Haploidy-linked cell proliferation defects limit larval growth in Zebrafish

  1. Kan Yaguchi
  2. Daiki Saito
  3. Triveni Menon
  4. Akira Matsura
  5. Miyu Hosono
  6. Takeomi Mizutani
  7. Tomoya Kotani
  8. Sreelaja Nair
  9. Ryota Uehara

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Abstract

Haploid larvae in non-mammalian vertebrates are lethal with characteristic organ growth retardation collectively called “haploid syndrome.” In contrast to mammals whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

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  1. Version published to 10.1101/2022.05.12.491746v3 on bioRxiv
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    Reply to the reviewers

    Reply to the Reviewers

    We sincerely thank the Referees for providing important and constructive comments. We have addressed their concerns point-by-point as described below.

    Associated to Reviewer#1's comments

    *- Diploid embryos are used as controls. Gynogenetic diploids seem to be better controls to ensure that the observed phenotypes are not related to loss of heterozygosity. To limit the amount of work, the use of gynogenetic diploids could be restricted to spindle polarity and centrosome number experiments. *

    Response 1-1

    __[Experimental plan] __Following the reviewer's suggestion, we will conduct immunostaining of a-tubulin and centrin (for visualizing the spindles and centrioles, respectively) in gynogenetic diploids that will be generated by applying heat shock to gynogenetic haploid embryos during the 1st - 2nd cleavage stage. We will observe the head area of gynogenetic diploid larvae at 3-dpf when the haploid counterparts suffer particularly drastic centrosome loss and spindle monopolarization.

    *- As the authors discuss, it would be necessary to rescue centrosome loss to establish a causal relationship between centrosome loss and haploid viability. I certainly acknowledge that this is difficult (if not impossible), but it currently limits the significance of the results. *

    Response 1-2

    We agree that rescuing centrosome loss would provide an important advancement in understanding the cause of haploid syndrome in the context of our study. However, as the reviewer also pointed out in the above comment, this poses a significant technical challenge. As described in *Discussion *in the original manuscript, we have attempted to restore normal centrosome number through cell cycle modulations. However, we have not found a condition that rescues centrosome loss without damaging larval viability. As an alternative approach, we have also tried to induce centriole amplification by injecting mRNA encoding plk4, an essential centriole duplication inducer. However, this caused earlier embryonic death, precluding us from observing its effects on larval morphology after 1 dpf. The main challenge is that any treatment to increase centrosome number can cause centrosome overduplication, which is as deleterious to development as centrosome loss. Efforts to identify a key factor enabling the rescue of centrosome loss in haploid larvae are underway in our laboratory, which requires new explorations over several years and is beyond the scope of the present study. Reflecting on the reviewer's comment, we added a new sentence explaining the situation on this issue (line 395, page 19). To further discuss possible contributions of centrosome loss and mitotic defects to haploidy-linked embryonic defects, we also added a citation of a previous study reporting that depletion of centrosomal proteins caused mitotic defects leading to embryonic defects similar to those observed in haploid embryos in zebrafish (Novorol et al., 2013 Open Biology; line 380, page 19).

    __[Experimental plan] __Meanwhile, as a new trial to induce centriole amplification in a scalable and temporally controllable manner, we plan the following experiment, which can be conducted within the time range of the revision schedule: We will investigate the effects of low dose treatment of a plk4 inhibitor centrinone B on tissue growth and viability of haploid larvae. A recent study reported that centrinone B had complicated effects on the centriole duplication process, which is highly dose-sensitive (Tkach et al., 2022 Elife, PMID: 35758262). While it blocks centriole duplication at sufficiently high concentrations for blocking plk4 activities, it paradoxically causes centriole amplification at suboptimal conditions, presumably though over-stabilizing plk4 by blocking its autophosphorylation-dependent degradation (while its centriole duplicating function remains active). Since a previous study showed that centrinone B is also effective in zebrafish embryos (Rathbun et al., 2020 Current Biology, PMID: 32916112), we try to find optimal centrinone B treatment condition that potentially restores tissue growth or viability of haploid embryos. If we find such a rescuing condition, we will address the principle of the rescuing effects by investigating the possession of centrioles in mitotic cells in these haploid larvae.

    * *

    *- Some experiments are not, or arguably, quantified/statistically analyzed. *

    o Figure 2, Active caspase level. Larvae are sorted into three categories, and no statistical test is performed on the obtained contingency table. A Fisher'*s exact test here, or much better, the active caspase-3 levels should be quantified, instead of sorting larvae into categories. *

    Response 1-3

    We apologize that we showed only "zoomed-out" images of the immunostained embryos in the original figures (Fig. 2A), which precluded a clear presentation of the haploidy-associated aggravation of apoptosis and mitotic arrest. We could clearly distinguish cleaved caspase-3- and pH3-positive cells from non-specific background staining with an enlarged view of the same immunostaining data. Therefore, to quantitatively evaluate the extent of the haploidy-linked apoptosis and mitotic arrest, we compared the density of these cells within the right midbrain. This new quantification demonstrated a statistically significant increase in cleaved caspase-3- or pH3-positive cells in haploids compared to diploids.

    In the revised manuscript, we added the enlarged views of cleaved-caspase and pH3 immunostaining (Fig. 2B) and new quantifications with statistical analyses (Fig. 2C). Accompanying these revisions, we omitted the categorization of the severeness of the apoptosis, which was pointed out to be subjective in the reviewer#2's comment (see Response 2-3). We rewrote the corresponding section of the manuscript to explain the new quantitative analyses (line 143, page 7).

    o Same comment for 3E-F. Larvae are scored as Scarce, Mild or Severe. Looking at Fig S3A, I see one mild p53MO embryo, but the two others are not that different from 'severe*' *cases, which would completely change the contingency table. Again, a proper quantification would be better.

    Response 1-4

    We also quantified the frequency of cleaved caspase-3-positive cells in control and p53MO larvae (original Fig. 3E and F) as described in Response 1-3. While conducting the cell counting with enlarged images, we realized that staining quality within the inner larval layers of morphants was relatively poor in these experiments. This problem precluded us from counting cleaved caspase-3-positive cells within the inner larval layers. Therefore, we tentatively quantified only the surface larval layers of these morphants and found that cleaved caspase-3-positive cells were significantly reduced in haploids upon depletion of p53. We currently show this quantification in Fig. 3G of the revised manuscript. While this quantification confirmed the trend of p53MO-dependent decrease in apoptosis, we think it more appropriate to newly conduct the same experiment with better quality of the staining to apply the same standard of quantification for Fig. 3 as Fig. 2.

    __[Experimental plan] __For the reason described above, we propose to re-conduct immunostaining of cleaved caspase-3 in control and p53MO-injected haploid larvae to improve the visibility of the inner layer of the larvae for better quality of the quantitation.

    Meanwhile, we revised Fig. 3 by adding an enlarged view of immunostaining in Fig. 3F and omitting the subjective categorization shown in the original Fig. 3F and S3A. We plan to replace these data with new images and quantification to be obtained during the next revision. We also rewrote the main text to update these changes (line 166, page 8).

    *o Figure 4D-E, no stats. *

    Response 1-5

    We conducted the ANOVA followed by the post-hoc Tukey test for new Fig. 4D and the Fisher exact test with Benjamini-Hochberg multiple testing correction for new Fig. 4E. Please note that statistical analyses were conducted after adding the data from original Fig. 6B-C following the reviewer's suggestion (see also Response 1-6).

    *o Figure 6, Reversine treated haploid should be compared to haploid embryos (on the graphs and statistically). If no specific controls have been quantified for this experiment, data could be reused from previous figures, provided this is stated. *

    Response 1-6

    The live imaging data shown in original Fig. 4C-E and Fig. 6A-C were obtained within the same experimental series conducted in parallel at the same period under the same experimental condition. In the original manuscript, we separated them into two different figures according to the logical flow. However, following the reviewers' comments (see also Response 2-1), we realized it more appropriate to show them as a single figure panel as in the original experimental design. Therefore, we moved the reversine-treated haploid data from the original Fig. 6A-C to Fig. 4C-E to facilitate direct comparison among conditions with statistical analyses (see also Response 1-5).

    *o Rescue by p53MO and Reversine, it would be nice to also include diploid measurements on the graphs, so that the reader can appreciate the extent of the rescue. *

    Response 1-7

    Following the reviewer's comment, we added control MO-injected or DMSO-treated diploid larval data in the corresponding graphs in Fig. 3I and 6G, respectively. Please refer to Response 2-6 for further discussion on the extent of the rescue.

    Minor comments:

    *- Lines 221-223, authors claim that centriole loss and spindle monopolarization commence earlier in the eyes and brain than in skin. I am note sure I see this in Fig. S5. It could as well be that the defect is less pronounced in skin. *

    Response 1-8

    We rewrote the manuscript to include the possible interpretation suggested by the reviewer on the result (line 225, page 11).

    - Lines 227-229, authors claim that 'The developmental stage when haploid larvae suffered the gradual aggravation of centrosome loss corresponded to the stage when larval cell size gradually decreased through successive cell divisions'. I did not get that. Doesn'*t cell size decrease since the first division? Fig 5D shows that cell size decreases all along development. *

    Response 1-9

    We agree that the original sentence implies, against our intention, that cell size does not decrease before the developmental stage mentioned here. To correct this problem, we rewrote the corresponding part of Discussion as below (line 230, page 11):

    "Since the first division, embryonic cell size continuously reduces through successive cell divisions during early development (Menon et al., 2020). Cell size reduction continued at the developmental stage when we observed the gradual aggravation of the centrosome loss in haploid larvae."

    *- Some correlations are used to draw conclusions: *

    o Line 301-303. "The correlation between centrosome loss and spindle monopolarization indicates that haploid larval cells fail to form bipolar spindle because of the haploidy-linked centrosome loss."*. As stated by the authors, this is a correlation only. I agree it points in this direction. *

    Response 1-10

    We added a note to the corresponding sentence to draw readers' attention to the discussion on the limitation of the study with respect to the lack of centrosome rescue experiment (line 332, page 16).

    *O Line 305-308. "Interestingly, centrosome loss occurred almost exclusively in haploid cells whose size became smaller than a certain border (Fig. 5), indicating that cell size is a key determinant of centrosome number homeostasis in the haploid state." This one is more problematic. There is no causal link established between cell size and centrosome number homeostasis. It could very well be that some unidentified problem induces both a reduction in cell size and the loss of centrioles. *

    Response 1-11

    To avoid an over-speculative description, we deleted the subsentence "indicating that cell size is a key determinant of centrosome number homeostasis in the haploid state." (line 336, page 17). We also added a new sentence, "Alternatively, it is also possible that other primary causes, such as the lack of second active allele producing sufficient protein pools induced cell size reduction and centrosome loss in parallel without causality between them." to discuss the possibility raised by the reviewer (line 348, page 17), in association with another comment from the reviewer #3 (see also Response 3-3).

    *I have concerns regarding the significance of the reported findings. Haploid zebrafish embryos show numerous developmental defects (some as early as gastrulation, as previously shown by the authors, Menon 2020), and they die by 4 dpf. That they experience massive apoptosis at day 3 does not seem very surprising, and that inhibiting p53 transiently improves the phenotype is not a big surprise. *

    Response 1-12

    Many reports have revealed tissue-level developmental abnormalities in haploid embryos since the discovery of haploid lethality in vertebrates more than 100 years ago. This has stimulated speculation of underlying causes of haploid intolerance for decades. However, there have been surprisingly few descriptions of cellular abnormalities underlying these tissue defects, precluding an evidence-based understanding of the principle that limits developmental ability in haploid embryos. Our findings of the haploidy-linked p53 upregulation and mitotic defects illustrate what happens in the dying haploid embryos at a cellular level. These findings would provide an evidence-based frame of reference for understanding why vertebrates cannot develop in the haploid state and also provide clues to controlling haploidy-linked embryonic defects in future studies. We added a new section in Discussion to discuss the importance of addressing the haploidy-linked defects at a cellular level (line 276, page 14).

    *This reminds me of the non-specific effects of morpholino injection, which can be partially rescued by knocking down p53. *

    Response 1-13

    We believe the reviewer refers to the previous findings that different morpholinos generally have off-target effects activating p53-mediated apoptosis (e.g., Robu et al., 2007 PLoS Genet, PMID:17530925). However, p53 upregulation and apoptosis aggravation were also observed in uninjected haploid embryos free from morpholinos' artificial effects (Fig. 2, Fig. 3A, and B). To further address this issue, we plan to compare the frequency of cleavage caspase-3-positive cells between uninjected and control MO-injected haploids after revising the immunostaining of morphants in the original Fig. 3E-F (see Response 1-4 for details).

    *The observation of mitotic arrest and mitotic defects and the observation that haploid cells often lack a centrosome is interesting. However, I felt that the manuscript suggested that these observations were novel and could explain the haploid syndrome specifically in non-mammalian embryos, when the authors reported the same observations in human haploid cells as well as in mouse haploid embryos (Yaguchi 2018). To me, this manuscript mainly confirms that their previous observation is not mammalian specific, but at least conserved in vertebrates. *

    Response 1-14

    As we originally wrote (line 341, page 17 in the original manuscript), we think these haploidy-linked cellular defects are conserved among mammalian and non-mammalian vertebrates. To improve the clarity of our interpretation, we rewrote a corresponding part of the manuscript (line 50, page 2).

    *While I am no expert at centrosome duplication, I find the observation that haploidy leads to centrosome loss very intriguing, but have the impression that this manuscript falls short of improving our understanding of this phenomenon. *

    Response 1-15

    We express our gratitude to the reviewer for being interested in our findings. We hope the revisions made in the manuscript and the new results provided by the planned experiments will strengthen the contribution of this study to our understanding of haploidy-linked cellular defects.

    Associated to Reviewer#2's comments

    * *

    - Lack of proper controls in many experiments. For example, in the experiments where the authors treated haploids with reversine to suppress the SAC, there was no no-treatment control (Fig. 6A-C).

    Response 2-1

    We addressed the same point in__ Response 1-6__. In the original manuscript, we separately presented control and experimental conditions in the same experiment series in Fig. 4 and Fig. 6. We rejoined them in Fig. 4 as in the original experimental design. Please refer to __Response 1-6 __for further details.

    • In Fig. 6D, when a DMSO control was included, the control fish were from 3 dpf while the reversine-treated fish were from 0.5-3 dpf. This is a big flaw in experimental design, especially considering the authors were looking at mitotic index, which is hugely impacted by developmental time. *

    Response 2-2

    In this experiment, we treated haploid larvae with either DMSO or reversine from 0.5 to 3 dpf, isolated cells from the larvae at 3 dpf, and subjected them to flow cytometry. Both DMSO- and reversine-treated larval cells were from 3-dpf larvae. Therefore, this experiment does not have the problem noted by the reviewer. To improve the clarity of the description of the experimental design, we rewrote the corresponding part of the figure legend (line 646, page 34).

    *- Subjective and inadequate data quantification. In the immunostaining experiments to detect caspase-3 and pH3, the authors either did not quantify at all and only showed single micrographs that might or might not be representative (for pH3), or only did very subjective and unconvincing quantification (for caspase-3). Objective measurements of fluorescence intensity could have been done, but the authors instead chose to categorize the staining into arbitrary categories with unclear standards. In example images they showed in the supplementary data, it is not obvious at all why some of the samples were classified as "mild" and others as "severe" when their staining did not appear to be very different. *

    Response 2-3

    We apologize that we showed only "zoomed-out" images of the immunostained embryos in the original figures (Fig. 2A, 3E, and 6F), in which the distribution of individual cleaved caspase-3- or pH3-positive cells could not be clearly recognized. We added the enlarged view of identical immunostaining where these cells were clearly visualized in a countable manner (Fig. 2B, 3F, and 6D). Following the reviewer's suggestion, we newly conducted quantification by comparing the density of these cells within the right midbrain in haploids and diploids.

    This new quantification demonstrated the haploidy-linked increase in cleaved caspase-3- or pH3-positive cells and a reversine-dependent decrease in pH3-positive cells. We added these new quantifications with statistical analyses to the revised manuscript (Fig. 2C and 6E). Accompanying these revisions, we omitted the categorization of the severeness of apoptosis, which was pointed out to be subjective. We rewrote the corresponding section of the manuscript to explain the new quantitative analyses (line 143, page 7; line 260, page 12).

    While we also quantified cleaved caspase-3-positive cells in control and p53MO larvae in the original Fig. 3E, we realized that the staining quality of the inner larval layers of these morphants was relatively poor and could not apply the same standard of quantification as Fig. 2. Though we confirmed a statistically significant reduction in cleaved caspase-3-positive cells upon p53 depletion by quantified limited number of confocal sections (shown in Fig. 3G, please see also Response 1-4 for details), we decided to re-conduct this experiment for improving the staining quality to apply the same criteria of quantification for Fig 3 as Fig. 2 (Experimental plan is provided in Response 1-4).

    Please note that we also tried to evaluate the extent of apoptosis and mitotic arrest based on the fluorescence intensity of organ areas. However, background staining outside the dead cell area precluded the precise quantification.

    *Additionally, the authors claimed that "clusters of apoptotic cells" were only present in haploids but not diploids or p53 MO haploids, but they did not show any quantification. From the few example images (Fig.S3A), apoptotic clusters can be seen in p53 MO treated fish. Also, in some cases, the clusters were visible only because those fish were mounted in an incorrect orientation. For example, in Fig. S3A, control #2, that fish was visualized from its side, thus exposing areas around its eye that contained such clusters. These areas are not visible in other images where the fish were visualized from the top. *

    __Response 2-4 __

    We agree that the definition of "apoptotic clusters" was ambiguous in the original manuscript. We also agree that the visuals of the clusters could be affected by sample conditions, making them less reliable criteria for judging the severity of apoptotic upregulation in larvae. Following the reviewer's suggestion, we newly conducted apoptotic cell counting (Response 2-3), which recapitulated more reliably ploidy- or condition-dependent changes in the extent of apoptosis. Therefore, we decided to omit the description of the clusters in the new version of the manuscript.

    *- Subpar data quality. Aside from issues with qualification, the IF data was not convincing as staining appeared to be inconsistent and uneven, with potential artefacts. *

    Response 2-5

    We apologize that the zoomed-out images in the original figures did not appropriately demonstrate the specific visualization of individual apoptotic or mitotic cells. As described in Response 2-3, we added enlarged views of the immunostaining to the revised manuscript, in which these individual cells are clearly distinguished from non-specific background staining (Fig. 2B, 3F, and 6D). Because of the poorer staining of inner layers of control and p53 morphants, we plan to re-conduct immunostaining for Fig. 3 and Fig. S3 (please refer to Response 1-4 for further detail). The current version of immunostaining and quantification in these figures will be replaced in the next revision.

    *- Unsupported and overstated claims. There were many overstatements. For one, in line 268, the authors claimed that "the haploidy-linked mitotic stress with SAC activation is a primary constraint for organ growth in haploid larvae", while what they were actually showed was that reversine treatment, which suppresses the SAC, was partially rescued 2 out of the 3 growth defects they assessed, to such a small extent that the difference between haploid and haploid rescue was only Response 2-6

    Following the reviewer's comment, we added control MO-injected or DMSO-treated diploid larval data in the corresponding graphs in Fig. 3I and 6G, respectively. We newly estimated the relative extent of the recovery in Results (line 174, page 8; line 268, page 13).

    Reflecting the estimation, we rewrote the manuscript to discuss that haploidy-linked cell death or mitotic defects are a partial cause of organ growth retardation but that there could be other unaddressed cellular defects that also contribute to the growth retardation (line 305, page 15). We also discussed the possibility that incomplete resolution of cell death by p53MO or mitotic defects by reversine treatment may have limited their rescue effects on organ growth retardation (line 303, page 15). We also toned down several descriptions in our manuscript (lines 48 and 50, page 2; line 111, page 5; line 271, page 13; line 298, page 15; line 403, page 20) to achieve a more balanced interpretation on the potential contributions of cell death and mitotic defects to the formation of haploid syndrome.

    In association with this issue, we also discussed the difficulty of assuming a priori "fully-rescued" haploid larval size in this context. This is because even normally developing haploid larvae in haplodiplontic species tend to be much smaller than their diploid counterparts. We newly cited a few cases of haplodiplontic species where haploids are smaller than or the same in size as diploids (line 307, page 15).

    *With so many fundamental flaws, the data seem unreliable and the paper does not meet publishable standards. *

    Response 2-7

    We express our gratitude to the reviewer for providing important suggestions to improve the quality of analyses, data presentations, and interpretations in this study. We sincerely hope that one-by-one verifications of the points raised by the reviewer have improved the credibility of the paper and made it suitable for publication.

    *The low quality of the analysis makes the significance low. *

    *Reviewers have expertise in vertebrate embryogenesis and ploidy manipulation. *

    Response 2-8

    We hope that by addressing and solving the concerns pointed out by the reviewer, we could have clarified the significance of the study.

    * *

    * *

    Associated to Reviewer#3's comments

    * *

    *There seem to be a discrepancy between the microscopic images from Figure 2A and the quantification of pH3 positive cells using flow cytometry in Figure 4. According to the flow cytometric results the proportion of pH3 positive cells is about 3 times higher in haploid larvae compared to the control. The increase in mitotic cells in the imaging results however seems much more drastic. It would be helpful if the authors explain here. *

    Response 3-1

    Following comments provided by other reviewers (see also Response 1-2, 1-4, and__ 2-3__), we newly compared the frequency of pH3 positive cells between the immunostained haploid and diploid larvae. In this new analysis, pH3-positive cells were 6.4 times more frequent in haploids than in diploids, which is a more substantial difference than the one estimated based on the flow cytometric analysis.

    The apparent discrepancy between the immunostaining and flow cytometric quantification would arise because pH3-positive mitotic cells tended to be more localized on the surface than in the inner region of larvae. This inevitably results in higher pH3-positive cell density in immunostaining, in which only larval surface is analyzed. To discuss this point, we newly conducted pH3 immunostaining in haploid larvae made transparent using RapiClear reagent and showed a vertical section of 3-d reconstituted larval image of pH3 immunostaining in Fig. S4E. We rewrote the manuscript to add our interpretation of this issue (line 652, page 34).

    *Mitotic slippage that the authors observe to be increased in the haploid larvae to up to 5% of cells should result in an increase in the number of aneuploid cells. I am wondering why this is not recapitulated in the analyses of the DNA content in Figure S1. *

    Response 3-2

    A possible interpretation would be that the limited viability of newly formed aneuploid progenies precluded the detection of these populations in flow cytometric analyses. We discussed the possible generation of aneuploid progenies with our interpretation of their absence in the flow cytometric analyses in *Discussion *(line 293, page 14).

    *Discussion: *

    *I find the explanation of centrosomal loss due to depletion of centrosomal protein pools in the cytoplasm during drastic cell reduction interesting. I wonder if the reduction in size is not necessarily caused by the reduction in cells, but rather the result of the absence of a second active allele that produces centrosomal proteins? *

    Response 3-3

    We added the possible interpretation provided by the reviewer to the corresponding part of Discussion, in association with another comment from reviewer #1 (line 348, page 17; see also Response 1-11).

    Reviewer #3 (Significance (Required)):

    *Overall, I find the study interesting even to a broader audience since diploid development is a fundamental feature of most animals. The authors also manage to discuss their findings on the consequences of haploidy in this bigger context of the restricted diploid development in animals. The study is very well-written even to non-experts. *

    Response 3-4

    We express our gratitude to the reviewer for providing positive comments on the significance of our findings. We sincerely hope that one-by-one verifications of the points raised by the reviewer further improve the quality of the paper.

    I am not an expert of the literature describing previous characterizations of the consequences associated with haploid cell development in animals, which is why I cannot comment on the novelty of their study. Based on my expertise on centromeres and genome organisation I can however assess the results regarding the mitotic defects observed in haploid larvae (see comments).

    Response 3-5

    We sincerely thank the reviewer for providing constructive suggestions and critiques based on the expertise.

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    Referee #3

    Evidence, reproducibility and clarity

    In this study the authors aim to shed light onto the molecular reasons why most animals are restricted to diploid cell generations. In mammals, haploid intolerance has been previously attributed to defects linked to genomic imprinting, but the molecular defects associated with haploidy in non-mammalian species are unknown. To fill these gaps, the authors in this study investigate defects associated with haploidy in zebrafish larvae. They found that haploid larvae show elevated numbers of apoptotic cells that could be partially rescued by inhibition of p53. The also detected many cells with prolonged mitosis reflected by an increase of cells positive for the mitotic histone modification phospho- histone H3 (pH3) as well as cell division defects specific to the haploid larvae. These defects are likely caused by the loss of centrosomes in haploid larval cells resulting in an increase of monopolar spindle formation. Loss of centrosomes was particularly pronounced in smaller cells and occurred concomitant with a reduction in cell size through continous cell divisions. The authors could rescue the increase of cells with prolonged mitosis by inhibiting the SAC. Both restoration of mitotic length and decreased apoptosis (by p53 inhibition) also improved some organ growth defects observed in haploid larvae.

    I only have some minor comments particularly regarding the mitotic defects.

    There seem to be a discrepancy between the microscopic images from Figure 2A and the quantification of pH3 positive cells using flow cytometry in Figure 4. According to the flow cytometric results the proportion of pH3 positive cells is about 3 times higher in haploid larvae compared to the control. The increase in mitotic cells in the imaging results however seems much more drastic. It would be helpful if the authors explain here. Mitotic slippage that the authors observe to be increased in the haploid larvae to up to 5% of cells should result in an increase in the number of aneuploid cells. I am wondering why this is not recapitulated in the analyses of the DNA content in Figure S1.

    Discussion:

    I find the explanation of centrosomal loss due to depletion of centrosomal protein pools in the cytoplasm during drastic cell reduction interesting. I wonder if the reduction in size is not necessarily caused by the reduction in cells, but rather the result of the absence of a second active allele that produces centrosomal proteins?

    Significance

    Overall, I find the study interesting even to a broader audience since diploid development is a fundamental feature of most animals. The authors also manage to discuss their findings on the consequences of haploidy in this bigger context of the restricted diploid development in animals. The study is very well-written even to non-experts.

    I am not an expert of the literature describing previous characterizations of the consequences associated with haploid cell development in animals, which is why I cannot comment on the novelty of their study. Based on my expertise on centromeres and genome organisation I can however assess the results regarding the mitotic defects observed in haploid larvae (see comments).

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    Referee #2

    Evidence, reproducibility and clarity

    This study examined cell proliferation and death in haploid and diploid zebrafish and attempted to provide insights into cellular mechanisms underlying haploidy-linked defects in non-mammalian vertebrates. While some of the ideas were potentially interesting, the experiments were not rigorous and inadequate data analyses were performed. Major issues include:

    • Lack of proper controls in many experiments. For example, in the experiments where the authors treated haploids with reversine to suppress the SAC, there was no no-treatment control (Fig. 6A-C). In Fig. 6D, when a DMSO control was included, the control fish were from 3 dpf while the reversine-treated fish were from 0.5-3 dpf. This is a big flaw in experimental design, especially considering the authors were looking at mitotic index, which is hugely impacted by developmental time.
    • Subjective and inadequate data quantification. In the immunostaining experiments to detect caspase-3 and pH3, the authors either did not quantify at all and only showed single micrographs that might or might not be representative (for pH3), or only did very subjective and unconvincing quantification (for caspase-3). Objective measurements of fluorescence intensity could have been done, but the authors instead chose to categorize the staining into arbitrary categories with unclear standards. In example images they showed in the supplementary data, it is not obvious at all why some of the samples were classified as "mild" and others as "severe" when their staining did not appear to be very different. Additionally, the authors claimed that "clusters of apoptotic cells" were only present in haploids but not diploids or p53 MO haploids, but they did not show any quantification. From the few example images (Fig.S3A), apoptotic clusters can be seen in p53 MO treated fish. Also, in some cases, the clusters were visible only because those fish were mounted in an incorrect orientation. For example, in Fig. S3A, control #2, that fish was visualized from its side, thus exposing areas around its eye that contained such clusters. These areas are not visible in other images where the fish were visualized from the top.
    • Subpar data quality. Aside from issues with qualification, the IF data was not convincing as staining appeared to be inconsistent and uneven, with potential artefacts.
    • Unsupported and overstated claims. There were many overstatements. For one, in line 268, the authors claimed that "the haploidy-linked mitotic stress with SAC activation is a primary constraint for organ growth in haploid larvae", while what they were actually showed was that reversine treatment, which suppresses the SAC, was partially rescued 2 out of the 3 growth defects they assessed, to such a small extent that the difference between haploid and haploid rescue was only <20% of that between haploid and diploid. Again, they did not include proper controls so haploid, haploid rescue, and diploid were never in one experiment together - they were in different figures, plotted in drastically different scales - and 20% is only an estimate. With so many fundamental flaws, the data seem unreliable and the paper does not meet publishable standards.

    Significance

    The low quality of the analysis makes the significance low.

    Reviewers have expertise in vertebrate embryogenesis and ploidy manipulation.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Yaguchi et al. investigate the causes of the "haploid syndrome" in the zebrafish embryo, the old observation that haploid embryos suffer from severe developmental defects and growth retardation of organs such as the brain and eyes (these defects are not simply a consequence of loss of heterozygosity, as they are rescued by forced diploidization of haploid larvae). Looking at apoptosis and proliferation, the authors show an increase in the number of apoptotic and mitotic cells in haploid larvae. Regarding apoptosis, they show an increase in p53 levels and demonstrate that knockdown of p53 limits apoptosis and leads to some phenotypic improvement. Regarding mitosis, they show an increase in mitotic delays and failures in haploid larvae. Inhibition of the spindle assembly checkpoint can reduce these defects and leads to some improvement in body axis length and eye size. Looking at the cause of the mitotic defects, the authors show that haploid cells often have monopolar spindles and loss of one centrosome, defects that appear to correlate with cell size.

    Major comments:

    While some experiments could be better quantified and/or statistically analyzed (see below), overall the results are convincing and clearly presented.

    • Diploid embryos are used as controls. Gynogenetic diploids seem to be better controls to ensure that the observed phenotypes are not related to loss of heterozygosity. To limit the amount of work, the use of gynogenetic diploids could be restricted to spindle polarity and centrosome number experiments.
    • As the authors discuss, it would be necessary to rescue centrosome loss to establish a causal relationship between centrosome loss and haploid viability. I certainly acknowledge that this is difficult (if not impossible), but it currently limits the significance of the results.
    • Some experiments are not, or arguably, quantified/statistically analyzed.
      • Figure 2, Active caspase level. Larvae are sorted into three categories, and no statistical test is performed on the obtained contingency table. A Fisher's exact test here, or much better, the active caspase-3 levels should be quantified, instead of sorting larvae into categories.
      • Same comment for 3E-F. Larvae are scored as Scarce, Mild or Severe. Looking at Fig S3A, I see one mild p53MO embryo, but the two others are not that different from 'severe' cases, which would completely change the contingency table. Again, a proper quantification would be better.
      • Figure 4D-E, no stats.
      • Figure 6, Reversine treated haploid should be compared to haploid embryos (on the graphs and statistically). If no specific controls have been quantified for this experiment, data could be reused from previous figures, provided this is stated.
      • Rescue by p53MO and Reversine, it would be nice to also include diploid measurements on the graphs, so that the reader can appreciate the extent of the rescue.

    Minor comments:

    • Lines 221-223, authors claim that centriole loss and spindle monopolarization commence earlier in the eyes and brain than in skin. I am note sure I see this in Fig. S5. It could as well be that the defect is less pronounced in skin.

    • Lines 227-229, authors claim that 'The developmental stage when haploid larvae suffered the gradual aggravation of centrosome loss corresponded to the stage when larval cell size gradually decreased through successive cell divisions'. I did not get that. Doesn't cell size decrease since the first division? Fig 5D shows that cell size decreases all along development.

    • Some correlations are used to draw conclusions:

    • Line 301-303. "The correlation between centrosome loss and spindle monopolarization indicates that haploid larval cells fail to form bipolar spindle because of the haploidy-linked centrosome loss.". As stated by the authors, this is a correlation only. I agree it points in this direction.

    • Line 305-308. "Interestingly, centrosome loss occurred almost exclusively in haploid cells whose size became smaller than a certain border (Fig. 5), indicating that cell size is a key determinant of centrosome number homeostasis in the haploid state." This one is more problematic. There is no causal link established between cell size and centrosome number homeostasis. It could very well be that some unidentified problem induces both a reduction in cell size and the loss of centrioles.

    Significance

    I have concerns regarding the significance of the reported findings. Haploid zebrafish embryos show numerous developmental defects (some as early as gastrulation, as previously shown by the authors, Menon 2020), and they die by 4 dpf. That they experience massive apoptosis at day 3 does not seem very surprising, and that inhibiting p53 transiently improves the phenotype is not a big surprise. This reminds me of the non-specific effects of morpholino injection, which can be partially rescued by knocking down p53. The observation of mitotic arrest and mitotic defects and the observation that haploid cells often lack a centrosome is interesting. However, I felt that the manuscript suggested that these observations were novel and could explain the haploid syndrome specifically in non-mammalian embryos, when the authors reported the same observations in human haploid cells as well as in mouse haploid embryos (Yaguchi 2018). To me, this manuscript mainly confirms that their previous observation is not mammalian specific, but at least conserved in vertebrates.

    While I am no expert at centrosome duplication, I find the observation that haploidy leads to centrosome loss very intriguing, but have the impression that this manuscript falls short of improving our understanding of this phenomenon.

  6. Version published to 10.1101/2022.05.12.491746v2 on bioRxiv
  7. Version published to 10.1101/2022.05.12.491746v1 on bioRxiv