H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes

Oikopleura dioica is a ubiquitous marine zooplankton of biological interest owing to features that include dioecious reproduction, a short life cycle, conserved chordate body plan, and a compact genome. It is an important tunicate model for evolutionary and developmental research, as well as investigations into marine ecosystems. The genome of north Atlantic O. dioica comprises three chromosomes. However, comparisons with the genomes of O. dioica sampled from mainland and southern Japan revealed extensive sequence differences. Moreover, historical studies have reported widely varying chromosome counts. We recently initiated a project to study the genomes of O. dioica individuals collected from the coastline of the Ryukyu (Okinawa) Islands in southern Japan. Given the potentially large extent of genomic diversity, we employed karyological techniques to count individual animals’ chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. Epifluorescence and confocal images were obtained of embryos and oocytes stained with two commercial anti-H3S28P antibodies (Abcam ab10543 and Thermo Fisher 07-145). The data lead us to conclude that diploid cells from Okinawan O. dioica contain three pairs of chromosomes, in line with the north Atlantic populations. The finding facilitates the telomere-to-telomere assembly of Okinawan O. dioica genome sequences and gives insight into the genomic diversity of O. dioica from different geographical locations. The data deposited in the EBI BioImage Archive provide representative images of the antibodies’ staining properties for use in epifluorescent and confocal based fluorescent microscopy.


Introduction
The larvacean, Oikopleura dioica, possesses a fascinating genome: it has reduced to a mere 70Mbp and exhibits unique characteristics such as non-canonical splicing and the scattering of Hox genes (Denoeud et al., 2010;Edvardsen et al., 2005;Marz et al., 2008;Seo et al., 2001).It is thought that a combination of large effective population size and high mutation rate per generation have led to fast evolution (Berná & Alvarez-Valin, 2014).The recently published genome sequence of a "Japanese O. dioica" from mainland Japan highlighted large sequence variations between the Pacific and Atlantic populations (Wang et al., 2020).In addition, we recently released a telomere-to-telomere genome sequence of an O. dioica individual collected from the Okinawan coastline in southern Japan (Bliznina et al., 2020), which, to our surprise, revealed large differences in synteny to the mainland Japanese genome despite the geographical proximity.The genetic map of the north Atlantic O. dioica is reported to contain three chromosomes (two autosomes, X and Y sex chromosomes; Denoeud et al., 2010); however, prior studies based on histochemical techniques reported three (Körner, 1952) and eight chromosomes (Colombera & Fernaux, 1973).Given the large sequence and synteny differences between the assembled O. dioica genomes, as well as the discrepancies among previous studies, we wished to assess the karyotype for the local Okinawan O. dioica population.
Karyotyping is a long-established histochemical method to visualize eukaryotic chromosomes (Hsu & Benirschke, 1967;Tjio & Levan, 1950).This rapid technique, involving the use of stains including methylene blue, eosin, and azure B, allows for observation of chromosomes with a simple light microscope, naturally lending itself to a first attempt for karyotyping analysis (Giemsa, 1904).However, we were unable to determine an accurate count for the Okinawan O. dioica by this method due to variability which ranged from 11-27 chromosomes per nucleus.
As an alternative approach, we decided to immunostain the centromere as a means of quantifying the number of chromosomes.Metaphase-specific histone 3 (H3) markers have been used to determine the structure and the segregation of genetic material during oogenesis in situ (Ganot et al., 2006;Schulmeister et al., 2007).One such marker that has been successfully visualized in O. dioica is histone H3 phosphorylated at Ser-28 (Kawajiri et al., 2003;Kurihara et al., 2006), whose localization depends on the phase of the cell cycle: during metaphase, sister chromatids were stained in a manner consistent with alignment along the metaphase plate, whereas in non-mitotic cells, spatially punctate signals were found evenly spread within the nuclear envelope (Campsteijn et al., 2012;Feng & Thompson, 2018;Feng et al., 2019;Olsen et al., 2018).A structure in which chromosomes are sequestered in a ∏-shaped conformation has also been observed during meiotic cell divisions between the final phases of oogenesis and mature oocytes (Ganot et al., 2008).In Table 1, we list the publications in which the H3S28P marker was applied to O. dioica: the studies were all performed using cultured strains originating from the north Atlantic Ocean.Here, we visualized anti-H3S28P stained embryos from two commercially available antibody  et al., 2020).Mature females were collected prior to spawning, individually washed with filtered autoclaved seawater (FASW) 3 times for 10 minutes and placed in separate 1.5 ml tubes containing 500 µl of FASW.Nearly mature males, full of sperm, were also washed 3 times in FASW.Mature males that successfully made it through the washes intact were placed in 100 µl of fresh FASW and allowed to spawn naturally.As soon as females spawned, each individual clutch of 100-200 eggs was washed three times for 10 minutes by moving eggs along with a pulled capillary micropipette from well to well in a 6-well dish, each containing 5 ml of FASW, and left in a fresh well of 5 ml FASW in the same dish.These were stored at 17 °C and set aside awaiting fertilization.Staged embryos were initiated by gently mixing 10 µl of the spawned male sperm with the awaiting eggs in FASW at 23 °C.Developing embryos were staged and collected by observation under a Leica M165C dissecting microscope.These embryos were quickly dechorionated using 0.1% sodium thioglycolate and 0.01% actinase in FASW for 2-3 minutes, then promptly washed with 2 washes with FASW prior to fixation and staining.Unfertilized eggs were treated similarly with three successive 10-minute washes.
Histochemical staining.Embryos were Giemsa stained as previously described in Shoguchi et al., 2005.Briefly, approximately 20-30 dechorionated embryos were treated with 0.04% colchicine in FASW for 30 minutes and then treated with decreasing amounts of KCl (50 mM and 25 mM) for five minutes each.Fixation was quickly performed with cold methanol:glacial acetic acid (3:1).The fixative was changed three times in the span of 18 hours while at -30 °C.The next morning, the fixed cells were quickly resuspended in 60% Acetic acid and methodically dropped from a height of 7 -8cm onto a 48°C pre-warmed slide (Matsunami Glass, S2441).The slides were incubated for an additional 2 hours at 48°C; then stained with 6% Gimesa in 67mM sodium phosphate pH 7.0 for 2 hours at room temperature and rinsed with double distilled H 2 O.These were dried for two hours at room temperature, mounted with DPX Mountant (Sigma, 06522) and covered with No.1 35 x 50 mm glass coverslips (Matsunami Glass, C035551).

Results
We initially attempted to visualize chromosomes using Giemsa staining on developing embryos.The spreads from 32-and 64-cell developmental stages, gave results with counts ranging between 11-27 stains per cell (BioImage Archive, S-BIAD21, Experiment A).Although cell-spreads were confined as a result of incomplete dechorionation with the enzymatic dissociation cocktail, we were still able to assign chromosomes to individual cells.Disappointingly, chromosome counts were unreliable due to the observed variability.
Consequently, we performed immunostaining of similarly staged embryos using a H3S28P-specific primary antibody and a secondary antibody conjugated to Alexa488 directed against the primary antibody.Signal-based thresholding was employed to determine the number of distinct 515 nm emission signals present in images acquired with epifluorescent and laser confocal microscopes (BioImage Archive, S-BIAD21, Experiment B & D).The data was analyzed using the Imaris SPOT DETECTION tool (Oxford Instruments).
Cells were manually classified into two types depending on the staining pattern visible in the nucleus: (i) those with intense clusters of signals in the center, considered to be in metaphase and (ii) those containing evenly distributed, clearly separated spots within a faint background of signal defining a region encompassed by the nuclear envelope, interpreted as non-mitotic (Figure 1A and 1B,blue circles;Figure 1A and 1B,red squares).Counts from these two classes of nuclei fall into separate distributions (Figure 1C and 1D), with both epifluorescence and confocal acquisitions in agreement with each other.We interpreted the nuclei with an average of six large, clustered signals as centromeric regions in metaphase (Figure 1B), however, we cannot explain the cell cycle state of those containing the average of 12 spatially distinct punctate signals.
To rule out polyploidy, which occurs in O. dioica somatic cells that give rise to the mucosal house (Ganot & Thompson, 2002), we also analyzed oocytes in metaphase I before fertilization (Schulmeister et al., 2007).We identified confined groupings of signals in unfertilized eggs (Figure 2A; BioImage Archive, S-BIAD21, Experiment E) and analyzed confocal images using the Imaris SPOT DETECTION tool to determine H3S28P signal counts (Figure 2B).Counts from the compact rosette-shaped chromatin structure averaged near 6.Visual inspection of individual Z-sections (Figure 2C) confirms the Imaris count analysis and annotation (Figure 2D).We interpreted each spot as representing a centromere from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).

Discussion
Our initial attempts at karyotyping by traditional Giemsa-staining gave us wildly varying counts which we unable to overcome with or without mitotic arrest.Giemsa-staining has been applied successfully to other organisms with small chromosomes such as the tunicate Ciona intestinalis (Shoguchi et al., 2005).The difference in outcome might be explained by the higher AT content of those genomes compared with O. dioica, since Giemsa preferentially stains AT-rich sequences.Although we do rule out Giemsa-staining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results.
Most karyotyping studies display a representative image to support the conclusion; however, given the variability in signal counts between nuclei, we decided to take a statistical approach that quantifies the uncertainty in the estimated chromosome count.Despite testing many different image acquisition settings, we were unable to eliminate the variability; we believe there are several possible reasons that explain the variance.(i) We applied uniform signal thresholds to all cells, so any spots below the threshold would have been missed.(ii) Spots displayed non-uniform signals, and individual centromeres may have occasionally contributed multiple counts.(iii) The H3S28P signal is not always confined to centromeres, and so may have caused multiple counts (see below).(iv) Finally, the threedimensional rosette structures in oocytes might not have always been captured reliably in the focal plane.It is worth noting that for O. dioica, immunostaining showed much smaller variabilities than Giemsa-staining.
An important consideration is what the H3S28P signal represents.It has been used to visualize centromeric regions in O. dioica (Table 1), but the signal is not confined to the centromere and its localization depends on the cellular state (Figure 1; Hake et al., 2005;Feng & Thompson, 2018).However, we are confident that the signals seen in Figure 1 labelled as metaphase and Figure 2 represent centromeres and their associated chromosome.Further, DNA-staining images of mature oocyte have previously been interpreted as chromosomes condensed in a structure resembling the Greek character ∏ (Ganot et al., 2007;Ganot et al., 2007b;Ganot et al., 2008).Since we did not perform DNA stains, our interpretation of the H3S28P signal in the oocyte does not preclude the previously reported ∏-structure.Additionally, the positions and numbers of crossovers between homologous pairs are unresolvable in this highly condensed state and the signal positions are not definitive of centromeric regions.
Currently, the nucleotide sequence of the centromeric region is unknown for O. dioica, although chromatin immunoprecipitation with a H3S28P antibody followed by long-read sequencing might be able to provide this information.However, our whole embryo staining data (Figure 1) and the previous literature (Table 1) show that the H3S28P antibody produces non-centromeric signals which may confound such analysis.Thus, alternative targets such as other centromeric histone 3 variants (Moosmann et al., 2011) might be preferable.Knowledge of centromeric sequences would also open the possibility of confirming these results with fluorescence in situ hybridization.
Despite the variations in signal counts between nuclei, a haploid chromosome count of three provides the most parsimonious explanation of the collected data and is consistent with previously published genome sequence assemblies (Denoeud et al., 2010).In summary, we conclude that the Okinawan Oikopleura dioica genome consists of three pairs of chromosomes in diploid cells.We believe that the images may be useful for examining cell cycle specific changes to chromosome structure and encourage the reuse and reanalysis of our data located in the EBI BioImage Archive (Ellenberg et al., 2018).
main aim of this study, I would like the authors to describe merits of this new method in further detail.Without sufficiently convincing explanations, the authors' method appears to be a less sophisticated alternative to the standard karyotyping methods.Particularly, discussion is required for the observation of seven or eight signals within a single nucleus.It will help if the authors explain why the standard methods are not applicable to O. dioica.
If the authors' main aim is to determine the number of chromosomes in Okinawan O. dioica, they should explain more about particularity of this species.Is there a hypothesis that Pacific and Atlantic O. dioica are different species?If not, is there the possibility that different populations (Pacific and Atlantic) have different numbers of chromosomes within the same species?The number of chromosomes is highly variable even between closely related species.However, to my knowledge, the number of chromosomes is essentially invariant within a species.Uncommon exceptions are chromosome reorganization in Ascaris embryos and Paramecium macronuclei.Although the authors discuss the discrepancy in the number of the O. dioica chromosomes (n = 3, or n = 8), I felt that the argument has already been settled (on n = 3) by the extensive genome sequencing (Denoeud et al., 2010).If the authors want to insist that the number of chromosomes in Pacific O. dioica may not be three, more detailed biological information (rationale) is necessary.

Minor points:
In Table 1,  I guess that "ddH2O" (page 4 line 17) is double-distilled H 2 O. Anyway, "ddH2O" is a laboratory-specific jargon.Similarly, I guess that "ON" (page 4 line 23) means "overnight"?These abbreviations cannot be recommended to be used in articles.

2.
Since the authors have knowledge that some somatic cells are polyploid in O. dioica.Therefore, they had better clearly state that the cells shown in Figure 1 are not the case.
Although the authors state that 32~64-cell embryos were used for Giemsa staining, they did not tell the developmental stages they used for the antibody staining (in the second paragraph of the Results section).Are they also the early embryos?And do they consist exclusively of diploid cells? 3.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 29 Jan 2021 Andrew W Liu, Okinawa Institute of Science and Technology, Onna-son, Japan We thank Dr Fujiwara's helpful feedback and critique on our We have done our best to address all the concerns and minor points he has brought to our attention, which are listed below.

Reviewer comment
This manuscript describes a new method for karyotyping using the antibody raised against Ser28-phosphorylated Histone H3 (H3S28P).Using this method, the authors obtained the results suggesting that Okinawan Oikopleura dioica somatic cells contain three sets of chromosomes.Specific detection of O. dioica's phosphorylated H3 by the antibody has been proven in other papers, shown in Table 1.The data presented in this article are therefore reliable, and the conclusion seems appropriate.However, after I read to the end of the article, I did not really understand what the main aim and novelty of this article were.Which is the main aim, development of a new karyotyping method or determination of the number of chromosomes in diploid O. dioica somatic cells?Although the article type is "BRIEF REPORT", clearer statements and more detailed explanations are required.I hope that the following comments are useful for the authors.All of my comments are for presentation and description.

Author response
We thank the referee for the feedback, which have helped improve the clarity and quality of the manuscript.To clarify, the aim of this paper is to determine the number of chromosomes for the Okinawan O. dioica genome.We have detailed the reasons for this in our response to Reviewer Comment 1.1 above.

Manuscript changes
We clarified the main aim of the paper and strengthened the justification for this in the

Reviewer comment
The Introduction section starts with the history of karyotyping.This implies that the development of a new karyotyping technique appears to be the main aim of this study.

Author response
We thank the referee for this comment.We have now substantially revised the Introduction to clarify that the main aim of the study is to determine the chromosome count.We have retained the description of the histochemical and immunostaining methods as two contrasting approaches, in order to explain why we chose the latter approach here; however, we hope that it is now clear that we are not implying the publication of a new karyotyping technique.dioica is about one-fifth of that in C. Intestinalis.Therefore, readers may feel that the average size of the O. dioica chromosomes is large enough to be examined by the standard methods.If the development of the new method is really the main aim of this study, I would like the authors to describe merits of this new method in further detail.Without sufficiently convincing explanations, the authors' method appears to be a less sophisticated alternative to the standard karyotyping methods.

Manuscript changes
It will help if the authors explain why the standard methods are not applicable to O. dioica.

Author response
We thank the referee for this comment.We were equally frustrated by the difficulties in performing Giemsa staining, which gave even larger variations in signal counts.
Anecdotally, this appears to be a similar experience in other laboratories studying O. dioica.
FISH is an attractive future possibility for further validation of the immunostaining and genome assembly results.

Manuscript changes
Please also see authors response to Reviewer 1 comment 3.1 -Interpretation of H3S28P signal locations above.
1. Paragraph 14 (Discussion)."Our initial attempts at karyotyping by traditional Giemsa staining gave us wildly varying counts which we unable to overcome with or without mitotic arrest.Giemsa-staining has been applied successfully to other organisms with small chromosomes such as the tunicate Ciona intestinalis (Shoguchi et al., 2005).The difference in outcome might be explained by the higher AT content of those genomes compared with O. dioica, since Giema preferentially stains AT-rich sequences.Although we do rule out Giemastaining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results." 2. Paragraph 11. "Consequently, we performed immunostaining of similarly staged embryos…" 3. Paragraph 13. "To rule out polyploidy, which occurs in O. dioica somatic cells that give rise to the mucosal house (Ganot & Thompson, 2002), we also analyzed oocytes in metaphase I before fertilization" Competing Interests: The authors disclose no competing interests with regard to F1000's review process or this individual's peer review report.Reviewer Expertise: cell cycle, oogenesis I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
It's interesting to know, though not surprising, that Japanese O. dioica has the same number of centromeres and chromosomes as that in Norwegian species.

Author response
We thank the reviewer for this comment.
1.After submission of this manuscript to F1000Research, two additional O. dioica genomes were published for (i) samples acquired in mainland Japan and (i) an individual from the Okinawa coastline.Preliminary comparison of the three O. dioica genomes have revealed very divergent sequences at single nucleotide and kilo/megabase scales (unpublished results).Given that mainland and Okinawan O. dioica are both "Japanese", we avoid the term "Japanese O. dioica" in the present manuscript.
2. Historical studies reported between 3 and 8 chromosomes for O. dioica.
3. For these reasons, it was not obvious to us that the Okinawan O. dioica would have the same number of chromosomes as the Norwegian and mainland Japanese O. dioica.
4. Therefore, we wished to confirm independently the chromosome number of the Okinawan O. dioica.

Manuscript changes
We rewrote the Abstract and Introduction to strengthen the justification for this study.
Abstract.Oikopleura dioica is a ubiquitous marine zooplankton of biological interest owing to features that include dioecious reproduction, a short life cycle, conserved chordate body plan, and a compact genome.It is an important tunicate model for evolutionary and developmental research, as well as investigations into marine ecosystems.The genome of north Atlantic O. dioica comprises three chromosomes.However, comparisons with the genomes of O. dioica sampled from mainland and southern Japan revealed extensive sequence differences.Moreover, historical studies have reported widely varying chromosome counts.We recently initiated a project to study the genomes of O. dioica individuals collected from the coastline of the Ryukyu (Okinawa) Islands in southern Japan.
Given the potentially large extent of genomic diversity, we employed karyological techniques to count individual animals' chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3.Introduction (paragraphs 1-3, complete).The larvacean, Oikopleura dioica, possesses a fascinating genome: it has reduced to a mere 70Mbp and exhibits unique characteristics such as non-canonical splicing and the scattering of Hox genes (Seo et al., 2001;Edvardsen et al., 2005;Marz et al., 2008;Denoeud et al., 2010).It is thought that a combination of large effective population size and high mutation rate per generation have led to fast evolution (Berná et al., 2014).The recently published genome sequence of a "Japanese O. dioica" from mainland Japan highlighted large sequence variations between the Pacific and Atlantic populations (Wang et al., 2020).In addition, we recently released a telomere-to-telomere genome sequence of an O. individual collected from the Okinawan coastline in southern Japan (Bliznina et al., 2020), which, to our surprise, revealed large differences in synteny to the mainland Japanese genome despite the geographical proximity.Colombera & Fernaux, 1973).Given the large sequence and synteny differences between the assembled O. dioicagenomes, as well as the discrepancies among previous studies, we wished to assess the karyotype for the local Okinawan O. dioica population.
Karyotyping is a long-established histochemical method to visualize eukaryotic chromosomes (Hsu & Benirschke, 1967;Tjio & Levan, 1950).This rapid technique, involving the use of stains including methylene blue, eosin, and azure B, allows for observation of chromosomes with a simple light microscope, naturally lending itself to a first attempt for karyotyping analysis.However, we were unable to determine an accurate count for the Okinawan O. dioica by this method due to variability which ranged from 11-27 chromosomes per nucleus.
As an alternative approach, we decided to immunostain the centromere as a means of quantifying the number of chromosomes.Metaphase-specific histone 3 (H3) markers have been used to determine the structure and the segregation of genetic material during oogenesis in situ (Ganot et al., 2006;Schulmeister et al., 2007).One such marker that has been successfully visualized in O. dioica is histone H3 phosphorylated at Ser-28 (Kawajiri et al., 2003;Kurihara et al., 2006), whose localization depends on the phase of the cell cycle: during metaphase, sister chromatids were stained in a manner consistent with alignment along the metaphase plate, whereas in non-mitotic cells, spatially punctate signals were found evenly spread within the nuclear envelope (Campsteijn et al., 2012;Feng & Thompson, 2018;Feng et al., 2019;Olsen et al., 2018).A structure in which chromosomes are sequestered in a ∏-shaped conformation has also been observed during meiotic cell divisions between the final phases of oogenesis and mature oocytes (Ganot et al., 2008).In Table 1, we list the publications in which the H3S28P marker was applied to O. dioica: the studies were all performed using cultured strains originating from the north Atlantic Ocean.
Here, we visualized anti-H3S28P stained embryos from two commercially available antibody sources and unfertilized oocytes to determine the chromosome count of the local Okinawan O. dioica.

Reviewer comment
This piece of work can boost broad interests in using O. dioica as a new model in epigenetics and cell cycle studies.However, some results are a bit confusing to me and may be misinterpreted.Ganot et al., 2007).Since we did not perform DNA stains, our interpretation of the H3S28P signal in the oocyte does not preclude the previously reported ∏-structure.Additionally, the positions and numbers of crossovers between homologous pairs are unresolvable in this highly condensed state and the signal positions are not definitive of centromeric-regions." Paragraph17."Currently, the nucleotide sequence of the centromeric region is unknown for O. dioica, although chromatin immunoprecipitation with a H3S28P antibody followed by long-read sequencing might be able to provide this information.However, our whole embryo staining data (Figure 1) and the previous literature (Table 1) show that the H3S28P antibody produces non-centromeric signals which may confound such analysis.The timing of sampling is not indicated, which makes it even harder to interpret the data.

Author response
We thank the referee for this comment.The process of rinsing the eggs took more than 15 min and so the oocytes were metaphase I. Changes were made in the methods section.

Manuscript changes
Paragraph 4. "Unfertilized eggs were treated similarly with three successive 10-minute washes."Paragraph 6. "Washed eggs, 32 and 64 cell embryos (described above) were immediately fixed…" _____________________ Reviewer 1 comment 3.3 -Schematic representation of chromosomes in embryos in Figure 2E Reviewer comment But again, it should one red dot between a pair of sister chromatids in Fig 2E.
Author response

Figure 2 .
Figure 2. Centromere counts from unfertilized eggs.A Maximum signal projection of a representative confocal Z-stack acquisition of anti-H3S28P rat monoclonal stained oocyte used for the count analysis (EBI Image Archive S-BIAD21, Experiment E 20200114_04.lsm).B Distribution of signal counts in each rosette-shaped chromatin structure, analyzed by Imaris software SPOT DETECTION tool (n = 23, mean 5.70, 95% CI 5.2 -6.2).C Individual Z-sections from same image acquisition showing the 3D structure of the chromatin.Each plane is 0.54 µm apart.D Imaris spot analysis and annotation of signal positions from Z-stack acquisition.E Schematic representation of our interpretation that each signal is a centromere from a pair of sister chromatids.Chromosomes have been drawn with equal lengths for simplicity.The positions of centromeric regions cannot be determined as chiasmata(s) are present along the homologous pairs of chromosomes in a highly condensed state.

Reviewer Report 05
August 2020 https://doi.org/10.5256/f1000research.27598.r68260© 2020 Feng H.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Haiyang Feng Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway It's interesting to know, though not surprising, that Japanese O. dioica has the same number of centromeres and chromosomes as that in Norwegian species.This piece of work can boost broad interests in using O. dioica as a new model in epigenetics and cell cycle studies.However, some results are a bit confusing to me and may be misinterpreted.In Fig 1, centromere counts at prophase are 12, and at metaphase are 6, which are inconsistent.H3S28p signals locate at inner centromeric regions, flanked by CenpA signals that mark kinetochores at metaphase in embryonic mitosis in Norwegian O. dioica.The counts of H3S28p signals should be the same at prophase and metaphase, which are 6.In addition, centromere is a piece of DNA sequence that holds a pair of sister chromatids in mitotic phase before they separate at anaphase.We can say that a chromosome has one centromere and a pair of sister chromatids at prophase.Thus, the schema representing prophase in Fig 1B should be a pair of sister chromatids is linked by one red dot at centromere.H3S28p signals in female meiosis of Norwegian O. dioica are a bit different from those in mitosis.It localizes on entire chromosomes in prophase, moves towards centromeric regions during prometaphase, and is enriched at centromeric regions (or accurately speaking, midline of a bivalent) at metaphase I. Since chromosomes are more condensed in meiosis, and the midline of a bivalent should be crossover site between homologous chromosomes, we don't know how far away it is between centromere and crossover in meiotic chromosomes of O. dioica, and how many crossovers a bivalent has.I would say centromeric region of H3S28p signals in female meiosis with caution.Actually, H3S28p shows several spots (more than 6) during prometaphase I, as can be seen Fig S4 in Feng and Thompson, 2018 cell cycle.The stages of meiosis depend on when the oocytes are collected.Just after spawning, the oocytes are before prometaphase I. Within 10 to 15 min after spawning, it is prometaphase I. Later, it should be at metaphase I.The timing of sampling is not indicated, which makes it even harder to interpret the data.But again, it should one red dot between a pair of sister chromatids in Fig 2E.Is the work clearly and accurately presented and does it cite the current literature?Yes Is the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes Competing Interests: No competing interests were disclosed.
Epifluorescence and confocal images were obtained of embryos and oocytes stained with two commercial anti-H3S28P antibodies (Abcam ab10543 and Thermo Fisher 07-145).The data lead us to conclude that diploid cells from Okinawan O. dioicacontain three pairs of chromosomes, in line with the north Atlantic populations.The finding facilitates the telomere-to-telomere assembly of Okinawan O. dioica genome sequences and give insight into the genomic diversity of O. dioica from different geographical locations.The data deposited in the EBI BioImage Archive provide representative images of the antibodies' staining properties for use in epifluorescent and confocal based fluorescent microscopy.

Figure 1
Figure 1 labelled as metaphase and Figure 2 represent centromeres and their associated chromosome.Further, DNA-staining images of mature oocyte have previously been interpreted as chromosomes condensed in a structure resembling the Greek character ∏ (Ganot et al., 2007).Since we did not perform DNA stains, our interpretation of the H3S28P signal in the oocyte does not preclude the previously reported ∏-structure.Additionally, the positions and numbers of crossovers between homologous pairs are unresolvable in this highly condensed state and the signal positions are not definitive of centromeric-regions." Paragraph17."Currently, the nucleotide sequence of the centromeric region is unknown for O. dioica, although chromatin immunoprecipitation with a H3S28P antibody followed by long-read sequencing might be able to provide this information.However, our whole embryo staining data (Figure1) and the previous literature (Table1) show that the H3S28P antibody produces non-centromeric signals which may confound such analysis.Thus, alternative targets such as other centromeric histone 3 variants(Moosmann et al., 2011)   might be preferable.Knowledge of centromeric sequences would also open the possibility of confirming these results with fluorescence in situhybridization." Figure 1 labelled as metaphase and Figure 2 represent centromeres and their associated chromosome.Further, DNA-staining images of mature oocyte have previously been interpreted as chromosomes condensed in a structure resembling the Greek character ∏ (Ganot et al., 2007).Since we did not perform DNA stains, our interpretation of the H3S28P signal in the oocyte does not preclude the previously reported ∏-structure.Additionally, the positions and numbers of crossovers between homologous pairs are unresolvable in this highly condensed state and the signal positions are not definitive of centromeric-regions." Paragraph17."Currently, the nucleotide sequence of the centromeric region is unknown for O. dioica, although chromatin immunoprecipitation with a H3S28P antibody followed by long-read sequencing might be able to provide this information.However, our whole embryo staining data (Figure1) and the previous literature (Table1) show that the H3S28P antibody produces non-centromeric signals which may confound such analysis.Thus, alternative targets such as other centromeric histone 3 variants(Moosmann et al., 2011)   might be preferable.Knowledge of centromeric sequences would also open the possibility of confirming these results with fluorescence in situhybridization."

2 comment 1.3 -Use of immunostaining over histochemical methods Reviewer comment
Epifluorescence and confocal images were obtained of embryos and oocytes stained with two commercial anti-H3S28P antibodies (Abcam ab10543 and Thermo Fisher 07-145).The data lead us to conclude that diploid cells from Okinawan O. dioica contain three pairs of chromosomes, in line with the north Atlantic populations.The finding facilitates the telomere-to-telomere assembly of Okinawan O. dioica genome sequences and give insight into the genomic diversity of O. dioica from different geographical locations.The data deposited in the EBI BioImage Archive provide representative images of the antibodies' staining properties for use in epifluorescent and confocal based fluorescent microscopy." 2. Paragraph 1 (Introduction)."…Giventhe large sequence and synteny differences between the assembled O. dioica genomes, as well as the discrepancies among previous studies, we wished to assess the karyotype for the local Okinawan O. dioica population." the conclusion; however, given the variability in signal counts between nuclei, we decided to take a statistical approach that quantifies the uncertainty in the estimated chromosome count.Despite testing many different image acquisition settings, we were unable to eliminate the variability; we believe there are several possible reasons that explain them.(i)Weapplied uniform signal thresholds to all cells, so any spots below the threshold would have been missed.(ii)Spotsdisplayed non-uniform signals, and individual centromeres may have occasionally contributed multiple counts.(iii)TheH3S28P signal is not always confined to centromeres, and so may have caused multiple counts (see below).(iv)Finally, the three-dimensional rosette structures in oocytes might not have always been captured reliably in the focal plane.It is worth noting that for O. dioica, immunostaining showed much smaller variabilities than Giemsa-staining."_____________________ReviewerShoguchiet al. (2005)(cited in this article) clearly showed 14 pairs of chromosomes of the Ciona intestinalis genome by means of Giemsa staining and FISH.While the size of the genome in O. dioica is a half of that in C. intestinalis, the number of chromosomes in O.
1. Abstract."Oikopleuradioica is a ubiquitous marine zooplankton of biological interest owing to features that include dioecious reproduction, a short life cycle, conserved chordate body plan, and a compact genome.It is an important tunicate model for evolutionary and developmental research, as well as investigations into marine ecosystems.The genome of north Atlantic O. dioica comprises three chromosomes.However, comparisons with the genomes of O. dioica sampled frommainland and southern Japan revealed extensive sequence differences.Moreover, historical studies have reported widely varying chromosome counts.We recently initiated a project to study the genomes of O. dioica individuals collected from the coastline of the Ryukyu (Okinawa) Islands in southern Japan.Given the potentially large extent of genomic diversity, we employed karyological techniques to count individual animals' chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3.3.Paragraph 2 (Introduction)."However, we were unable to resolve individual O. dioica chromosomes by this method [Giemsa staining]..." support The genetic map of the north Atlantic O. dioica is reported to contain three chromosomes (two autosomes, X and Y sex chromosomes; Denoeud et al., 2010); however, prior studies based on histochemical techniques reported three(Körner, 1952)and eight chromosomes (