if this was not clear and hope to have made it clear this time.
We must agree with the reviewer that chemosensing is not excluded, in fact we had provided a paragraph in the discussion about EV secretion rates to tone down our claim and did acknowledge that secretion could still overcome the dilution we are causing. We think we had already addressed this problem in the previous eLife manuscript but now we have discussed the possibilities and the experimental evidence that supports each of them (see page 28, last paragraph). The key experiment that does not fit with secretion is pointed out in the end, and we ask the reviewer to read it in the context of wildtype animals. We agree both scenarios must be discussed and leave space for future data on mmp21 and CIROP. However, so far, in zebrafish we cannot favor chemosensing as much as mechanosensing, we can only wait for more discoveries and be open.
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eLife assessment:
Sampaio and colleagues utilize an elegant approach to manipulate fluid dynamics in zebrafish Kupffer's vesicle to ask if fluid movement or something in the fluid governs the break in symmetry. These valuable results support a role for fluid movement and detection as important in breaking symmetry in a ciliated left-right organizer and help set a time window when fluid flow is critical for this process. However, as the fluid extraction experiments affect both chemical and physical features, the authors need to provide further convincing evidence to support their mechanosensory hypothesis or temper the claims.
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Reviewer #1 (Public Review):
In this manuscript, Sampaio et al. tackle the role of fluid flow during left-right axis symmetry breaking. The left-right axis is broken in the left-right organiser (LRO) where cilia motility generates a directional flow that permit to dictate the left from the right embryonic side. By manipulating the fluid moved by cilia in zebrafish, the authors conclude that key symmetry breaking event occurs within 1 hour through a mechanosensory process.
Overall, while the study undeniably represents a huge amount of work, the conclusions are not sufficiently backed up by the experiments. Furthermore, the results provided present a limited advance to the field: the transient activity of the LRO is well established, and narrowing down this activity to 1 hour (even though unclear from the presented data that it is a …
Reviewer #1 (Public Review):
In this manuscript, Sampaio et al. tackle the role of fluid flow during left-right axis symmetry breaking. The left-right axis is broken in the left-right organiser (LRO) where cilia motility generates a directional flow that permit to dictate the left from the right embryonic side. By manipulating the fluid moved by cilia in zebrafish, the authors conclude that key symmetry breaking event occurs within 1 hour through a mechanosensory process.
Overall, while the study undeniably represents a huge amount of work, the conclusions are not sufficiently backed up by the experiments. Furthermore, the results provided present a limited advance to the field: the transient activity of the LRO is well established, and narrowing down this activity to 1 hour (even though unclear from the presented data that it is a valid conclusion) does not help to understand better the mechanism of symmetry breaking. Importantly, the authors do not provide any convincing experiments to back up the mechanosensory hypothesis because the fluid extraction experiments affect both the chemical and physical features of the LRO, so it is impossible to disentangle the two with this approach.
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Reviewer #2 (Public Review):
The manuscript by Sampaio and colleagues utilizes an elegant and delicate approach to manipulate fluid dynamics in zebrafish Kupffer's vesicle (KV) to answer a long-standing question in the field - is it fluid movement or something in the fluid that governs the break in symmetry?
The researchers extract fluid from KV at different times during somitogenesis and find this procedure results in left-right organ defects when fluid is removed from the 3 to 5 somite stage, peaking at 5 somites. The effect on left-right patterning by this manipulation is not significant from the 6 somite stage onward. This technique is non-trivial and the researchers have used it with great effect.
Fluid extraction in this sensitive time window (3-5 somites) did not affect cilia number, length, or distribution within KV suggesting …
Reviewer #2 (Public Review):
The manuscript by Sampaio and colleagues utilizes an elegant and delicate approach to manipulate fluid dynamics in zebrafish Kupffer's vesicle (KV) to answer a long-standing question in the field - is it fluid movement or something in the fluid that governs the break in symmetry?
The researchers extract fluid from KV at different times during somitogenesis and find this procedure results in left-right organ defects when fluid is removed from the 3 to 5 somite stage, peaking at 5 somites. The effect on left-right patterning by this manipulation is not significant from the 6 somite stage onward. This technique is non-trivial and the researchers have used it with great effect.
Fluid extraction in this sensitive time window (3-5 somites) did not affect cilia number, length, or distribution within KV suggesting the effect on left-right patterning is due to disruption of the fluid. There is a clear effect of the manipulation on dand5 RNA asymmetry as expected. Manipulated embryos that developed left-right defects also showed a decrease in angular velocity of particle movement in the anterior LRO. Increasing the viscosity of the fluid in KV with methylcellulose also results in left-right patterning defects. Taken together, these results are in strong support of fluid movement and detection being important in breaking symmetry in a ciliated left-right organizer. They also argue against the idea that there are signals in the fluid that are being moved asymmetrically to signal to the "left" to break the symmetry. Importantly, they help set a time window when fluid flow is critical for this process.
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