Comment on a PREreview of "On theindependent irritabilityof goldfish eggs and embryos – a living communication on the rhythmic yolk contractions in goldfish"
Creators: name: Paul Gerald Layague Sanchez
Publisher: name: Zenodo
Year: 2024

description: This Zenodo record is a permanently preserved version of a comment on a PREreview. You can view the complete PREreview and comments at https://prereview.org/reviews/11444773.


This work presents the first modern descriptions of the rhythmic contractility of the yolk of the domesticated goldfish Carassius auratus. The authors use image analysis tools to quantify the contractions using the mean pixel intensity of the yolk as it oscillates over time. They demonstrate that these oscillations are not present in either zebrafish or carp embryos. They then go on to show that contractions are an autonomous property of the goldfish yolk using carp/goldfish hybrids and unfertilized eggs. Finally, they show that manipulations of early embryonic patterning correlate with changes to speed and/or the magnitude of yolk contractions.
Overall this study of early goldfish development is a valuable contribution to the existing body of work on early teleost development and highlights the variation in the evolution of embryo and egg behaviour that we can observe when we sample widely across taxa. However, the data currently in this manuscript is not sufficient to support the more detailed conclusions of the authors.
Major points:


The various metrics used to quantify yolk contractility in Figure 1 are a great starting point however, the focus on the mean pixel intensity of the yolk/embryo as the central metric for quantifying yolk contractions throughout the paper is a cause for concern. My concern is that the mean pixel intensity metric is too tangentially related to the biological phenomena of interest to be a consistent readout. It is not clear what high or low values of this metric would mean biologically or whether they would even be the same between imaging sessions. This is in contrast with a metric like circularity which has a well-defined meaning for shape analysis. The reasoning in the first section of the results on page 2 for using this metric, that frames at similar phases of oscillation will have similar intensities, does not seem to account for any irregularity in the contractions (particularly as they get stronger) which might cause differences in where the light hits the yolk. Similarly, there is clear variation in the illumination of the yolk between different embryos (see supplementary movies M1 and M5). While the detrending performed on the oscillating trace adjusts for the absolute intensity differences between embryos it will not account for differences in the range of pixel intensities, an example of which would be in movie M1 where some embryos have completely saturated pixels and others do not. Instead, I would suggest that the authors use circularity or a similar metric as their main readout of yolk contraction. While the circularity trace shown in Figure 1 is less smooth than the mean pixel intensity trace it is likely to be a much more consistent and biologically meaningful quantification. Alternatively, if the authors wanted to quantify the contractions in even greater detail, I would suggest generating a set number of radii from the centre of mass of the embryo/yolk and measure their change in length as the yolk contracts. This would pick up more of the subtleties of contraction and would give a nice way to investigate its symmetry.
Response:
Thank you for this comment. We understand that the reason for the choice of quantification method was not elaborated in the first version of the manuscript. We expanded our discussion on this in the Extracting the mean pixel value of an egg / embryo over time allows quantification of the period of yolk contractions from simple timelapse stereomicroscopy section of the second version of the manuscript, which we quote here: 
"To quantify these contractions, we segmented the yolk and determined its circularity, its perimeter, and its projected area over time. However, we immediately realized that quantifying yolk contractions from measurements of the shape of the yolk requires precise image segmentation (with the yolk as foreground) over long periods of time (spanning > 12 hours post-fertilization, from cleavage to epiboly stages). This is challenging in our simple experimental and imaging conditions as the yolk has low contrast compared to the background and to the ooplasm / embryo proper, the yolk changes its shape and position in 3D, and the entire egg / embryo is not fixed in one axis. Accordingly, we also extracted the timeseries from the mean pixel value of the entire egg / embryo, circumscribed by its chorion, over time (Figure 1, Supplementary Movie M2, reasoning that still-frames of an egg / embryo after a full contraction of the yolk would look most similar, and hence would have most similar mean pixel values (Figure 1 1 and 3, 2 and 4, 5 and 8). For the analysis, we detrended the extracted timeseries via sinc-filter detrending after specifying a cut-off period. We subjected the detrended timeseries to continuous wavelet transform using the Morlet wavelet as mother wavelet (Mönke et al., 2020), allowing us to recover the instantaneous period along a ridge tracing wavelet with maximum power for every timepoint. Comparing the timeseries and the evolution of period over time, we noted that the mean pixel value of the embryo is a proxy of the projected area of the segmented yolk. However, as it does not rely on image segmentation, extracting the timeseries of yolk contractions from the mean pixel value of the entire egg / embryo is additionally more robust, at least in quantifying the period of contractions (which does not rely on sample orientation, unlike quantifying phase and amplitude), compared to measurements of the perimeter, circularity, or projected area of the yolk when some segmentation is possible. As exemplified in Figure 1, the timeseries extracted from the mean pixel value provides a suitable approximation of the period of yolk contractions, matching the actual period of a full contraction (e.g. Figure 1, timepoints 1 and 3, or 2 and 4, or 5 and 8) determined from manual inspection of timelapse imaging (i.e. the ground truth). Thus, the method from mean pixel value provides a simple and flexible measure of the yolk contractions, and we henceforth used this method in our quantification."

While I am not too familiar with the details of the wavelet transformation method, I can see the use for extracting the period and amplitude etc of the oscillating trace in question. However, I do not understand why it would be applied to a non-oscillating trace such as those acquired from the zebrafish or carp embryos. In this case I would assume that the resulting properties of the waveform do not actually correspond to true changes in their behaviour but rather minor variation in quantification/imaging over time that has been erroneously amplified. This undermines the conclusion, which would appear to be otherwise strongly supported from the live movies presented, that yolk contractions are found in goldfish but not carp or zebrafish. Instead of the wavelet analysis I would suggest presenting the traces currently found in supplementary figure 1 which clearly show a lack of oscillation in these species.
Response:
We now included comparisons of the timeseries from the embryos to those of background regions in close proximity to the fish embryos (Supplementary Figure F2B) in the second version of the manuscript. We showed that the rhythms we detect are not an artefact of image acquisition or due to background effects, as we reported in the Goldfish, unlike closely-related common carp and zebrafish, exhibit persistent rhythmic yolk contractions during embryonic development section of the second version: 
"The rhythms we had detected for the three fish species had high wavelet powers (Supplementary Figure F1A), correlating well with considered wavelets and not with white noise (Mönke et al., 2020) at least at specific timepoints during development (see differences in temporal profile of wavelet power between the three fish species as represented by black bars in Supplementary Figure F1A). Comparison with timeseries from background regions (in close proximity to analyzed fish embryos) verified that the detected embryo rhythms were not an artefact of the image acquisition nor were due to background effects (Figure 2B versus Supplementary Figure F2B)." 
In addition, we actually also detect a similar long (slow) period rhythmic trend in goldfish, as we also report:
"Interestingly, we also observed a trend in the timeseries of goldfish embryos (see for e.g. raw timeseries of representative goldfish sample in Figure 2B) seemingly matching the slow (long-period) rhythms detected in common carp and zebrafish. For some goldfish samples (e.g. 15/61 goldfish samples plotted in Figure 2C), this slow rhythm could even register higher wavelet power than the faster yolk contractions when the cut-off period was set at 850 seconds. We are still not certain what these slow rhythms are."

The data presented in Figure 2 and 3 very nicely demonstrate, in two different ways, that yolk contractions are an autonomous property of the yolk cell itself. However, my major point about these figures is the need for more samples in order to be able to conclude that (a) unfertilized eggs begin contracting at the same time as fertilised ones (one embryo does, while the other begins contracting later), and (b) the period of unfertilised yolk contraction is meaningfully faster than in fertilised eggs (there is considerable variation in period between the samples presented). If the latter remains true with further repeats then this result could relate to the increased period of contraction seen when treating with nocodazole or the oranda mutant, it could be possible that changes to the embryonic development generally may alter the dynamics of contraction. Additionally, the authors should consider whether these manipulations don't just change the period or amplitude but are actually affecting the consistency of the oscillations.
Response:
Since the previous version, we have significantly increased the number of the samples. Whenever possible, we also recovered eggs from the same clutches and performed simultaneous imaging using multi-well dishes to minimize the confounding effect of slight differences in environmental conditions. Additionally, we extended the duration of our timelapse imaging and now report how the period of contractions change during development. Briefly, in wild-type conditions, we found that the contractions emerge at the 4-cell stage and become stable at the early blastula stages before gradually slowing down and then ceasing towards the end of epiboly. In the second version, we also include quantification of the onset of yolk contractions (Figure 3F) and show more quantitatively that the onset is similar for fertilized (n = 27) and unfertilized (n = 30) goldfish eggs. Regarding the differences in period, with our higher sample numbers, we found that the period of yolk contractions in unfertilized samples (n = 62) are actually slower than in fertilized counterparts (n = 45). We currently do not know if this slowing down of period has biological implications or is merely a consequence of the unfertilized eggs eventually dying.

The results presented in Figure 4 show that treatment with nocodozole prior to the first division causes an increase in the proportion of embryos that develop a ventralised phenotype which is consistent with the effect of nocodozole treatment on microtubule polymerisation and the transport of dorsal determinants during a similar phase of zebrafish development. As noted by the authors the continued presence of contractions at later stages suggests that they are not sufficient to distribute the dorsal determinants. However, I disagree with the related conclusion that this data indicates that the yolk contractions do not rely on microtubules. As nocodozole is only transiently administered prior to the first cell division and the contractions do not begin until the 4-cell stage it is possible that the microtubules have re-polymerised in this time. While it is more likely that the contractile force is generated by actomyosin than microtubules, this cannot be assumed based on the results presented here. To further reinforce this data the authors should treat the embryos with nocodozole during the onset of contractions and verify the microtubule loss using immunohistochemistry (assuming this is possible in the goldfish). As the authors note, it would then seem like a natural next step to investigate the role of actomyosin contractility as the potential motor for yolk oscillations.
Response:
Thank you for this comment. We agree that the timing of the nocodazole treatment does not eliminate the possibility that the yolk contractions rely on microtubules. We have to emphasize that the main aim of the nocodazole experiment is to relate yolk contractions to one of the initial symmetry breaking events in DV patterning of fish embryos, i.e. formation of parallel microtubule array prior to the first cleavage resulting to asymmetric distribution of dorsal determinants (Jesuthasan and Strähle, 1997), and the consequent ventralization of the embryo, and not to investigate the importance of microtubules per se in driving the contractions. In the first version, we observed that the period of yolk contractions in treated embryos (n = 5) were faster than in controls (n = 2), and we interpreted this as evidence of fast contractions correlating with ventralized phenotypes. In the second version (Figure 5, Table 5), with much higher sample numbers, we actually observed a slower period in treated embryos (n = 15) than in controls (n = 18), opposite the result we previously reported. It could be that the ventralizing effect of disrupting microtubule polymerization prior to the first cleavage supersedes any patterning influence from the yolk contractions. Alternatively, this could mean that the period of yolk contractions do not have any direct effect on the ventralization of the embryo at all. We also did different pharmacological perturbations of actomyosin and calcium, and saw a slowing down of contractions in all these perturbations. While we considered timepoints where the embryos are still alive (and the contractions register a median wavelet power > 3, greater than the minimum wavelet power corresponding to 95% confidence interval in case of white noise (Mönke et al., 2020)), at the moment, it remains unclear if the slowing down of contraction period is evidence of the dependence of yolk contractions to these said molecular machineries or is the consequence of drug effects on embryo viability (Supplementary Figure F3).

The final conclusion on the function of yolk contractions shown in Figure 6, which posits that the yolk contractions act as a "stirrer" of dorsal determinants which dilutes their action and leads to the increased prevalence of ventralised phenotypes, appears to contradict the observations of the authors as well as the literature cited in the manuscript. Firstly, as the authors note in the discussion there is existing literature that suggests that dorsal determinants are already asymmetrically localised by the 4-cell stage and the onset of contractions. Similarly, it is early treatment with nocodozole that disrupts dorsal-ventral patterning, well before the 4-cell stage as shown by the authors in Figure 4. Secondly, while the data does appear to show a correlation between the two treatments (nocodozole and the oranda mutant) causing ventralised phenotypes and changes to the contraction dynamics, this would primarily suggest that these treatments affect the contractions themselves rather than the other way around. Finally, what these two treatments have in common is that they both result in a ventralised phenotypes, however, this phenotype is caused in distinct ways by each treatment. In the case of nocodozole, presumably, there is a loss of microtubule polymerisation which reduces the quantity of dorsal determinants which reach the embryo. In the oranda mutant there is a mutation in chordin, one of the dorsal determinants, that presumably affects its function in dorsal specification. Crucially, the mutant is not an example of defective transport of dorsal determinants and therefore seems unlikely to be an example of how yolk "stirring" might lead to greater prevalence of ventralisation phenotypes through dilution of dorsal determinants. Importantly, the discussion of this model tends to frame the hypothesised "stirring" of dorsal determinants as a potential function of yolk contractions. It seems more likely that if this process does occur it is a consequence of the true function of contractions, as the primary outcome of this model would appear to only sensitise the embryo to developing patterning defects. Taken together I feel that the authors do not present enough evidence to propose their "stirring" model, instead I suggest that the authors mention the "stirring" model as a potential hypothesis to explore in the future, along with plausible alternate hypotheses as part of their discussion. I would encourage to authors to fully discuss and clarify where possible, the above noted conflicts between the model and the data. If the authors wish to present this model as the main conclusion of the paper, then this would require further experiments as noted in the future work section below.
Response:
Thank you for this comment. In the first version, we intended to pose the last part of the Discussion and open questions and the last figure as a question / framework guiding future studies and serving as a starting point for further discussion. In the second version, we have framed this more explicitly (e.g. see caption of Figure 9). As discussed in response to major point 5, with much more sample numbers, we found that the yolk contractions are actually slower in nocodazole-treated embryos (n = 15), which end up more ventralized, than controls (n = 18). This shows that the loss of critical microtubule polymerization prior to the first cleavage results in ventralization, which likely supersedes any influence from the yolk contractions. In the second version, we have also increased the numbers of wild-type (n = 56 in the second version, versus n = 2 in the first version) versus Oranda (n = 25 in the second version, versus n = 5 in the first version). Additionally, we now included quantifications of contractions from embryos with different ChdS genotypes. We confirm that the contractions are faster in Oranda than in wild-type counterparts (Table 9), but now report that the period of contractions is actually independent of the ChdS genotype (Table 10). With this, we think that yolk contractions might have more general effects to embryo development and patterning, which could be co-opted to perturb DV patterning. More generally, yolk contractions could lower the robustness during embryo development and patterning, permitting (rather than instructing?) the emergence of novel phenotypes (e.g. twin-tail and dorsal finless strains) under strong selective pressures (e.g. domestication) which is just later on fixed by acquisition of genetic mutations (e.g. ChdS(-) allele). Please find here the relevant part of the Discussion and open questions section:
"Embryos of the even closer related Carassius auratus indigentiaus subsp. nov. have been shown to also exhibit rhythmic yolk contractions (Zhang et al., 2023). It thus seem likely that the ancestral Carassius auratus goldfish initially used in domestication also exhibited rhythmic yolk contractions. Could yolk contractions then be permissive of the emergence of median fin morphotypes in goldfish? We observed a distribution of periods of yolk contractions in each clutch of goldfish eggs, with some contracting slightly faster than the rest. It could be that development and patterning is less robust in these fast-contracting embryos, and the lowering of robustness could then allow emergence of atypical morphotypes especially when these embryos are subjected to selective pressures like domestication. We found here that the presence and the period of yolk contractions is a trait that is maternal in origin. If the period of contractions is something that can be passed on from one generation to the next, then the fast contractions in present-day twin-tail Oranda embryos reflect the faster yolk contractions among goldfish that first showed the twin-tail morphotype versus their wild-type counterparts / siblings. That novel morphotypes could have a higher likelihood of emergence in goldfish that have fast yolk contractions implies that faster-contracting twin-tail goldfish strains could further acquire other novel morphotypes. Indeed, it has been documented that goldfish strains showing the even more ventralized dorsal finless morphotype (e.g. the Ranchu strain) were actually derived from twin-tail goldfish (Matsui, 1935; Ota, 2021; Chen et al., 2022). Considering this, more generally, could yolk contractions be perturbing the robustness of early fish development and patterning (e.g. disrupting early DV patterning), permitting the emergence of novel phenotypes (e.g. twin-tail morphotype) under strong selective pressures (e.g. goldfish domestication) which are then only later fixed by acquisition of genetic mutations (e.g. ChdS(-) allele)?"
Suggestions for future work:


Does yolk size affect contraction strength/ability? Removal of yolk could be performed as in the zebrafish and detailed here: https://doi.org/10.1242/dev.161257.
Response:
Thank you for this question. We did a related experiment where we performed equatorial bisection of goldfish embryos to investigate if the rhythmic contractions are maintained by signals coming from either the animal or the vegetal poles (Figure 4, Supplementary Movies M6-M7). From these experiments, we observed that rhythmic movements of the yolk persist in both animal (i.e. embryo proper and a bit of yolk) and vegetal (i.e. isolated yolk) portions, lasting for at least 8 hours post-fertilization. This indicates that the persistent yolk contractions in goldfish are not maintained by signals coming from either the vegetal or the animal poles. Since the first version, we have refrained from interpreting contraction strength/ability as it relates to amplitudes which we could not accurately quantify with our simple quantification method. Nonetheless, we could quantify the period of contractions confidently and we saw a negative correlation between period of contractions and projected area of the surgically manipulated sample, more especially for the vegetal portions. However, we postpone further interpretation of this result as we observed peculiar dynamics in vegetal portions (i.e. apparent rotation) which we believe merit a more thorough quantification. We cite here the relevant part of the The rhythmic contractions are an emergent property of the goldfish yolk, persisting even after dechorionation and equatorial bisection section:
"There was no clear match in period between animal-vegetal partners, i.e. animal and vegetal portions recovered from the same bisected goldfish embryo (Figure 4E). However, we saw a negative correlation between size (i.e. projected area) of the sample and the period of rhythmic contractions, i.e. the smaller the sample, the longer the period, especially for the vegetal portions (Figure 4F). That is, we observe a relationship opposite to what might be expected if there was scaling between sample size and yolk contraction period. As we observed peculiar dynamics in these samples (e.g. apparent rotations in vegetal portions), more sophisticated imaging modalities (e.g. to image in 3D) and quantification methods (e.g. to capture shape, area, and volume changes) will be be needed to describe them more precisely."

If you tie off the yolk prior to contraction onset, do the two lobes oscillate and do they oscillate in unison or separately? Tying the yolk could be performed as in classical embryology papers noted in this review in Box1 https://doi.org/10.1242/dev.177709.
Response:
Thank you for this suggestion. We did not perform this particular experiment this past spawning season, but this would certainly be an exciting prospective experiment. As suggested, earlier work bisected/tied goldfish embryos along the first cleavage plane (Tung and Tung, 1943; Tung, Lee, and Tung, 1955; Mizuno et al., 1997). These studies provided early evidence for asymmetric distribution of a material, which is important for normal development and patterning, in the yolk during the early cleavage stages, i.e. until the 4-cell stage. It would indeed then be curious to investigate if the period/onset of yolk contractions only depends on the amount of the yolk (and the amount of "immobile" embryo, then the two separate lobes would contract within the same period) or if it is affected by the asymmetric distribution of material in the yolk (then the periods of the two separate lobes could be entirely different, and one might not even contract at all). Additionally, it would be interesting to investigate if the yolk contractions (if present) would give an impression of a wave traveling from one lobe to the other despite physical separation. We added this prospective experiment in the Future perspectives section, which we cite here:
"Another prospective experiment could involve tying or bisecting goldfish embryos along the first cleavage plane prior to the onset of yolk contractions, as previously done by Mizuno et al. (1997); Tung et al. (1955); Tung and Tung (1943) and as recently suggested by Saunders (2024). The separated samples could then be imaged and the dynamics of yolk contractions (if present) between the pairs be compared."

While this process may or may not have a functional role (at least at this stage of development) how are processes that govern embryo patterning, growth, and morphogenesis able to cope with these large-scale movements of the yolk cell?
Response:
We currently do not have the technology/resolution to address this precisely. However, we think that the yolk contractions generally perturb (lessen the robustness of) these processes during embryo development and patterning, at least until before they cease towards the end of epiboly, and by doing so may permit the emergence of atypical patterning phenotypes. Indeed, it would be interesting to investigate how the yolk contractions affect e.g. the coordinated cell movements during epiboly, as it is clear that the exposed yolk continues to contract during this entire morphogenetic process while it is progressively being covered by the blastoderm. Additionally, It would be curious to investigate if the dynamic yolk could be altering later processes like the yolk extension (and axial elongation) in cypriniform fishes.

In order to provide evidence the "stirring" model, I would recommend the authors quantify the occurrence of ventralised phenotypes in wildtype goldfish clutches and compared this to ventralisation occurrence in carp and zebrafish. Additionally, the authors could quantify the severity/occurrence of ventralised phenotypes in nocodozole treated or chd mutant fish, again comparing between goldfish and carp/zebrafish. If the authors are able to find a method to inhibit contractions then a natural prediction of the model would be that instances of ventralisation decrease in the resulting embryos.
Response:
We did not make these specific comparisons of phenotypes ourselves, but some quantifications on phenotypes in wild-type goldfish, twin-tail goldfish, carp, and zebrafish had already been done by our lab and others. We list some of the data below. For data citing our current work, we categorized the phenotypes as: 0 = no axis defect, 1 = has well developed posterior, but shows head phenotype, 2 = has two axes, 3 = has head phenotype and missing or truncated posterior, 4 = has anterior mass and some segmentation in posterior, 5 = no anterior mass, but has some segmentation in posterior, 6 = no axis, or dead (Supplementary Figure F3).


Wild-type goldfish, ZWJ-ChdS(+/+) strain; DMSO control of nocodazole treatment; 0 = 114/123, 1 = 0/123, 2 = 0/123, 3 = 4/123, 4 = 0/123, 5 = 1/123, 6 = 0/123, dead = 4/123, at 1dpf; current work, version 2 (Supplementary Figure F3)

Wild-type goldfish, ZWJ-ChdS(+/+) strain; DMSO control of latrunculin treatment; 0 = 106/147, 1 = 4/147, 2 = 0/147, 3 = 9/147, 4 = 0/147, 5 = 0/147, 6 = 3/147, dead = 25/147, at 1 dpf; current work, version 2 (Supplementary Figure F3)

Wild-type goldfish, ZWJ-ChdS(+/+) strain; No injection control; 82/82 normal at 2-3 dpf; Abe et al., 2016 (Figure S5)

Wild-type goldfish, ZWJ-ChdS(+/+) strain; Injection (control morpholino) control; 149/149 normal at 2-3 dpf; Abe et al., 2016 (Figure S5)

Wild-type goldfish, ZWJ-ChdS(+/+) strain; Nocodazole treatment for ~ 4 mins at 10 mins post-fertilization; 0 = 1/140, 1 = 9/140, 2 = 3/140, 3 = 6/140, 4 = 58/140, 5 = 4/140, 6 = 0/140, dead = 59/140, at 1 dpf; current work, version 2 (Supplementary Figure F3)

Single-tail ChdS(-/-) goldfish, ZWJ-ChdS(-/-) strain; Tap water control; 0 = 1/13, 1 = 0/13, 2 = 0/13, 3 = 0/13, 4 = 1/13, 5 = 0/13, 6 = 0/13, dead = 11/13, at 1dpf; current work, version 2 (Supplementary Figure F3)

Twin-tail goldfish, ChdS(-/-); No injection control; 60/60 ventralized, bifurcated caudal fin fold at 2 dpf; Abe et al., 2014 (Figure 3E)

Twin-tail goldfish, ChdS(-/-); Injection (water) control; 60/60 ventralized, bifurcated caudal fin fold at 2 dpf; Abe et al., 2014 (Figure 3E)

Common carp; No injection control; 76/76 normal at 2-3 dpf; Abe et al., 2016 (Figure S8)

Common carp; Injection (water) control; 81/81 normal at 2-3 dpf; Abe et al., 2016 (Figure S8)

Zebrafish; Wild-type; 1253/1259 normal at 1 dpf; Ge et al., 2014 (Table 1)
In summary, comparison of these data shows higher frequency of ventralized phenotypes (generally lack head / anterior structures, but still has some posterior elongation / segmentation) in wild-type goldfish (~4%) than in carp (0%) and zebrafish (~0.5%) embryos. Also, 100% of twin-tail goldfish embryos show ventralized, bifurcated caudal fin fold at 2-3 dpf. Only ~1% of surviving nocodazole-treated embryos had no axis defect.
In the second version, we also report pharmacological perturbation of actomyosin (by latrunculin A and by blebbistatin(-)) and calcium (by EDTA). The drug treatments we did all resulted in slower yolk contractions in treated embryos. However, we currently do not know if these results indicate importance of the perturbed molecular machinery in the maintenance of yolk contractions or instead reflect the effect of the drugs on embryo viability, as each resulted in mostly dead embryos (Supplementary Figure F3).
We welcome the likelihood that the yolk contractions are mainly permissive, instead of being instructive, of atypical phenotypes, e.g. the ventralized twin-tail morphotype. We cite here the last part of the Discussion and open questions section which we also already cited as response to major point 5:
"Embryos of the even closer related Carassius auratus indigentiaus subsp. nov. have been shown to also exhibit rhythmic yolk contractions (Zhang et al., 2023). It thus seem likely that the ancestral Carassius auratus goldfish initially used in domestication also exhibited rhythmic yolk contractions. Could yolk contractions then be permissive of the emergence of median fin morphotypes in goldfish? We observed a distribution of periods of yolk contractions in each clutch of goldfish eggs, with some contracting slightly faster than the rest. It could be that development and patterning is less robust in these fast-contracting embryos, and the lowering of robustness could then allow emergence of atypical morphotypes especially when these embryos are subjected to selective pressures like domestication. We found here that the presence and the period of yolk contractions is a trait that is maternal in origin. If the period of contractions is something that can be passed on from one generation to the next, then the fast contractions in present-day twin-tail Oranda embryos reflect the faster yolk contractions among goldfish that first showed the twin-tail morphotype versus their wild-type counterparts / siblings. That novel morphotypes could have a higher likelihood of emergence in goldfish that have fast yolk contractions implies that faster-contracting twin-tail goldfish strains could further acquire other novel morphotypes. Indeed, it has been documented that goldfish strains showing the even more ventralized dorsal finless morphotype (e.g. the Ranchu strain) were actually derived from twin-tail goldfish (Matsui, 1935; Ota, 2021; Chen et al., 2022). Considering this, more generally, could yolk contractions be perturbing the robustness of early fish development and patterning (e.g. disrupting early DV patterning), permitting the emergence of novel phenotypes (e.g. twin-tail morphotype) under strong selective pressures (e.g. goldfish domestication) which are then only later fixed by acquisition of genetic mutations (e.g. ChdS(-) allele)?"
Minor points:


In Figure 1 it may be useful to show the trace prior to the onset of contraction to emphasise the difference between the non-contracting and contracting regimes (as done in Figure 3B)
Response:
Thank you for this suggestion. We alternatively now show the temporal evolution of the period of contractions for the entire duration of the experiment, spanning at least 10 hours for most experiments. We have plotted the median wavelet power of the samples, where wavelet power threshold = 3 corresponds to 95% confidence interval in case of white noise (Mönke et al., 2020). This allows a better summary of non-contracting (i.e. generally below the wavelet power threshold) and contracting (i.e. generally above the wavelet power threshold) regimes for numerous samples and a more effective side-by-side comparison of multiple conditions.

The diagram in Figure 2A could be reduced in half, both the top and bottom halves convey the same information.
Response:
Thank you for this comment. We followed this suggestion and revised the figure accordingly (Figure 8A in the second version).

Figure 2 and 3, it could be more impactful to combine these two figures into one (with maybe an additional supplement for any data that does not fit into the resulting figure) as they effectively answer the same question.
Response:
Thank you for this suggestion. We decided to reorder the figures and now put the figure corresponding to experiments with the reciprocal hybrids (Figure 8) towards the end of the Results section. We believe the experiment corresponding to the said figure best shows that the contractions are maternal in origin, which nicely follows our new findings that the period of contractions of the yolk is independent of the ChdS genotype but is dependent on the mother (batch of egg) instead (Table 10, Figure 7).
Overall, the manuscript in its current state provides a solid foundation for understanding the novel behaviour of yolk contractions in goldfish embryos and with some revisions will be a valuable contribution to the field.
References cited: 
Abe, G., Lee, S.-H., Chang, M., Liu, S.-C., Tsai, H.-Y., and Ota, K. G. (2014). The origin of the bifurcated axial skeletal system in the twin-tail goldfish.
Abe, G., Lee, S.-H., Li, I.-J., Chang, C.-J., Tamura, K., and Ota, K. G. (2016). Open and closed evolutionary paths for drastic morphological changes, involving serial gene duplication, sub-functionalization and selection.
Chen, H.-C., Wang, C., Li, I.-J., Abe, G., and Ota, K. G. (2022). Pleiotropic functions of chordin gene causing drastic morphological changes in ornamental goldfish.
Ge, X., Grotjahn, D., Welch, E., Lyman-Gingerich, J., Holguin, C., Dimitrova, E., Abrams, E. W., Gupta, T., Marlow, F. L., Yabe, T., et al. (2014). Hecate/grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction.
Jesuthasan, S. and Strähle, U. (1997). Dynamic microtubules and specification of the zebrafish embryonic axis.
Matsui, Y. (1935). Kagaku to shumi kara mita kingyo no kenkyū.
Mizuno, T., Yamaha, E., and Yamazaki, F. (1997). Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus.
Mönke, G., Sorgenfrei, F. A., Schmal, C., and Granada, A. E. (2020). Optimal time frequency analysis for biological data - pyBOAT.
Ota, K. G. (2021). Goldfish development and evolution.
Saunders, D. (2024). PREreview of "On the independent irritability of goldfish eggs and embryos – a living communication on the rhythmic yolk contractions in goldfish".
Tung, T.-C., Lee, C.-Y., and Tung, Y.-F.-Y. (1955a). Further research on the developmental ability of fish eggs.
Tung, T.-C. and Tung, Y.-F.-Y. (1943). Experimental studies on the development of goldfish.

          
Competing interests

          

            The author of this comment declares that they have no competing interests.