Centromere-specific antibody-mediated karyotyping of Okinawan <i>Oikopleura dioica</i>&nbsp;suggests the presence of three chromosomes

Andrew W. Liu; Yongkai Tan; Aki Masunaga; Charles Plessy; Nicholas M. Luscombe

doi:10.12688/f1000research.25019.1

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Brief Report

Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioica suggests the presence of three chromosomes

[version 1; peer review: 2 approved with reservations]

Andrew W. Liu ¹^*, Yongkai Tan ¹^*, Aki Masunaga¹, Charles Plessy¹, Nicholas M. Luscombe^1-3

Andrew W. Liu ¹^*, Yongkai Tan ¹^*, [...] Aki Masunaga¹, Charles Plessy¹, Nicholas M. Luscombe^1-3

^* Equal contributors

PUBLISHED 28 Jul 2020

Author details Author details

¹ Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, Okinawa, 904-0324, Japan
² Francis Crick Institute, London, NW1 1AT, UK
³ Department of Genetics, Evolution and Environment, University College London, London, WC1E 6BT, UK

Andrew W. Liu
Roles: Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation

Yongkai Tan
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Visualization

Aki Masunaga
Roles: Visualization, Writing – Review & Editing

Charles Plessy
Roles: Conceptualization, Project Administration, Writing – Review & Editing

Nicholas M. Luscombe
Roles: Conceptualization, Funding Acquisition, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals’ chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies’ staining properties for use in epifluorescent and confocal based fluorescent microscopy.

Keywords

karyotype, chromosome, centromere, histone H3, Oikopleura, oocyte, embryo, H3S28P

Corresponding authors: Andrew W. Liu, Yongkai Tan

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2020 Liu AW et al. This is an open access article 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.

How to cite: Liu AW, Tan Y, Masunaga A et al. Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioica suggests the presence of three chromosomes [version 1; peer review: 2 approved with reservations]. F1000Research 2020, 9:780 (https://doi.org/10.12688/f1000research.25019.1) First published: 28 Jul 2020, 9:780 (https://doi.org/10.12688/f1000research.25019.1) Latest published: 01 Mar 2021, 9:780 (https://doi.org/10.12688/f1000research.25019.2)

Introduction

Karyotyping is a long-established histochemical method to visualize chromosomes of eukaryotes (Hsu & Benirschke, 1967; Tjio & Levan, 1950). A multi-dye reagent developed at the turn of the 20^th century for the diagnosis of infections in human histological preparations (Giemsa, 1902; Giemsa, 1904) was later used to stain chromosomes themselves in order to study their numbers, translocations, and other aberrations. 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.

Although individual chromosomes have been resolved by histochemical techniques in O. dioica, the reported results differ in numbers from n=3 (Körner, 1952) to n=8 (Colombera & Fernaux, 1973). More recently, 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) while providing greater detail and resolution. One such marker is histone H3 phosphorylated at Ser-28 (Kawajiri et al., 2003); although it is typically used to identify centromeres during metaphase (Kurihara et al., 2006), we observed in data presented in previous studies that signals were not confined to centromeres. More importantly, the localization of the H3S28P signal depends on the phase of the cell cycle: spatially punctate signals were found evenly spread within the nuclear envelope during prophase, while condensed chromatin gave an outlined staining of the sister chromatids during metaphase in a manner consistent with alignment along the metaphase plate (Table 1; Campsteijn et al., 2012; Feng & Thompson, 2018; Feng et al., 2019; Olsen et al., 2018). Moreover, a structure in which genetic material is sequestered in a ∏-shaped conformation has been observed during meiotic cell divisions between the final phases of oogenesis and mature oocytes (Ganot et al., 2008). However, these results were all obtained from the same laboratory strain originating from the Atlantic Ocean. Considering the discrepancy of past findings, and the fact that our laboratory strain originates from a geographically distinct ocean, we applied H3S28P staining on intact embryos and oocytes to confirm the chromosome count and validate our genome sequencing assemblies of Okinawan O. dioica marine populations among the Ryukyu Islands of southern Japan.

Table 1. Reference to images cited in this study.

Author	Date	Journal	H3S28P information	Figure information	Target sample
Spada et al	2005	Journal of Cellular Biochemistry	H3S28P, Thermo Fisher 07-145	Fig. 3 & Fig. 6	Day 3
Schulmeister et al	2007	Chromosome Research	H3S28P, Abcam, ab10543	Fig. 3 & Fig. 5	Male gonad/female coenocyte
Ganot et al	2008	Developmental Biology	H3S28P, Thermo Fisher 07-145	Fig. 4, Fig. 7 & Fig. 8	Maturing oocytes
Campsteijn et al	2012	Molecular Biology and Evolution	H3S28P, Abcam, ab10543	Fig. 1	Hatched larvae
Øvrebø et al	2015	Cell Cycle	H3S28P, Abcam, ab10543	Fig. 1, Fig. 4, Fig. 5, Fig. 7 & Fig. S2A	Maturing oocytes (P3, P4)
Feng et al	2018	Cell Cycle	H3S28P, Abcam, ab10543	Fig. 1, Fig. 2 & Fig. 7	P4 ovaries
Olsen et al	2018	BMC Developmental Biology	H3S28P, Abcam, ab10543	Fig. 5 & Addition file3	4, 8, 16, 32 cell
Feng et al	2019	Cell Cycle	H3S28P, Abcam ab10543	Fig. 1, Fig. 3, Fig. 4, Fig. 5 & Fig. 6	Hatched larvae

Methods

Oikopleura dioica culture, staging & preparation of biological material

Histochemical staining. Live specimens were collected from Ishikawa Harbor (26 °25'39.3 "N, 127 °49'56.6 "E) by a hand-held plankton net and cultured in the lab (Masunaga 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 for 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 filtered autoclaved seawater prior to fixation and staining. Unfertilized eggs were treated similarly.

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 slide was 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 ddH2O. 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).

Immunostaining. Washed eggs and embryos were immediately fixed in 4% w/v paraformaldehyde, 100 mM MOPS pH 7.5, 0.5 M NaCl, 0.1% triton-X100 at 23 °C ON (Campsteijn et al., 2012). The samples were then washed for 10 minutes once with PBSTE (PBS supplemented with 1 mM EDTA) and 3 times for 10 min with PBSTEG (PBS supplemented with 1 mM EDTA and 0.1 M glycine). The samples were blocked using PBSTE supplemented with 3% bovine serum albumin at 4 °C overnight. Rabbit polyclonal (Thermo Fisher Scientific Cat# 720099, RRID:AB_2532807) or rat monoclonal (Abcam Cat# ab10543, RRID:AB_2295065) primaries directed against H3S28P were diluted 1:100 in PBSTE 3% BSA and incubated at 4 °C for 3 days. The next morning, these were washed in PBSTE for 10 minutes 3 times and incubated with anti-rabbit (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217) or anti-rat (Molecular Probes Cat# A-11006, RRID:AB_141373) Alexa488 conjugated secondary antibodies diluted 1:500 with PBSTE 3% BSA at 4 °C ON. The following morning, samples were washed 3 times for 10 min with PBSTE. The samples were mounted on cleaned glass slides (Matsunami Glass, S2441) with fluorescence preserving mounting medium (ProLong. Fluoromount G Mounting Medium, RRID:SCR_015961) covered with No.1 35 x 50 mm glass coverslips (Matsunami Glass, C035551) and sealed with nail polish.

Image acquisition

Both a Nikon Ni-E epifluorescent and a Zeiss LSM 510 Meta confocal microscopes were used to acquire Z-stack images of eggs and embryos. Brightfield images were obtained using a 20x/0.75 CFI Plan Apo λ objective (Nikon, MRD00205) for histochemical staining. Epifluorescent immunofluorescent images were obtained with both 20x/0.75 and 40x/0.95 CFI Plan Apo λ air objectives (Nikon, MRD00405); each sample acquisition was Z-stacked with each plane set at an interval of 1 µm. Confocal images were acquired using a 40x/0.75 EC Plan-Neofluar M27 (Zeiss, 420360-9900-000) and 63x/1.4 Plan-Apochromat M27 oil immersion (Zeiss, 420782-9900-79) objectives; each sample acquisition was Z-stacked, line averaged twice with each plane set at an interval of 0.6 and 0.27 µm, respectively.

Image processing and analysis

Images acquired from a Nikon Ni-E epifluorescent were deconvoluted with Nikon Elements-AR v5.0 software. Images for both epifluorescent and confocal acquisitions were analyzed using Imaris software SPOT DETECTION tool (Imaris, RRID:SCR_007370) for embryos and unfertilized eggs, parameters set at 0.5 and 0.43 µm spot detection size, respectively, and software preset to QUALITY auto signal threshold for each individual cell within a sample. Alternatively, ImageJ v1.51 3D Objects Counter may be employed to count signals. Epifluorescent and confocal acquisitions of embryos and their subsequent analysis were performed independently by different researchers to exclude bias.

Statistical analysis

Confidence intervals were calculated with Prism 8 (GraphPad) and histograms plotted with R (v3.6.3).

Results

Initial attempts at visualizing individual chromosomes were done with developing embryos and Giemsa staining. The spreads from two time points, 32- and 64-cell developmental stages, gave results with counts ranging between 11–27 stains per cell (BioImage Archive, S-BIAD21, Experiment A). Although hypotonic-induced cell spreads were confined as a result of incomplete dechorionation and digestion with the enzymatic dissociation cocktail, groups of chromosomes were easily associated to a single cell. However, individual chromosomes were difficult to resolve due to the low resolution of images. In order to eliminate possible miscounts and other Giemsa staining artifacts, immunostaining was used to count individual chromosomes using a centromere-specific primary antibody directed against H3S28P 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 acquired images from epifluorescent and laser confocal microscopes (BioImage Archive, S-BIAD21, Experiment B & D). The data was analyzed using the Imaris SPOT DETECTION tool (Oxford Instruments). Two types of nuclei were apparent within each embryo: nuclei containing evenly distributed, clearly separated spots that were interpreted as being in prophase (Figure 1A and 1B, blue circles) and nuclei with intense clusters of signals in the center, considered to be in metaphase (Figure 1A and 1B, red squares). Counts from these two classes of nuclei fall into separate distributions (Figure 1C and 1D). Both epifluorescent and confocal acquisitions were in near agreement, epifluorescence n = 20, mean 6.2 , 95% CI 5.6 – 6.8; confocal n = 13, mean 6.4, 95% CI 5.7 – 7.1 and epifluorescence n = 20, mean 12, 95% CI 11.0 – 13.0; confocal, n = 14, mean 14.1, 95% CI 12.9 – 15.3. We interpret the results as a count of 12 distinct centromeres in prophase cells and a count of 6 larger spots identifying pairs of centromeres in metaphase (Figure 1B).

Figure 1. Centromere counts from embryos.

Anti-H3S28P rabbit-derived polyclonal stained 64-cell whole-embryo chromosomal imaging data analyzed by Imaris software SPOT DETECTION tool using different microscopy techniques. A Maximum projection of confocal image of an embryo demonstrating the differences in signal localization appearance and signal count, which was inferred to represent distinct cell cycle phases. Red box, metaphase; blue circle, prophase. B Schematic interpretation of signals with respect to chromatin structure during prophase and metaphase cell cycle states. As a simplification, all chromosomes have been drawn at an equal length although they actually vary in O. dioica. C Distribution of signal counts within individual cells using epifluorescent (n = 40) and D confocal (n = 27) microscopes. Two distinct populations were observed in a bimodal distribution, which corresponded with cell cycle stage. Red, metaphase; blue, prophase.

To confirm our observations on germ cells and therefore rule out polyploidy, which is frequent in O. dioica’s somatic cells (Ganot & Thompson, 2002), we also analyzed oocytes in prometaphase I before fertilization (Schulmeister et al., 2007). We identified confined groupings of signals in unfertilized eggs (Figure 2A; BioImage Archive, S-BIAD21, Experiment E). Images were analyzed using the Imaris SPOT DETECTION tool to determine chromosome counts and their distributions (Figure 2B). Counts from the compact rosette-shaped genetic material averaged near 6 (n = 23, mean 5.70, 95% CI 5.2 – 6.2). Visual inspection of individual Z-sections (Figure 2C) confirm agreement with the Imaris count analysis and annotation (Figure 2D). We interpret these results as each spot corresponding to a pair of centromeres from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).

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. B Distribution of signal counts from centromere-stained oocyte genetic material, analyzed by Imaris software SPOT DETECTION tool (n = 23). C Individual Z-sections from same image acquisition showing the 3D structure of the genetic material, 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 pair of closely associated centromeres from a pair of sister chromatids. As a simplification, all chromosomes have been drawn at an equal length although they actually vary in O. dioica.

Discussion

Despite the variation in signal counts across different image acquisitions settings, a haploid chromosomal count of three provides the most parsimonious explanation of the collected data and agrees with previously published assemblies (Denoeud et al., 2010).

Oocyte staining with rat anti-H3S28P and a conjugated secondary fluorophore gave rise to a compact area in which signals appear to stack on top of one another (Figure 2A). Previously, DNA stains at this stage have been interpreted as a structure resembling the Greek character ∏ (Ganot et al., 2007), representing condensed chromosomes seen in mature oocytes arrested in meiosis I. Our data does not include DNA stains and therefore our illustration (Figure 2E) should not be interpreted as precluding the previously reported ∏-structure.

Currently, the sequence of the centromeres is not known, 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 and the previous literature (Table 1) show that non-centromeric signal present outside metaphase stages may introduce noise. Thus, alternative targets such as other centromeric histone 3 variants (Moosmann et al., 2011) might be preferable. Availability of centromeric sequences would open the possibility of confirming our results with fluorescence in situ hybridization.

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).

Data availability

Underlying data

Image acquisitions: Image data are available in the BioImage Archive

Centromere specific antibody mediated karyotyping of Okinawan Oikopleura dioica. Accession number S-BIAD21. https://www.ebi.ac.uk/biostudies/preview/studies/S-BIAD21

Acknowledgements

We thank Drs. Daniel Chourrout, Hiroki Nishida & Eiichi Shoguchi for discussions and suggestions regarding the subject matter. Additionally, great appreciation is given the staff (Drs. Toshiaki Mochizuki, Shinya Komoro & Paolo Barzaghi) in the Imaging Section of the Research Support Division at OIST for providing technical assistance. Finally, we are grateful for Dr. Michael Mansfield and Charlotte West comments on the manuscript’s draft and appearance.

Faculty Opinions recommended

References

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Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 28 Jul 2020

Author details Author details

Andrew W. Liu
Roles: Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation

Yongkai Tan
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Visualization

Aki Masunaga
Roles: Visualization, Writing – Review & Editing

Charles Plessy
Roles: Conceptualization, Project Administration, Writing – Review & Editing

Nicholas M. Luscombe
Roles: Conceptualization, Funding Acquisition, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (2)

version 2

Revised

Published: 01 Mar 2021, 9:780

https://doi.org/10.12688/f1000research.25019.2

version 1

Published: 28 Jul 2020, 9:780

https://doi.org/10.12688/f1000research.25019.1

© 2020 Liu AW et al. This is an open access article 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.

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Liu AW, Tan Y, Masunaga A et al. Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioica suggests the presence of three chromosomes [version 1; peer review: 2 approved with reservations]. F1000Research 2020, 9:780 (https://doi.org/10.12688/f1000research.25019.1)

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Open Peer Review

Version 1

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PUBLISHED 28 Jul 2020

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Reviewer Report 12 Aug 2020

Shigeki Fujiwara, Department of Chemistry and Biotechnology, Faculty of Science and Technology, Kochi University, Kochi, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.27598.r68262

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.

Major concerns:

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. The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres). Shoguchi et 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. 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. 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, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”.
I guess that “ddH2O” (page 4 line 17) is double-distilled H₂O. 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.
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?

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.

Reviewer Expertise: Tunicate embryogenesis, asexual reproduction, and evolutionary developmental biology. Transcriptional regulation.

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.

CITE

Report a concern

Author Response 01 Mar 2021

Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan

01 Mar 2021

Author Response

We thank Dr Fujiwara’s helpful feedback and critique on our manuscript. We have done our best to address all the concerns and minor points he has brought to our attention, ... Continue reading We thank Dr Fujiwara’s helpful feedback and critique on our manuscript. We have done our best to address all the concerns and minor points he has brought to our attention, which are listed below.

Reviewer 2 synopsis

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 Abstract and Introduction (please see authors response to Reviewer Comment 1.1).
_____________________
Major concerns

Reviewer 2 comment 1.1 – Clarification of study aim

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.

Manuscript changes
1. 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 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. 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). “… 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.”

3. Paragraph 2 (Introduction). “However, we were unable to resolve individual O. dioica chromosomes by this method [Giemsa staining]...”

4. Paragraph 3 (Introduction). “As an alternative approach, we decided to immunostain the centromere as a means of quantifying the numbers of chromosomes… 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 2 comment 1.2 – Variability of data

Reviewer comment
The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres).

Particularly, discussion is required for the observation of seven or eight signals within a single nucleus.

Author response
We thank the referee for this comment.

1. Despite the apparent certainty in chromosome numbers, variability in signal counts does not appear to be unusual. For instance, Fenaux and Colombera noted (1973) reported “In another five anaphase plates, presumably because of chromosome losses during the squashing, a lower number was found.” However, most karyological papers generally present a very small number of representative images; therefore, we cannot comment on whether the variation we observe is unusually large compared with other studies. It is worth noting that in our hands, Giemsa-staining yielded even larger variability than immunostaining in our hands.

2. It is because of this variability that we decided to use a statistical approach: calculating confidence intervals allows us to quantify the uncertainty in the conclusions that we draw from each set of experiments, fully accounting for the variability.

3. We have added a discussion of the possible sources of variation in the number of H3S28P signals. Specifically, we believe that nuclei containing 7-8 counts, arise from non-uniform spots being split into multiple counts; for individual nuclei, this could be resolved by adjusting the signal threshold, but this is not possible if a uniform threshold is applied across all nuclei.

4. From a statistical perspective, the intervals are all narrow and centre on a mean of 6 across three different experimental set ups (Figure 1: mean 6.2, 95% CI 5.6 – 6, mean 6.4, 95% CI 5.7 – 7.1; Figure 2: mean 5.70, 95% CI 5.2 – 6.2). From a biological perspective, the observation is consistent with our genome sequence assembly. Together, these give us reasonable confidence that we have reached the correct conclusion that there are three chromosomes.

Manuscript changes
Paragraph 15 (Discussion). “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 them. (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 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.”
_____________________
Reviewer 2 comment 1.3 – Use of immunostaining over histochemical methods

Reviewer comment
Shoguchi et 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. 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.

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 Giema-staining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results.”

2. Paragraph 17 (Discussion). “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.”
_____________________
Reviewer 2 comment 2.1 – Rationale of study

Reviewer comment
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.

Author response
We thank the referee for this comment. Please also see authors response to Reviewer 1 Comment 1.1.

Briefly, we observe large genome sequence variations between north Atlantic, mainland Japanese and Okinawan O. dioica samples, both at nucleotide level and kilo-megabase scale. This is why we decided to check the number of chromosomes in the Okinawan O. dioica. We feel it’s too early to conclude whether they represent distinct species.

Regarding the earlier literature, since Colombera and Fenaux (1973) reported 8 chromosomes, it is possible that they examined a different species of Oikopleura.

Manuscript changes
Please see authors response to Reviewer 1 Comment 1.1
_____________________
Minor points

Reviewer 2 minor point 1

Reviewer comment
In Table 1, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”.

Author response
We have made the changes to the citations in the manuscript as suggested.

Manuscript changes
1. Feng et al. (2018) was changed to “Feng & Thompson”.

2. Ganot et al. (2008) was left unchanged as it refers to Ganot P, Schulmeister A, Thompson EM, (2008).
___________________
Reviewer 2 minor point 2

Reviewer comment
I guess that “ddH2O” (page 4 line 17) is double-distilled H₂O. 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.

Author response
We have replaced jargon and abbreviations with full terminology in the methods sections.

Manuscript changes
Changes made in paragraph 5 and 6.
_____________________
Reviewer 2 minor point 3

Reviewer comment
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?

Author response
We updated the methods section to indicate the developmental stage of the stained embryos (“32 and 64-cell embryos”) and underlined that the same stage was used for Giemsa and antibody staining by adding the words “similarly staged embryos” in the first paragraph of the results section. It is our understanding that the polyploid cells outlined in Ganot & Thompson, 2002 which are responsible for the extrusion of the mucosal house are present in the later stages of development.

Manuscript changes
1. Paragraph 6. “Washed eggs, 32 and 64 cell embryos (described above) were immediately fixed…”

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”
We thank Dr Fujiwara’s helpful feedback and critique on our manuscript. We have done our best to address all the concerns and minor points he has brought to our attention, which are listed below.

Reviewer 2 synopsis

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 Abstract and Introduction (please see authors response to Reviewer Comment 1.1).
_____________________
Major concerns

Reviewer 2 comment 1.1 – Clarification of study aim

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.

Manuscript changes
1. 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 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. 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). “… 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.”

3. Paragraph 2 (Introduction). “However, we were unable to resolve individual O. dioica chromosomes by this method [Giemsa staining]...”

4. Paragraph 3 (Introduction). “As an alternative approach, we decided to immunostain the centromere as a means of quantifying the numbers of chromosomes… 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 2 comment 1.2 – Variability of data

Reviewer comment
The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres).

Particularly, discussion is required for the observation of seven or eight signals within a single nucleus.

Author response
We thank the referee for this comment.

1. Despite the apparent certainty in chromosome numbers, variability in signal counts does not appear to be unusual. For instance, Fenaux and Colombera noted (1973) reported “In another five anaphase plates, presumably because of chromosome losses during the squashing, a lower number was found.” However, most karyological papers generally present a very small number of representative images; therefore, we cannot comment on whether the variation we observe is unusually large compared with other studies. It is worth noting that in our hands, Giemsa-staining yielded even larger variability than immunostaining in our hands.

2. It is because of this variability that we decided to use a statistical approach: calculating confidence intervals allows us to quantify the uncertainty in the conclusions that we draw from each set of experiments, fully accounting for the variability.

3. We have added a discussion of the possible sources of variation in the number of H3S28P signals. Specifically, we believe that nuclei containing 7-8 counts, arise from non-uniform spots being split into multiple counts; for individual nuclei, this could be resolved by adjusting the signal threshold, but this is not possible if a uniform threshold is applied across all nuclei.

4. From a statistical perspective, the intervals are all narrow and centre on a mean of 6 across three different experimental set ups (Figure 1: mean 6.2, 95% CI 5.6 – 6, mean 6.4, 95% CI 5.7 – 7.1; Figure 2: mean 5.70, 95% CI 5.2 – 6.2). From a biological perspective, the observation is consistent with our genome sequence assembly. Together, these give us reasonable confidence that we have reached the correct conclusion that there are three chromosomes.

Manuscript changes
Paragraph 15 (Discussion). “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 them. (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 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.”
_____________________
Reviewer 2 comment 1.3 – Use of immunostaining over histochemical methods

Reviewer comment
Shoguchi et 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. 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.

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 Giema-staining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results.”

2. Paragraph 17 (Discussion). “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.”
_____________________
Reviewer 2 comment 2.1 – Rationale of study

Reviewer comment
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.

Author response
We thank the referee for this comment. Please also see authors response to Reviewer 1 Comment 1.1.

Briefly, we observe large genome sequence variations between north Atlantic, mainland Japanese and Okinawan O. dioica samples, both at nucleotide level and kilo-megabase scale. This is why we decided to check the number of chromosomes in the Okinawan O. dioica. We feel it’s too early to conclude whether they represent distinct species.

Regarding the earlier literature, since Colombera and Fenaux (1973) reported 8 chromosomes, it is possible that they examined a different species of Oikopleura.

Manuscript changes
Please see authors response to Reviewer 1 Comment 1.1
_____________________
Minor points

Reviewer 2 minor point 1

Reviewer comment
In Table 1, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”.

Author response
We have made the changes to the citations in the manuscript as suggested.

Manuscript changes
1. Feng et al. (2018) was changed to “Feng & Thompson”.

2. Ganot et al. (2008) was left unchanged as it refers to Ganot P, Schulmeister A, Thompson EM, (2008).
___________________
Reviewer 2 minor point 2

Reviewer comment
I guess that “ddH2O” (page 4 line 17) is double-distilled H₂O. 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.

Author response
We have replaced jargon and abbreviations with full terminology in the methods sections.

Manuscript changes
Changes made in paragraph 5 and 6.
_____________________
Reviewer 2 minor point 3

Reviewer comment
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?

Author response
We updated the methods section to indicate the developmental stage of the stained embryos (“32 and 64-cell embryos”) and underlined that the same stage was used for Giemsa and antibody staining by adding the words “similarly staged embryos” in the first paragraph of the results section. It is our understanding that the polyploid cells outlined in Ganot & Thompson, 2002 which are responsible for the extrusion of the mucosal house are present in the later stages of development.

Manuscript changes
1. Paragraph 6. “Washed eggs, 32 and 64 cell embryos (described above) were immediately fixed…”

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. Close
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COMMENTS ON THIS REPORT

Author Response 01 Mar 2021

Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan

01 Mar 2021

Author Response

We thank Dr Fujiwara’s helpful feedback and critique on our manuscript. We have done our best to address all the concerns and minor points he has brought to our attention, ... Continue reading We thank Dr Fujiwara’s helpful feedback and critique on our manuscript. We have done our best to address all the concerns and minor points he has brought to our attention, which are listed below.

Reviewer 2 synopsis

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 Abstract and Introduction (please see authors response to Reviewer Comment 1.1).
_____________________
Major concerns

Reviewer 2 comment 1.1 – Clarification of study aim

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.

Manuscript changes
1. 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 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. 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). “… 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.”

3. Paragraph 2 (Introduction). “However, we were unable to resolve individual O. dioica chromosomes by this method [Giemsa staining]...”

4. Paragraph 3 (Introduction). “As an alternative approach, we decided to immunostain the centromere as a means of quantifying the numbers of chromosomes… 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 2 comment 1.2 – Variability of data

Reviewer comment
The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres).

Particularly, discussion is required for the observation of seven or eight signals within a single nucleus.

Author response
We thank the referee for this comment.

1. Despite the apparent certainty in chromosome numbers, variability in signal counts does not appear to be unusual. For instance, Fenaux and Colombera noted (1973) reported “In another five anaphase plates, presumably because of chromosome losses during the squashing, a lower number was found.” However, most karyological papers generally present a very small number of representative images; therefore, we cannot comment on whether the variation we observe is unusually large compared with other studies. It is worth noting that in our hands, Giemsa-staining yielded even larger variability than immunostaining in our hands.

2. It is because of this variability that we decided to use a statistical approach: calculating confidence intervals allows us to quantify the uncertainty in the conclusions that we draw from each set of experiments, fully accounting for the variability.

3. We have added a discussion of the possible sources of variation in the number of H3S28P signals. Specifically, we believe that nuclei containing 7-8 counts, arise from non-uniform spots being split into multiple counts; for individual nuclei, this could be resolved by adjusting the signal threshold, but this is not possible if a uniform threshold is applied across all nuclei.

4. From a statistical perspective, the intervals are all narrow and centre on a mean of 6 across three different experimental set ups (Figure 1: mean 6.2, 95% CI 5.6 – 6, mean 6.4, 95% CI 5.7 – 7.1; Figure 2: mean 5.70, 95% CI 5.2 – 6.2). From a biological perspective, the observation is consistent with our genome sequence assembly. Together, these give us reasonable confidence that we have reached the correct conclusion that there are three chromosomes.

Manuscript changes
Paragraph 15 (Discussion). “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 them. (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 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.”
_____________________
Reviewer 2 comment 1.3 – Use of immunostaining over histochemical methods

Reviewer comment
Shoguchi et 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. 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.

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 Giema-staining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results.”

2. Paragraph 17 (Discussion). “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.”
_____________________
Reviewer 2 comment 2.1 – Rationale of study

Reviewer comment
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.

Author response
We thank the referee for this comment. Please also see authors response to Reviewer 1 Comment 1.1.

Briefly, we observe large genome sequence variations between north Atlantic, mainland Japanese and Okinawan O. dioica samples, both at nucleotide level and kilo-megabase scale. This is why we decided to check the number of chromosomes in the Okinawan O. dioica. We feel it’s too early to conclude whether they represent distinct species.

Regarding the earlier literature, since Colombera and Fenaux (1973) reported 8 chromosomes, it is possible that they examined a different species of Oikopleura.

Manuscript changes
Please see authors response to Reviewer 1 Comment 1.1
_____________________
Minor points

Reviewer 2 minor point 1

Reviewer comment
In Table 1, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”.

Author response
We have made the changes to the citations in the manuscript as suggested.

Manuscript changes
1. Feng et al. (2018) was changed to “Feng & Thompson”.

2. Ganot et al. (2008) was left unchanged as it refers to Ganot P, Schulmeister A, Thompson EM, (2008).
___________________
Reviewer 2 minor point 2

Reviewer comment
I guess that “ddH2O” (page 4 line 17) is double-distilled H₂O. 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.

Author response
We have replaced jargon and abbreviations with full terminology in the methods sections.

Manuscript changes
Changes made in paragraph 5 and 6.
_____________________
Reviewer 2 minor point 3

Reviewer comment
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?

Author response
We updated the methods section to indicate the developmental stage of the stained embryos (“32 and 64-cell embryos”) and underlined that the same stage was used for Giemsa and antibody staining by adding the words “similarly staged embryos” in the first paragraph of the results section. It is our understanding that the polyploid cells outlined in Ganot & Thompson, 2002 which are responsible for the extrusion of the mucosal house are present in the later stages of development.

Manuscript changes
1. Paragraph 6. “Washed eggs, 32 and 64 cell embryos (described above) were immediately fixed…”

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”
We thank Dr Fujiwara’s helpful feedback and critique on our manuscript. We have done our best to address all the concerns and minor points he has brought to our attention, which are listed below.

Reviewer 2 synopsis

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 Abstract and Introduction (please see authors response to Reviewer Comment 1.1).
_____________________
Major concerns

Reviewer 2 comment 1.1 – Clarification of study aim

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.

Manuscript changes
1. 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 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. 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). “… 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.”

3. Paragraph 2 (Introduction). “However, we were unable to resolve individual O. dioica chromosomes by this method [Giemsa staining]...”

4. Paragraph 3 (Introduction). “As an alternative approach, we decided to immunostain the centromere as a means of quantifying the numbers of chromosomes… 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 2 comment 1.2 – Variability of data

Reviewer comment
The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres).

Particularly, discussion is required for the observation of seven or eight signals within a single nucleus.

Author response
We thank the referee for this comment.

1. Despite the apparent certainty in chromosome numbers, variability in signal counts does not appear to be unusual. For instance, Fenaux and Colombera noted (1973) reported “In another five anaphase plates, presumably because of chromosome losses during the squashing, a lower number was found.” However, most karyological papers generally present a very small number of representative images; therefore, we cannot comment on whether the variation we observe is unusually large compared with other studies. It is worth noting that in our hands, Giemsa-staining yielded even larger variability than immunostaining in our hands.

2. It is because of this variability that we decided to use a statistical approach: calculating confidence intervals allows us to quantify the uncertainty in the conclusions that we draw from each set of experiments, fully accounting for the variability.

3. We have added a discussion of the possible sources of variation in the number of H3S28P signals. Specifically, we believe that nuclei containing 7-8 counts, arise from non-uniform spots being split into multiple counts; for individual nuclei, this could be resolved by adjusting the signal threshold, but this is not possible if a uniform threshold is applied across all nuclei.

4. From a statistical perspective, the intervals are all narrow and centre on a mean of 6 across three different experimental set ups (Figure 1: mean 6.2, 95% CI 5.6 – 6, mean 6.4, 95% CI 5.7 – 7.1; Figure 2: mean 5.70, 95% CI 5.2 – 6.2). From a biological perspective, the observation is consistent with our genome sequence assembly. Together, these give us reasonable confidence that we have reached the correct conclusion that there are three chromosomes.

Manuscript changes
Paragraph 15 (Discussion). “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 them. (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 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.”
_____________________
Reviewer 2 comment 1.3 – Use of immunostaining over histochemical methods

Reviewer comment
Shoguchi et 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. 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.

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 Giema-staining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results.”

2. Paragraph 17 (Discussion). “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.”
_____________________
Reviewer 2 comment 2.1 – Rationale of study

Reviewer comment
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.

Author response
We thank the referee for this comment. Please also see authors response to Reviewer 1 Comment 1.1.

Briefly, we observe large genome sequence variations between north Atlantic, mainland Japanese and Okinawan O. dioica samples, both at nucleotide level and kilo-megabase scale. This is why we decided to check the number of chromosomes in the Okinawan O. dioica. We feel it’s too early to conclude whether they represent distinct species.

Regarding the earlier literature, since Colombera and Fenaux (1973) reported 8 chromosomes, it is possible that they examined a different species of Oikopleura.

Manuscript changes
Please see authors response to Reviewer 1 Comment 1.1
_____________________
Minor points

Reviewer 2 minor point 1

Reviewer comment
In Table 1, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”.

Author response
We have made the changes to the citations in the manuscript as suggested.

Manuscript changes
1. Feng et al. (2018) was changed to “Feng & Thompson”.

2. Ganot et al. (2008) was left unchanged as it refers to Ganot P, Schulmeister A, Thompson EM, (2008).
___________________
Reviewer 2 minor point 2

Reviewer comment
I guess that “ddH2O” (page 4 line 17) is double-distilled H₂O. 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.

Author response
We have replaced jargon and abbreviations with full terminology in the methods sections.

Manuscript changes
Changes made in paragraph 5 and 6.
_____________________
Reviewer 2 minor point 3

Reviewer comment
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?

Author response
We updated the methods section to indicate the developmental stage of the stained embryos (“32 and 64-cell embryos”) and underlined that the same stage was used for Giemsa and antibody staining by adding the words “similarly staged embryos” in the first paragraph of the results section. It is our understanding that the polyploid cells outlined in Ganot & Thompson, 2002 which are responsible for the extrusion of the mucosal house are present in the later stages of development.

Manuscript changes
1. Paragraph 6. “Washed eggs, 32 and 64 cell embryos (described above) were immediately fixed…”

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. Close
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Reviewer Report 05 Aug 2020

Haiyang Feng, Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway

Approved with Reservations

https://doi.org/10.5256/f1000research.27598.r68260

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.

Reviewer Expertise: cell cycle, oogenesis

CITE

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Author Response 01 Mar 2021

Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan

01 Mar 2021

Author Response

We thank Dr Feng’s helpful feedback on our manuscript. We have done our best to address all the comments, which are listed below.

An aspect of the data that ... Continue reading We thank Dr Feng’s helpful feedback on our manuscript. We have done our best to address all the comments, which are listed below.

An aspect of the data that puzzles us is the evenly distributed, clearly separated H3S28P signal observed in non-mitotic cells, which we incorrectly referred to as prophase in the previous version of the manuscript (please see responses 2.1 and 3.1). We have consulted cell cycle experts who were unable to explain the results. Given Dr Feng’s extensive experience with H3S28P staining, we would like to ask if these patterns have been observed in his laboratory? We’d be grateful to organize a videoconference with Dr Feng to discuss the data archived at the EBI BioArchive to share our observations in detail.
_____________________
Reviewer 1 comment 1.1 – Comparison of chromosome numbers between Japanese and Norwegian O. dioica

Reviewer comment
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. dioicaindividuals 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. 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.

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. 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. 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 1 comment 1.2 – Misinterpretation of data

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.

Author response
We thank the referee for the detailed comments below. We agree that we misinterpreted some of the results and we have now revised the manuscript to correct this.

Manuscript changes
Specific instances of misinterpretations (response to comments 2.1 & 3.1) and changes in schematics have been addressed below (response to comments 2.2 & 3.3). Clarification of timing of oocyte collection has explained in more detail (response to comment 3.2).
_____________________
Reviewer 1 comment 2.1 – Cell cycle state of cells containing 12 spots

Reviewer comment
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.

Author response
We thank the reviewer for this comment. We agree that the cells containing ~12 spots cannot be in prophase. In fact, we cannot explain the cell cycle state of these cells, so we now refer to them as “non-mitotic”.

Manuscript changes
Paragraph 12. “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.”
_____________________
Reviewer 1 comment 2.2 – Schematic representation of chromosomes in embryos in Figure 1B

Reviewer comment
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.

Author response
We thank the referee for this comment. We have corrected our use of “centromere” and redrawn Figure 1B.

Manuscript changes
1. We have changed all instances of “a pair of centromeres” to “centromere”.

2. Figure 1B. We have corrected the schematic representation of metaphase and non-mitotic nuclei in Figure 1B.

3. We have updated our manuscript to replace “centromere” with “centromeric region” when referring to the DNA sequence regardless of the state of assembly of the centromere, and removed mentions of “a pair of” from the remaining occurences of “centromere”.
_____________________
Reviewer 1 comment 3.1 – Interpretation of H3S28P signal locations

Reviewer comment
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.

Author response
We thank the referee for this comment. To make a clearer distinction between observation and interpretation, we now refer to the spots in the imaging data as “H3S28P signal” and only equate them to the centromeric region in specific instances. We have also included caveats to the interpretation of the oocyte data in the discussion section.

Manuscript changes
1. We now refer to the image spots as “H3S28P” signal, and only equate them to centromere in specific, appropriate contexts.

2. Paragraph 12. “.... 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. ”

3. Paragraph 13. “.... We interpreted each spot as representing a centromere from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).”

4. Updated Paragraph 16-17.
Paragraph 16. “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 and 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). 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. 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.”

5. Figure 2 legend, last sentence. “… 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 1 comment 3.2 – Timing of oocyte collection

Reviewer comment
Actually, H3S28p shows several spots (more than 6) during prometaphase I, as can be seen Fig S4 in Feng and Thompson, 2018 Cell Cycle publication. 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.

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
We thank the referee for this comment and we have corrected Figure 2E.

Manuscript changes
1. Figure 2E. Schematic corrected so there is one red spot between each pair of sister chromatid.
We thank Dr Feng’s helpful feedback on our manuscript. We have done our best to address all the comments, which are listed below.

An aspect of the data that puzzles us is the evenly distributed, clearly separated H3S28P signal observed in non-mitotic cells, which we incorrectly referred to as prophase in the previous version of the manuscript (please see responses 2.1 and 3.1). We have consulted cell cycle experts who were unable to explain the results. Given Dr Feng’s extensive experience with H3S28P staining, we would like to ask if these patterns have been observed in his laboratory? We’d be grateful to organize a videoconference with Dr Feng to discuss the data archived at the EBI BioArchive to share our observations in detail.
_____________________
Reviewer 1 comment 1.1 – Comparison of chromosome numbers between Japanese and Norwegian O. dioica

Reviewer comment
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. dioicaindividuals 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. 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.

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. 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. 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 1 comment 1.2 – Misinterpretation of data

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.

Author response
We thank the referee for the detailed comments below. We agree that we misinterpreted some of the results and we have now revised the manuscript to correct this.

Manuscript changes
Specific instances of misinterpretations (response to comments 2.1 & 3.1) and changes in schematics have been addressed below (response to comments 2.2 & 3.3). Clarification of timing of oocyte collection has explained in more detail (response to comment 3.2).
_____________________
Reviewer 1 comment 2.1 – Cell cycle state of cells containing 12 spots

Reviewer comment
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.

Author response
We thank the reviewer for this comment. We agree that the cells containing ~12 spots cannot be in prophase. In fact, we cannot explain the cell cycle state of these cells, so we now refer to them as “non-mitotic”.

Manuscript changes
Paragraph 12. “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.”
_____________________
Reviewer 1 comment 2.2 – Schematic representation of chromosomes in embryos in Figure 1B

Reviewer comment
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.

Author response
We thank the referee for this comment. We have corrected our use of “centromere” and redrawn Figure 1B.

Manuscript changes
1. We have changed all instances of “a pair of centromeres” to “centromere”.

2. Figure 1B. We have corrected the schematic representation of metaphase and non-mitotic nuclei in Figure 1B.

3. We have updated our manuscript to replace “centromere” with “centromeric region” when referring to the DNA sequence regardless of the state of assembly of the centromere, and removed mentions of “a pair of” from the remaining occurences of “centromere”.
_____________________
Reviewer 1 comment 3.1 – Interpretation of H3S28P signal locations

Reviewer comment
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.

Author response
We thank the referee for this comment. To make a clearer distinction between observation and interpretation, we now refer to the spots in the imaging data as “H3S28P signal” and only equate them to the centromeric region in specific instances. We have also included caveats to the interpretation of the oocyte data in the discussion section.

Manuscript changes
1. We now refer to the image spots as “H3S28P” signal, and only equate them to centromere in specific, appropriate contexts.

2. Paragraph 12. “.... 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. ”

3. Paragraph 13. “.... We interpreted each spot as representing a centromere from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).”

4. Updated Paragraph 16-17.
Paragraph 16. “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 and 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). 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. 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.”

5. Figure 2 legend, last sentence. “… 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 1 comment 3.2 – Timing of oocyte collection

Reviewer comment
Actually, H3S28p shows several spots (more than 6) during prometaphase I, as can be seen Fig S4 in Feng and Thompson, 2018 Cell Cycle publication. 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.

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
We thank the referee for this comment and we have corrected Figure 2E.

Manuscript changes
1. Figure 2E. Schematic corrected so there is one red spot between each pair of sister chromatid.
Competing Interests: We disclose no competing interests with regard to F1000's review process or this individual's peer review report. Close
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COMMENTS ON THIS REPORT

Author Response 01 Mar 2021

Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan

01 Mar 2021

Author Response

We thank Dr Feng’s helpful feedback on our manuscript. We have done our best to address all the comments, which are listed below.

An aspect of the data that ... Continue reading We thank Dr Feng’s helpful feedback on our manuscript. We have done our best to address all the comments, which are listed below.

An aspect of the data that puzzles us is the evenly distributed, clearly separated H3S28P signal observed in non-mitotic cells, which we incorrectly referred to as prophase in the previous version of the manuscript (please see responses 2.1 and 3.1). We have consulted cell cycle experts who were unable to explain the results. Given Dr Feng’s extensive experience with H3S28P staining, we would like to ask if these patterns have been observed in his laboratory? We’d be grateful to organize a videoconference with Dr Feng to discuss the data archived at the EBI BioArchive to share our observations in detail.
_____________________
Reviewer 1 comment 1.1 – Comparison of chromosome numbers between Japanese and Norwegian O. dioica

Reviewer comment
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. dioicaindividuals 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. 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.

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. 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. 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 1 comment 1.2 – Misinterpretation of data

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.

Author response
We thank the referee for the detailed comments below. We agree that we misinterpreted some of the results and we have now revised the manuscript to correct this.

Manuscript changes
Specific instances of misinterpretations (response to comments 2.1 & 3.1) and changes in schematics have been addressed below (response to comments 2.2 & 3.3). Clarification of timing of oocyte collection has explained in more detail (response to comment 3.2).
_____________________
Reviewer 1 comment 2.1 – Cell cycle state of cells containing 12 spots

Reviewer comment
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.

Author response
We thank the reviewer for this comment. We agree that the cells containing ~12 spots cannot be in prophase. In fact, we cannot explain the cell cycle state of these cells, so we now refer to them as “non-mitotic”.

Manuscript changes
Paragraph 12. “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.”
_____________________
Reviewer 1 comment 2.2 – Schematic representation of chromosomes in embryos in Figure 1B

Reviewer comment
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.

Author response
We thank the referee for this comment. We have corrected our use of “centromere” and redrawn Figure 1B.

Manuscript changes
1. We have changed all instances of “a pair of centromeres” to “centromere”.

2. Figure 1B. We have corrected the schematic representation of metaphase and non-mitotic nuclei in Figure 1B.

3. We have updated our manuscript to replace “centromere” with “centromeric region” when referring to the DNA sequence regardless of the state of assembly of the centromere, and removed mentions of “a pair of” from the remaining occurences of “centromere”.
_____________________
Reviewer 1 comment 3.1 – Interpretation of H3S28P signal locations

Reviewer comment
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.

Author response
We thank the referee for this comment. To make a clearer distinction between observation and interpretation, we now refer to the spots in the imaging data as “H3S28P signal” and only equate them to the centromeric region in specific instances. We have also included caveats to the interpretation of the oocyte data in the discussion section.

Manuscript changes
1. We now refer to the image spots as “H3S28P” signal, and only equate them to centromere in specific, appropriate contexts.

2. Paragraph 12. “.... 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. ”

3. Paragraph 13. “.... We interpreted each spot as representing a centromere from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).”

4. Updated Paragraph 16-17.
Paragraph 16. “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 and 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). 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. 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.”

5. Figure 2 legend, last sentence. “… 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 1 comment 3.2 – Timing of oocyte collection

Reviewer comment
Actually, H3S28p shows several spots (more than 6) during prometaphase I, as can be seen Fig S4 in Feng and Thompson, 2018 Cell Cycle publication. 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.

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…”
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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
We thank the referee for this comment and we have corrected Figure 2E.

Manuscript changes
1. Figure 2E. Schematic corrected so there is one red spot between each pair of sister chromatid.
We thank Dr Feng’s helpful feedback on our manuscript. We have done our best to address all the comments, which are listed below.

An aspect of the data that puzzles us is the evenly distributed, clearly separated H3S28P signal observed in non-mitotic cells, which we incorrectly referred to as prophase in the previous version of the manuscript (please see responses 2.1 and 3.1). We have consulted cell cycle experts who were unable to explain the results. Given Dr Feng’s extensive experience with H3S28P staining, we would like to ask if these patterns have been observed in his laboratory? We’d be grateful to organize a videoconference with Dr Feng to discuss the data archived at the EBI BioArchive to share our observations in detail.
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Reviewer 1 comment 1.1 – Comparison of chromosome numbers between Japanese and Norwegian O. dioica

Reviewer comment
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. dioicaindividuals 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. 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.

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. 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. 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 1 comment 1.2 – Misinterpretation of data

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.

Author response
We thank the referee for the detailed comments below. We agree that we misinterpreted some of the results and we have now revised the manuscript to correct this.

Manuscript changes
Specific instances of misinterpretations (response to comments 2.1 & 3.1) and changes in schematics have been addressed below (response to comments 2.2 & 3.3). Clarification of timing of oocyte collection has explained in more detail (response to comment 3.2).
_____________________
Reviewer 1 comment 2.1 – Cell cycle state of cells containing 12 spots

Reviewer comment
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.

Author response
We thank the reviewer for this comment. We agree that the cells containing ~12 spots cannot be in prophase. In fact, we cannot explain the cell cycle state of these cells, so we now refer to them as “non-mitotic”.

Manuscript changes
Paragraph 12. “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.”
_____________________
Reviewer 1 comment 2.2 – Schematic representation of chromosomes in embryos in Figure 1B

Reviewer comment
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.

Author response
We thank the referee for this comment. We have corrected our use of “centromere” and redrawn Figure 1B.

Manuscript changes
1. We have changed all instances of “a pair of centromeres” to “centromere”.

2. Figure 1B. We have corrected the schematic representation of metaphase and non-mitotic nuclei in Figure 1B.

3. We have updated our manuscript to replace “centromere” with “centromeric region” when referring to the DNA sequence regardless of the state of assembly of the centromere, and removed mentions of “a pair of” from the remaining occurences of “centromere”.
_____________________
Reviewer 1 comment 3.1 – Interpretation of H3S28P signal locations

Reviewer comment
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.

Author response
We thank the referee for this comment. To make a clearer distinction between observation and interpretation, we now refer to the spots in the imaging data as “H3S28P signal” and only equate them to the centromeric region in specific instances. We have also included caveats to the interpretation of the oocyte data in the discussion section.

Manuscript changes
1. We now refer to the image spots as “H3S28P” signal, and only equate them to centromere in specific, appropriate contexts.

2. Paragraph 12. “.... 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. ”

3. Paragraph 13. “.... We interpreted each spot as representing a centromere from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).”

4. Updated Paragraph 16-17.
Paragraph 16. “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 and 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). 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. 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.”

5. Figure 2 legend, last sentence. “… 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 1 comment 3.2 – Timing of oocyte collection

Reviewer comment
Actually, H3S28p shows several spots (more than 6) during prometaphase I, as can be seen Fig S4 in Feng and Thompson, 2018 Cell Cycle publication. 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.

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…”
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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
We thank the referee for this comment and we have corrected Figure 2E.

Manuscript changes
1. Figure 2E. Schematic corrected so there is one red spot between each pair of sister chromatid.
Competing Interests: We disclose no competing interests with regard to F1000's review process or this individual's peer review report. Close
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Haiyang Feng, University of Bergen, Bergen, Norway
Shigeki Fujiwara, Kochi University, Kochi, Japan

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08 Mar 2021 | for Version 2

Shigeki Fujiwara, Department of Chemistry and Biotechnology, Faculty of Science and Technology, Kochi University, Kochi, Japan

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Approved

All of the concerns I had raised were carefully addressed. I feel that the revised version of the manuscript became much clearer and convincing.

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No competing interests were disclosed.

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Tunicate embryogenesis, asexual reproduction, and evolutionary developmental biology. Transcriptional regulation.

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02 Mar 2021 | for Version 2

Haiyang Feng, Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway

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Overall, I'm satisfied with the revision. As to the H3S28p signals in embryonic cells not at metaphase, it seems that these cells are at early prophase, since interphase during 32C to 64C stages should be very short. I would interpret the H3S28p dots of 8 to 18 early on at prophase are on chromosomes, but not necessarily on centromeres.

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cell cycle, oogenesis

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12 Aug 2020 | for Version 1

Shigeki Fujiwara, Department of Chemistry and Biotechnology, Faculty of Science and Technology, Kochi University, Kochi, Japan

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Approved With Reservations

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. The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres). Shoguchi et 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. 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. 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, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”.
I guess that “ddH2O” (page 4 line 17) is double-distilled H₂O. 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.
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?

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

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No competing interests were disclosed.

Reviewer Expertise

Tunicate embryogenesis, asexual reproduction, and evolutionary developmental biology. Transcriptional regulation.

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Author Response

01 Mar 2021

Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan

We thank Dr Fujiwara’s helpful feedback and critique on our manuscript. We have done our best to address all the concerns and minor points he has brought to our attention, which are listed below.

Reviewer 2 synopsis

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 Abstract and Introduction (please see authors response to Reviewer Comment 1.1).
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Major concerns

Reviewer 2 comment 1.1 – Clarification of study aim

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.

Manuscript changes
1. 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 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. 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). “… 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.”

3. Paragraph 2 (Introduction). “However, we were unable to resolve individual O. dioica chromosomes by this method [Giemsa staining]...”

4. Paragraph 3 (Introduction). “As an alternative approach, we decided to immunostain the centromere as a means of quantifying the numbers of chromosomes… 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 2 comment 1.2 – Variability of data

Reviewer comment
The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres).

Particularly, discussion is required for the observation of seven or eight signals within a single nucleus.

Author response
We thank the referee for this comment.

1. Despite the apparent certainty in chromosome numbers, variability in signal counts does not appear to be unusual. For instance, Fenaux and Colombera noted (1973) reported “In another five anaphase plates, presumably because of chromosome losses during the squashing, a lower number was found.” However, most karyological papers generally present a very small number of representative images; therefore, we cannot comment on whether the variation we observe is unusually large compared with other studies. It is worth noting that in our hands, Giemsa-staining yielded even larger variability than immunostaining in our hands.

2. It is because of this variability that we decided to use a statistical approach: calculating confidence intervals allows us to quantify the uncertainty in the conclusions that we draw from each set of experiments, fully accounting for the variability.

3. We have added a discussion of the possible sources of variation in the number of H3S28P signals. Specifically, we believe that nuclei containing 7-8 counts, arise from non-uniform spots being split into multiple counts; for individual nuclei, this could be resolved by adjusting the signal threshold, but this is not possible if a uniform threshold is applied across all nuclei.

4. From a statistical perspective, the intervals are all narrow and centre on a mean of 6 across three different experimental set ups (Figure 1: mean 6.2, 95% CI 5.6 – 6, mean 6.4, 95% CI 5.7 – 7.1; Figure 2: mean 5.70, 95% CI 5.2 – 6.2). From a biological perspective, the observation is consistent with our genome sequence assembly. Together, these give us reasonable confidence that we have reached the correct conclusion that there are three chromosomes.

Manuscript changes
Paragraph 15 (Discussion). “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 them. (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 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.”
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Reviewer 2 comment 1.3 – Use of immunostaining over histochemical methods

Reviewer comment
Shoguchi et 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. 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.

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 Giema-staining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results.”

2. Paragraph 17 (Discussion). “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.”
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Reviewer 2 comment 2.1 – Rationale of study

Reviewer comment
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.

Author response
We thank the referee for this comment. Please also see authors response to Reviewer 1 Comment 1.1.

Briefly, we observe large genome sequence variations between north Atlantic, mainland Japanese and Okinawan O. dioica samples, both at nucleotide level and kilo-megabase scale. This is why we decided to check the number of chromosomes in the Okinawan O. dioica. We feel it’s too early to conclude whether they represent distinct species.

Regarding the earlier literature, since Colombera and Fenaux (1973) reported 8 chromosomes, it is possible that they examined a different species of Oikopleura.

Manuscript changes
Please see authors response to Reviewer 1 Comment 1.1
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Minor points

Reviewer 2 minor point 1

Reviewer comment
In Table 1, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”.

Author response
We have made the changes to the citations in the manuscript as suggested.

Manuscript changes
1. Feng et al. (2018) was changed to “Feng & Thompson”.

2. Ganot et al. (2008) was left unchanged as it refers to Ganot P, Schulmeister A, Thompson EM, (2008).
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Reviewer 2 minor point 2

Reviewer comment
I guess that “ddH2O” (page 4 line 17) is double-distilled H₂O. 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.

Author response
We have replaced jargon and abbreviations with full terminology in the methods sections.

Manuscript changes
Changes made in paragraph 5 and 6.
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Reviewer 2 minor point 3

Reviewer comment
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?

Author response
We updated the methods section to indicate the developmental stage of the stained embryos (“32 and 64-cell embryos”) and underlined that the same stage was used for Giemsa and antibody staining by adding the words “similarly staged embryos” in the first paragraph of the results section. It is our understanding that the polyploid cells outlined in Ganot & Thompson, 2002 which are responsible for the extrusion of the mucosal house are present in the later stages of development.

Manuscript changes
1. Paragraph 6. “Washed eggs, 32 and 64 cell embryos (described above) were immediately fixed…”

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”

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Reviewer Report

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05 Aug 2020 | for Version 1

Haiyang Feng, Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway

63 Views Cite this report Responses(1)

Approved With Reservations

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.

Reviewer Expertise

cell cycle, oogenesis

Respond to this report

Responses (1)

Author Response

01 Mar 2021

Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan

We thank Dr Feng’s helpful feedback on our manuscript. We have done our best to address all the comments, which are listed below.

An aspect of the data that puzzles us is the evenly distributed, clearly separated H3S28P signal observed in non-mitotic cells, which we incorrectly referred to as prophase in the previous version of the manuscript (please see responses 2.1 and 3.1). We have consulted cell cycle experts who were unable to explain the results. Given Dr Feng’s extensive experience with H3S28P staining, we would like to ask if these patterns have been observed in his laboratory? We’d be grateful to organize a videoconference with Dr Feng to discuss the data archived at the EBI BioArchive to share our observations in detail.
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Reviewer 1 comment 1.1 – Comparison of chromosome numbers between Japanese and Norwegian O. dioica

Reviewer comment
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. dioicaindividuals 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. 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.

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. 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. 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.
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Reviewer 1 comment 1.2 – Misinterpretation of data

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.

Author response
We thank the referee for the detailed comments below. We agree that we misinterpreted some of the results and we have now revised the manuscript to correct this.

Manuscript changes
Specific instances of misinterpretations (response to comments 2.1 & 3.1) and changes in schematics have been addressed below (response to comments 2.2 & 3.3). Clarification of timing of oocyte collection has explained in more detail (response to comment 3.2).
_____________________
Reviewer 1 comment 2.1 – Cell cycle state of cells containing 12 spots

Reviewer comment
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.

Author response
We thank the reviewer for this comment. We agree that the cells containing ~12 spots cannot be in prophase. In fact, we cannot explain the cell cycle state of these cells, so we now refer to them as “non-mitotic”.

Manuscript changes
Paragraph 12. “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.”
_____________________
Reviewer 1 comment 2.2 – Schematic representation of chromosomes in embryos in Figure 1B

Reviewer comment
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.

Author response
We thank the referee for this comment. We have corrected our use of “centromere” and redrawn Figure 1B.

Manuscript changes
1. We have changed all instances of “a pair of centromeres” to “centromere”.

2. Figure 1B. We have corrected the schematic representation of metaphase and non-mitotic nuclei in Figure 1B.

3. We have updated our manuscript to replace “centromere” with “centromeric region” when referring to the DNA sequence regardless of the state of assembly of the centromere, and removed mentions of “a pair of” from the remaining occurences of “centromere”.
_____________________
Reviewer 1 comment 3.1 – Interpretation of H3S28P signal locations

Reviewer comment
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.

Author response
We thank the referee for this comment. To make a clearer distinction between observation and interpretation, we now refer to the spots in the imaging data as “H3S28P signal” and only equate them to the centromeric region in specific instances. We have also included caveats to the interpretation of the oocyte data in the discussion section.

Manuscript changes
1. We now refer to the image spots as “H3S28P” signal, and only equate them to centromere in specific, appropriate contexts.

2. Paragraph 12. “.... 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. ”

3. Paragraph 13. “.... We interpreted each spot as representing a centromere from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).”

4. Updated Paragraph 16-17.
Paragraph 16. “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 and 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). 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. 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.”

5. Figure 2 legend, last sentence. “… The positions of centromeric regions cannot be determined as chiasmata(s) are present along the homologous pairs of chromosomes in a highly condensed state.”
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Reviewer 1 comment 3.2 – Timing of oocyte collection

Reviewer comment
Actually, H3S28p shows several spots (more than 6) during prometaphase I, as can be seen Fig S4 in Feng and Thompson, 2018 Cell Cycle publication. 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.

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
We thank the referee for this comment and we have corrected Figure 2E.

Manuscript changes
1. Figure 2E. Schematic corrected so there is one red spot between each pair of sister chromatid.

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Competing Interests

We disclose no competing interests with regard to F1000's review process or this individual's peer review report.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioica suggests the presence of three chromosomes

Abstract

Keywords

Introduction

Table 1. Reference to images cited in this study.

Methods

Oikopleura dioica culture, staging & preparation of biological material

Image acquisition

Image processing and analysis

Statistical analysis

Results

Figure 1. Centromere counts from embryos.

Figure 2. Centromere counts from unfertilized eggs.

Discussion

Data availability

Underlying data

Acknowledgements

References

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