PREreview of On theindependent irritabilityof goldfish eggs and embryos – a living communication on the rhythmic yolk contractions in goldfish

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:

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

Suggestions for future work:

1. 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.

2. 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.

3. 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?

4. 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.

Minor points:

1. 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)

2. The diagram in Figure 2A could be reduced in half, both the top and bottom halves convey the same information.

3. 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.

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.

Competing interests

The author declares that they have no competing interests.