Keywords
karyotype, chromosome, centromere, histone H3, Oikopleura, oocyte, embryo, H3S28P
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Liu AW, Tan Y, Masunaga A et al. H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.12688/f1000research.25019.2)
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Brief Report
Revised
H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes
[version 2; peer review: 2 approved]
Previously titled: 'Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioica suggests the presence of three chromosomes'
PUBLISHED 01 Mar 2021
OPEN PEER REVIEW
REVIEWER STATUS
Abstract
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:
© 2021 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. H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.12688/f1000research.25019.2)
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)
Revised Amendments from Version 1
The revision incorporates structural changes to the manuscript and corrects misinterpretations in the data that we made. For the structural changes, wished to draw more attention to the rationale behind our desire to obtain a chromosome count which was done in an attempt to guide our concurrent telomere to telomere assembly of the Okinawa O. dioica genome. The new version deemphasizes our use of an antibody to obtain chromosome counts as a replacement for traditional histochemical methods. Figures which included schematics of the chromosome state and number of centromeres at different cell cycles were corrected. Misinterpretation of prophase chromosome structures have been changed to non-mitotic cell cycles. Further explanations of use of statistical methods to validate our data are presented. The title has also been amended.
See the authors' detailed response to the review by Haiyang Feng
See the authors' detailed response to the review by Shigeki Fujiwara
Introduction
The larvacean, Oikopleura dioica, possesses a fascinating genome: it has reduced to a mere 70Mbp and exhibits unique characteristics such as non-canonical splicing and the scattering of Hox genes (Denoeud et al., 2010; Edvardsen et al., 2005; Marz et al., 2008; Seo et al., 2001). It is thought that a combination of large effective population size and high mutation rate per generation have led to fast evolution (Berná & Alvarez-Valin, 2014). The recently published genome sequence of a “Japanese O. dioica” from mainland Japan highlighted large sequence variations between the Pacific and Atlantic populations (Wang et al., 2020). In addition, we recently released a telomere-to-telomere genome sequence of an O. dioica individual collected from the Okinawan coastline in southern Japan (Bliznina et al., 2020), which, to our surprise, revealed large differences in synteny to the mainland Japanese genome despite the geographical proximity. The genetic map of the north Atlantic O. dioica is reported to contain three chromosomes (two autosomes, X and Y sex chromosomes; Denoeud et al., 2010); however, prior studies based on histochemical techniques reported three (Körner, 1952) and eight chromosomes (Colombera & Fernaux, 1973). Given the large sequence and synteny differences between the assembled O. dioica genomes, as well as the discrepancies among previous studies, we wished to assess the karyotype for the local Okinawan O. dioica population.
Karyotyping is a long-established histochemical method to visualize eukaryotic chromosomes (Hsu & Benirschke, 1967; Tjio & Levan, 1950). This rapid technique, involving the use of stains including methylene blue, eosin, and azure B, allows for observation of chromosomes with a simple light microscope, naturally lending itself to a first attempt for karyotyping analysis (Giemsa, 1904). However, we were unable to determine an accurate count for the Okinawan O. dioica by this method due to variability which ranged from 11–27 chromosomes per nucleus.
As an alternative approach, we decided to immunostain the centromere as a means of quantifying the number of chromosomes. Metaphase-specific histone 3 (H3) markers have been used to determine the structure and the segregation of genetic material during oogenesis in situ (Ganot et al., 2006; Schulmeister et al., 2007). One such marker that has been successfully visualized in O. dioica is histone H3 phosphorylated at Ser-28 (Kawajiri et al., 2003; Kurihara et al., 2006), whose localization depends on the phase of the cell cycle: during metaphase, sister chromatids were stained in a manner consistent with alignment along the metaphase plate, whereas in non-mitotic cells, spatially punctate signals were found evenly spread within the nuclear envelope (Campsteijn et al., 2012; Feng & Thompson, 2018; Feng et al., 2019; Olsen et al., 2018). A structure in which chromosomes are sequestered in a ∏-shaped conformation has also been observed during meiotic cell divisions between the final phases of oogenesis and mature oocytes (Ganot et al., 2008). In Table 1, we list the publications in which the H3S28P marker was applied to O. dioica: the studies were all performed using cultured strains originating from the north Atlantic Ocean. Here, we visualized anti-H3S28P stained embryos from two commercially available antibody sources and unfertilized oocytes to determine the chromosome count of the local Okinawan O. dioica.
Table 1. Reference to images cited in this study.
| Author | Date | Journal | H3S28P source | Figure(s) | Target sample |
|---|---|---|---|---|---|
| Spada et al. | 2005 | Journal of Cellular Biochemistry | Thermo Fisher 07-145 | 3 & 6 | Day 3 |
| Schulmeister et al. | 2007 | Chromosome Research | Abcam, ab10543 | 3 & 5 | Male gonad/female coenocyte |
| Ganot et al. | 2008 | Developmental Biology | Thermo Fisher 07-145 | 4, 7 & 8 | Maturing oocytes |
| Campsteijn et al. | 2012 | Molecular Biology and Evolution | Abcam, ab10543 | 1 | Hatched larvae |
| Øvrebø et al. | 2015 | Cell Cycle | Abcam, ab10543 | 1, 4, 5, 7 & S2A | Maturing oocytes (P3, P4) |
| Feng & Thompson | 2018 | Cell Cycle | Abcam, ab10543 | 1, 2 & 7 | P4 ovaries |
| Olsen et al. | 2018 | BMC Developmental Biology | Abcam, ab10543 | 5 & Addendum 3 | 4, 8, 16, 32 cell |
| Feng et al. | 2019 | Cell Cycle | Abcam ab10543 | 1, 3, 4, 5 & 6 | Hatched larvae |
Methods
Oikopleura dioica culture, staging & preparation of biological material
Sample preparation. 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 awaiting fertilization. Staged embryos were initiated by gently mixing 10 µl of the spawned male sperm with the awaiting eggs in FASW at 23 °C. Developing embryos were staged and collected by observation under a Leica M165C dissecting microscope. These embryos were quickly dechorionated using 0.1% sodium thioglycolate and 0.01% actinase in FASW for 2–3 minutes, then promptly washed with 2 washes with FASW prior to fixation and staining. Unfertilized eggs were treated similarly with three successive 10-minute washes.
Histochemical staining. Embryos were Giemsa stained as previously described in Shoguchi et al., 2005. Briefly, approximately 20–30 dechorionated embryos were treated with 0.04% colchicine in FASW for 30 minutes and then treated with decreasing amounts of KCl (50 mM and 25 mM) for five minutes each. Fixation was quickly performed with cold methanol:glacial acetic acid (3:1). The fixative was changed three times in the span of 18 hours while at -30 °C. The next morning, the fixed cells were quickly resuspended in 60% Acetic acid and methodically dropped from a height of 7 – 8cm onto a 48°C pre-warmed slide (Matsunami Glass, S2441). The slides were incubated for an additional 2 hours at 48°C; then stained with 6% Gimesa in 67mM sodium phosphate pH 7.0 for 2 hours at room temperature and rinsed with double distilled H2O. 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, 32 and 64 cell embryos (described above) 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 overnight (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 (Figure 1; Thermo Fisher Scientific Cat# 720099, RRID:AB_2532807) or rat monoclonal (Figure 2; 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 overnight. 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.
Figure 1. H3S28P signal counts in O. dioica embryos.
Anti-H3S28P rabbit-derived polyclonal stained 64-cell whole-embryo chromosomal imaging data collected by epifluorescence & confocal microscopy and analyzed by Imaris software SPOT DETECTION tool. A Maximum projection of confocal image of an embryo demonstrating the differences in signal localization and count, which was inferred to represent distinct cell cycle phases. (Red box, metaphase; blue circle, non-mitotic; EBI Image Archive S-BIAD21, Experiment D 20191125_01.lsm). B Schematic interpretation of signals with respect to chromatin structure during non-mitotic and metaphase cell cycle states. All chromosomes have been drawn with equal lengths for simplicity. C Distribution of signal counts within individual cells using epifluorescent (n = 40) and D confocal (n = 27) microscopes. The bimodal distribution suggests two distinct populations of cells with different chromosome counts (metaphase, red: epifluorescence n = 20, mean 6.2 , 95% CI 5.6 – 6.8; confocal n = 13, mean 6.4, 95% CI 5.7 – 7.1; non-mitotic, blue: epifluorescence n = 20, mean 12, 95% CI 11.0 – 13.0; confocal, n = 14, mean 14.1, 95% CI 12.9 – 15.3).
Figure 2. Centromere counts from unfertilized eggs.
A Maximum signal projection of a representative confocal Z-stack acquisition of anti-H3S28P rat monoclonal stained oocyte used for the count analysis (EBI Image Archive S-BIAD21, Experiment E 20200114_04.lsm). B Distribution of signal counts in each rosette-shaped chromatin structure, analyzed by Imaris software SPOT DETECTION tool (n = 23, mean 5.70, 95% CI 5.2 – 6.2). C Individual Z-sections from same image acquisition showing the 3D structure of the chromatin. Each plane is 0.54 µm apart. D Imaris spot analysis and annotation of signal positions from Z-stack acquisition. E Schematic representation of our interpretation that each signal is a centromere from a pair of sister chromatids. Chromosomes have been drawn with equal lengths for simplicity. The positions of centromeric regions cannot be determined as chiasmata(s) are present along the homologous pairs of chromosomes in a highly condensed state.
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.
Results
We initially attempted to visualize chromosomes using Giemsa staining on developing embryos. The spreads from 32- and 64-cell developmental stages, gave results with counts ranging between 11–27 stains per cell (BioImage Archive, S-BIAD21, Experiment A). Although cell-spreads were confined as a result of incomplete dechorionation with the enzymatic dissociation cocktail, we were still able to assign chromosomes to individual cells. Disappointingly, chromosome counts were unreliable due to the observed variability.
Consequently, we performed immunostaining of similarly staged embryos using a H3S28P-specific primary antibody and a secondary antibody conjugated to Alexa488 directed against the primary antibody. Signal-based thresholding was employed to determine the number of distinct 515 nm emission signals present in images acquired with epifluorescent and laser confocal microscopes (BioImage Archive, S-BIAD21, Experiment B & D). The data was analyzed using the Imaris SPOT DETECTION tool (Oxford Instruments).
Cells were manually classified into two types depending on the staining pattern visible in the nucleus: (i) those with intense clusters of signals in the center, considered to be in metaphase and (ii) those containing evenly distributed, clearly separated spots within a faint background of signal defining a region encompassed by the nuclear envelope, interpreted as non-mitotic (Figure 1A and 1B, blue circles; Figure 1A and 1B, red squares). Counts from these two classes of nuclei fall into separate distributions (Figure 1C and 1D), with both epifluorescence and confocal acquisitions in agreement with each other. We interpreted the nuclei with an average of six large, clustered signals as centromeric regions in metaphase (Figure 1B), however, we cannot explain the cell cycle state of those containing the average of 12 spatially distinct punctate signals.
To rule out polyploidy, which occurs in O. dioica somatic cells that give rise to the mucosal house (Ganot & Thompson, 2002), we also analyzed oocytes in metaphase I before fertilization (Schulmeister et al., 2007). We identified confined groupings of signals in unfertilized eggs (Figure 2A; BioImage Archive, S-BIAD21, Experiment E) and analyzed confocal images using the Imaris SPOT DETECTION tool to determine H3S28P signal counts (Figure 2B). Counts from the compact rosette-shaped chromatin structure averaged near 6. Visual inspection of individual Z-sections (Figure 2C) confirms the Imaris count analysis and annotation (Figure 2D). We interpreted each spot as representing a centromere from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).
Discussion
Our initial attempts at karyotyping by traditional Giemsa-staining gave us wildly varying counts which we unable to overcome with or without mitotic arrest. Giemsa-staining has been applied successfully to other organisms with small chromosomes such as the tunicate Ciona intestinalis (Shoguchi et al., 2005). The difference in outcome might be explained by the higher AT content of those genomes compared with O. dioica, since Giemsa preferentially stains AT-rich sequences. Although we do rule out Giemsa-staining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results.
Most karyotyping studies display a representative image to support the conclusion; however, given the variability in signal counts between nuclei, we decided to take a statistical approach that quantifies the uncertainty in the estimated chromosome count. Despite testing many different image acquisition settings, we were unable to eliminate the variability; we believe there are several possible reasons that explain the variance. (i) We applied uniform signal thresholds to all cells, so any spots below the threshold would have been missed. (ii) Spots displayed non-uniform signals, and individual centromeres may have occasionally contributed multiple counts. (iii) The H3S28P signal is not always confined to centromeres, and so may have caused multiple counts (see below). (iv) Finally, the 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.
An important consideration is what the H3S28P signal represents. It has been used to visualize centromeric regions in O. dioica (Table 1), but the signal is not confined to the centromere and its localization depends on the cellular state (Figure 1; Hake et al., 2005; Feng & Thompson, 2018). However, we are confident that the signals seen in Figure 1 labelled as metaphase and Figure 2 represent centromeres and their associated chromosome. Further, DNA-staining images of mature oocyte have previously been interpreted as chromosomes condensed in a structure resembling the Greek character ∏ (Ganot et al., 2007; Ganot et al., 2007b; Ganot et al., 2008). Since we did not perform DNA stains, our interpretation of the H3S28P signal in the oocyte does not preclude the previously reported ∏-structure. Additionally, the positions and numbers of crossovers between homologous pairs are unresolvable in this highly condensed state and the signal positions are not definitive of centromeric regions.
Currently, the nucleotide sequence of the centromeric region is unknown for O. dioica, although chromatin immunoprecipitation with a H3S28P antibody followed by long-read sequencing might be able to provide this information. However, our whole embryo staining data (Figure 1) and the previous literature (Table 1) show that the H3S28P antibody produces non-centromeric signals which may confound such analysis. Thus, alternative targets such as other centromeric histone 3 variants (Moosmann et al., 2011) might be preferable. Knowledge of centromeric sequences would also open the possibility of confirming these results with fluorescence in situ hybridization.
Despite the variations in signal counts between nuclei, a haploid chromosome count of three provides the most parsimonious explanation of the collected data and is consistent with previously published genome sequence assemblies (Denoeud et al., 2010). In summary, we conclude that the Okinawan Oikopleura dioica genome consists of three pairs of chromosomes in diploid cells. We believe that the images may be useful for examining cell cycle specific changes to chromosome structure and encourage the reuse and reanalysis of our data located in the EBI BioImage Archive (Ellenberg et al., 2018).
Data availability
Underlying data
Image acquisitions: Image data are available from the BioImage Archive Accession number S-BIAD21 (https://www.ebi.ac.uk/biostudies/studies/S-BIAD21)
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how to cite this article
Liu AW, Tan Y, Masunaga A et al. H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved] F1000Research 2021, 9:780 (https://doi.org/10.12688/f1000research.25019.2)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Version 2
VERSION 2
PUBLISHED 01 Mar 2021
Revised
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How to cite this report:
Fujiwara S. Reviewer Report For: H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.5256/f1000research.54241.r80403)
The direct URL for this report is:
https://f1000research.com/articles/9-780/v2#referee-response-80403
https://f1000research.com/articles/9-780/v2#referee-response-80403
NOTE: it is important to ensure the information in square brackets after the title is included in this citation.
Reviewer Report 08 Mar 2021
Approved
VIEWS 8
All of the concerns I had raised were carefully addressed. I feel
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HOW TO CITE THIS REPORT
Fujiwara S. Reviewer Report For: H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.5256/f1000research.54241.r80403)
The direct URL for this report is:
https://f1000research.com/articles/9-780/v2#referee-response-80403
https://f1000research.com/articles/9-780/v2#referee-response-80403
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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How to cite this report:
Feng H. Reviewer Report For: H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.5256/f1000research.54241.r80404)
The direct URL for this report is:
https://f1000research.com/articles/9-780/v2#referee-response-80404
https://f1000research.com/articles/9-780/v2#referee-response-80404
NOTE: it is important to ensure the information in square brackets after the title is included in this citation.
Reviewer Report 02 Mar 2021
Approved
VIEWS 9
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
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Feng H. Reviewer Report For: H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.5256/f1000research.54241.r80404)
The direct URL for this report is:
https://f1000research.com/articles/9-780/v2#referee-response-80404
https://f1000research.com/articles/9-780/v2#referee-response-80404
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Version 1
VERSION 1
PUBLISHED 28 Jul 2020
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How to cite this report:
Fujiwara S. Reviewer Report For: H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.5256/f1000research.27598.r68262)
The direct URL for this report is:
https://f1000research.com/articles/9-780/v1#referee-response-68262
https://f1000research.com/articles/9-780/v1#referee-response-68262
NOTE: it is important to ensure the information in square brackets after the title is included in this citation.
Reviewer Report 12 Aug 2020
Approved with Reservations
VIEWS 31
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
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HOW TO CITE THIS REPORT
Fujiwara S. Reviewer Report For: H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.5256/f1000research.27598.r68262)
The direct URL for this report is:
https://f1000research.com/articles/9-780/v1#referee-response-68262
https://f1000research.com/articles/9-780/v1#referee-response-68262
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Author Response 01 Mar 2021Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan01 Mar 2021Author ResponseWe 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
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Author Response 01 Mar 2021Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan01 Mar 2021Author ResponseWe 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
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How to cite this report:
Feng H. Reviewer Report For: H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.5256/f1000research.27598.r68260)
The direct URL for this report is:
https://f1000research.com/articles/9-780/v1#referee-response-68260
https://f1000research.com/articles/9-780/v1#referee-response-68260
NOTE: it is important to ensure the information in square brackets after the title is included in this citation.
Reviewer Report 05 Aug 2020
Approved with Reservations
VIEWS 62
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
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HOW TO CITE THIS REPORT
Feng H. Reviewer Report For: H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes [version 2; peer review: 2 approved]. F1000Research 2021, 9:780 (https://doi.org/10.5256/f1000research.27598.r68260)
The direct URL for this report is:
https://f1000research.com/articles/9-780/v1#referee-response-68260
https://f1000research.com/articles/9-780/v1#referee-response-68260
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Author Response 01 Mar 2021Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan01 Mar 2021Author ResponseWe 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
COMMENTS ON THIS REPORT
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Author Response 01 Mar 2021Andrew W Liu, Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology, Onna-son, 904-0324, Japan01 Mar 2021Author ResponseWe 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
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