Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells

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Notch signaling is a key signaling pathway involved in retinal development. Although the involvement of this signaling pathway in the differentiation of retinal ganglion cells has been documented, less is known about its involvement in earlier stages of retinal progenitor cell differentiation. We aimed to clarify the timing of Notch receptor expression in undifferentiated retinal progenitor cells and elucidate the possible involvement of chromatin remodeling in the regulation of Notch receptor expressions. Methods We re-analyzed publicly available human fetal retina single-cell RNA-seq and ATAC-seq data (GSE183684) using Seurat/Signac pipelines. Results On days 59, 74, and 78, we observed NOTCH1 mRNA expression in early retinal progenitor cells, which diminished at later stages of differentiation. Integration of single-cell RNA-seq and ATAC-seq revealed that chromatin remodeling in part of the NOTCH1 locus was accompanied by transitions in its mRNA expression. Importantly, chromatin accessibility in the region upstream of NOTCH1 depended on the differentiated cell types. Conclusions These results suggest that chromatin remodeling may be involved in the differential expression of NOTCH1, although another type of Notch mRNA expression regulation may exist. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-31/v1", "name": "Expression status transition ofNOTCH1accompanies chromatin remodeling..." } } ] } Home Browse Expression status transition ofNOTCH1accompanies chromatin remodeling... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Watabe Y, Kobayashi S, Nakayama T et al. Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.12688/f1000research.159630.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Brief Report Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] Yoshitoku Watabe https://orcid.org/0009-0007-4611-8193 1,2 , Sakurako Kobayashi https://orcid.org/0009-0001-1242-2097 1,2 , Takahiro Nakayama https://orcid.org/0000-0002-4023-4560 1,2 , Satoru Takahashi https://orcid.org/0000-0002-8540-7760 2,3 , Masaharu Yoshihara https://orcid.org/0000-0002-0212-0909 2,4 Yoshitoku Watabe https://orcid.org/0009-0007-4611-8193 1,2 , Sakurako Kobayashi https://orcid.org/0009-0001-1242-2097 1,2 , [...] Takahiro Nakayama https://orcid.org/0000-0002-4023-4560 1,2 , Satoru Takahashi https://orcid.org/0000-0002-8540-7760 2,3 , Masaharu Yoshihara https://orcid.org/0000-0002-0212-0909 2,4 PUBLISHED 06 Jan 2025 Author details Author details 1 Department of Anatomy and Embryology, Institute of Medicine, University of Tsukuba, Tsukuba, Japan 2 College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Ibaraki Prefecture, Japan 3 Transborder Medical Center, Institute of Medicine, University of Tsukuba, Tsukuba, Japan 4 Department of Primary Care and Medical Education, Institute of Medicine, University of Tsukuba, Tsukuba, Japan Yoshitoku Watabe Roles: Data Curation, Formal Analysis, Software, Visualization, Writing – Original Draft Preparation Sakurako Kobayashi Roles: Writing – Original Draft Preparation Takahiro Nakayama Roles: Writing – Original Draft Preparation Satoru Takahashi Roles: Supervision, Writing – Review & Editing Masaharu Yoshihara Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Software, Writing – Original Draft Preparation OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Japan Institutional Gateway gateway. This article is included in the Cell & Molecular Biology gateway. Abstract Background The regulation of receptor expression is crucial for fine-tuned signal transduction. Notch signaling is a key signaling pathway involved in retinal development. Although the involvement of this signaling pathway in the differentiation of retinal ganglion cells has been documented, less is known about its involvement in earlier stages of retinal progenitor cell differentiation. We aimed to clarify the timing of Notch receptor expression in undifferentiated retinal progenitor cells and elucidate the possible involvement of chromatin remodeling in the regulation of Notch receptor expressions. Methods We re-analyzed publicly available human fetal retina single-cell RNA-seq and ATAC-seq data (GSE183684) using Seurat/Signac pipelines. Results On days 59, 74, and 78, we observed NOTCH1 mRNA expression in early retinal progenitor cells, which diminished at later stages of differentiation. Integration of single-cell RNA-seq and ATAC-seq revealed that chromatin remodeling in part of the NOTCH1 locus was accompanied by transitions in its mRNA expression. Importantly, chromatin accessibility in the region upstream of NOTCH1 depended on the differentiated cell types. Conclusions These results suggest that chromatin remodeling may be involved in the differential expression of NOTCH1 , although another type of Notch mRNA expression regulation may exist. READ ALL READ LESS Keywords differentiation, epigenetics, eye development, single-cell ATAC-seq, single-cell RNA-seq Corresponding Author(s) Masaharu Yoshihara ( [email protected] ) Close Corresponding author: Masaharu Yoshihara Competing interests: No competing interests were disclosed. Grant information: This study was supported by the JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (grant no. JP23K14429) to M.Y. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Watabe Y 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: Watabe Y, Kobayashi S, Nakayama T et al. Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.12688/f1000research.159630.1 ) First published: 06 Jan 2025, 14 :31 ( https://doi.org/10.12688/f1000research.159630.1 ) Latest published: 17 Apr 2026, 14 :31 ( https://doi.org/10.12688/f1000research.159630.4 )  There is a newer version of this article available. Suppress this message for one day. Introduction Signal transduction depends on the expression of receptors regulated at multiple levels. These regulations include chromatin remodeling and DNA-binding proteins, such as transcription factors and transcriptional repressors. Because fine-tuned signal transduction is necessary for development, it is important to clarify the regulatory mechanisms of receptor expression. Notch signaling in mammals is dependent on the binding of five canonical DSL ligands and four Notch receptors. 1 It has been suggested that Notch loci are subject to chromatin remodeling under both normal 2 – 7 and pathological 8 – 16 conditions. In terms of developmental biology, the retina is a good model for investigating cellular differentiation involving Notch signaling. 17 The developed retina is composed of multiple cell types, including retinal ganglion cells, bipolar cells, photoreceptors, amacrine cells, horizontal cells, and Müllar glial cells, all of which originate from the retinal progenitor cells (RPCs). RPC is characterized by the expression of Lhx2 , Pax6 , Rax , and Vsx2 while there are some additional marker genes for developing horizontal cells/retinal ganglion cells ( Onecut1 / 2 ), 18 developing amacrine cells ( Elavl 2/4), 19 developing photoreceptors/amacrine cells/Müllar glial cells ( Eef1a1 ), 20 developing retinal ganglion cells ( Meis2 ), 21 developing horizontal cells ( Lhx1 , Ptf1a ), 22 and glial cells ( Pax2 ). 23 Of the four Notch receptors in mammals, Notch1, has been suggested to be involved in the maintenance of RPCs and differentiation into retinal ganglion cells. 24 However, it is unclear when and how Notch receptor expression is switched on and off in RPCs during early differentiation. Here, we re-analyzed a public multi-omics dataset of single-cell RNA-seq and single-cell ATAC-seq from three human fetal retinas 25 to clarify the timing of Notch receptor expression and examine the involvement of chromatin remodeling in this receptor expression switch. Methods A single-cell multi-omics dataset (GSE183684) 25 was downloaded from the Gene Expression Omnibus ( https://www.ncbi.nlm.nih.gov/geo/ ); data from days 59, 74, and 78 in the dataset were selectively used because they contained many undifferentiated retinal progenitor cells. The data were processed in Seurat version 5.1.0 26 and Signac version 1.14.0 27 pipelines in R version 4.4.1 on the Ubuntu 22.04.4 LTS environment. Pseudotime analysis and integration of the single-cell RNA-seq data and the single-cell ATAC-seq data was conducted by using the “FindTransferAnchors” function of Signac and Monocle3 version 1.3.7, 28 respectively. The conditions used in the analysis are provided in the GitHub repository. Results Re-analysis day 59 human fetal retina First, early gestational stage samples were characterized (day 59). Uniform manifold approximation and projection (UMAP) analysis of single-cell RNA-seq identified 13 clusters ( Figure 1A ), which were further characterized by marker gene expression ( Figure 1B ). This sample primarily contained RPCs with various differentiation statuses except for PAX2 -expressing glial cells. Early RPCs (RPC1-3 and MKI67 -expressing Proliferating RPC) were characterized by LHX2 , PAX6 , RAX , and VSX2. In addition, pseudotime analysis suggested that these RPCs differentiated into either ONECUT1 / 2 -expressing, ELAVL2 / 4 -expressing, or EEF1A1 -expressing RPCs ( Figure 1C ). Next, we examined Notch mRNA expression and found that NOTCH1 - 3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes ( Figure 1D ). Then, we re-analyzed single-cell ATAC-seq data for this day 59 sample, which was mathematically integrated with its single-cell RNA-seq data by using the “FindTransferAnchors” function of Signac ( Figure 1E ). In the early RPCs (RPC1/2 and MKI67 -expressing Proliferating RPC), we observed peaks between Chr9 136510000-136520000 of the NOTCH1 gene, which diminished in the other RPC clusters. These results imply that Notch expression transition during RPC differentiation might be associated with the chromatin remodeling of NOTCH1 ( Figure 1F ). However, we noted that chromatin accessibility of the upstream region of the NOTCH1 locus remained high in ONECUT1/2 -expressing RPCs. In contrast to these populations, chromatin accessibility of the NOTCH1 locus in ELAVL2/4 -expressing RPCs was very low, which was consistent with the low mRNA expression in these RPC populations. In addition, the chromatin accessibility of the NOTCH3 locus remained unchanged during RPC differentiation, although its mRNA expression diminished ( Figure 1G ). Since NOTCH2 and NOTCH4 were less prominent, the feature plots of the marker genes and the dot plots of the Notch genes from single-cell RNA-seq along with coverage plots of NOTCH2 and NOTCH4 from single-cell ATAC-seq are available in “Additional_file_1” in our GitHub repository. Figure 1. NOTCH1 mRNA expression decrease and concomitant chromatin remodeling of the day 59 sample. (A) UMAP analysis of the single-cell RNA-seq data identified 13 clusters. (B) Dot plot of marker genes. Note that early RPC clusters expressed LHX2 , PAX6 , RAX , and VSX2. (C) Monocle3 pseudotime analysis for clarifying the differentiation status. (D) Feature plot of NOTCH1 - 4. Note that NOTCH1 and NOTCH3 expression was prominent in the early RPC clusters. (E) UMAP analysis of the single-cell ATAC-seq data, which was integrated with the single-cell RNA-seq data. (F) The coverage plot of the NOTCH1 locus showing the chromatin accessibility. Note that the upstream of the gene is on the right. (G) The coverage plot of the NOTCH3 locus. Note that the upstream of the gene is on the right. Re-analysis day 74 human fetal retina To examine whether the changes in Notch mRNA expression and chromatin accessibility of the Notch loci in the day 59 sample were representative, we investigated another early gestational stage sample (day 74). UMAP analysis identified 15 clusters ( Figure 2A ) that were further characterized by marker gene expression ( Figure 2B ). This sample primarily contained RPCs with various differentiation statuses. Early RPCs (RPC1-6) were characterized by LHX2 , PAX6 , RAX , and VSX2. In addition, pseudotime analysis suggested that these RPCs differentiated into either ELAVL2 / 4 -expressing, ONECUT1 / MEIS2 -expressing, or ONECUT2 -expressing RPCs ( Figure 2C ). ONECUT1 / MEIS2 -expressing RPCs differentiated into RPCs that markedly expressed VSX2. Next, we examined Notch mRNA expression and found that NOTCH1 - 3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes, similar to the day 59 sample ( Figure 2D ). We then re-analyzed the single-cell ATAC-seq data for this sample, which were integrated with the single-cell RNA-seq data ( Figure 2E ). In the early RPCs (RPC1-3), we observed peaks between Chr9 136510000-136520000 of the NOTCH1 gene, which diminished in the other RPC clusters. These results confirmed that Notch expression transition during RPC differentiation might be associated with the chromatin remodeling of NOTCH1 ( Figure 2F ). However, we noted that the chromatin accessibility of the upstream region of the NOTCH1 locus remained high in ONECUT -expressing RPCs. In contrast to these populations, the chromatin accessibility of the NOTCH1 locus in ELAVL2/4 -expressing RPCs was very low, which was consistent with the low mRNA expression in these RPC populations. In addition, the chromatin accessibility of the NOTCH3 locus remained unchanged during RPC differentiation ( Figure 2G ). In summary, similar to the day 59 sample, in the day 74 sample, a concomitant mRNA decrease and chromatin remodeling in NOTCH1 were observed, while chromatin accessibility in the upstream region of the NOTCH1 locus remained high in ONECUT -expressing RPCs. The feature plots of the marker genes and dot plots of the Notch genes from single-cell RNA-seq along with coverage plots of NOTCH2 and NOTCH4 from single-cell ATAC-seq are available in “Additional_file_2” in our GitHub repository. Figure 2. NOTCH1 mRNA expression decrease and concomitant chromatin remodeling of the day 74 sample. (A) UMAP analysis of the single-cell RNA-seq data identified 15 clusters. (B) Dot plot of marker genes. Note that early RPC clusters expressed LHX2 , PAX6 , RAX , and VSX2. (C) Monocle3 pseudotime analysis. (D) Feature plot of NOTCH1 - 4. Note that NOTCH1 and NOTCH3 expression were prominent in the early RPC clusters. (E) UMAP analysis of the single-cell ATAC-seq data which was integrated with the single-cell RNA-seq data. (F) The coverage plot of the NOTCH1 locus. (G) The coverage plot of the NOTCH3 locus. Re-analysis day 78 human fetal retina To further confirm the developmental changes in Notch mRNA expression and chromatin accessibility at the Notch loci, we investigated an additional early gestational stage sample (day 78). UMAP analysis identified 17 clusters ( Figure 3A ) that were further characterized by marker gene expression ( Figure 3B ). The sample primarily contained RPCs with various differentiation statuses. Early RPCs (RPC1-3 and MKI67 -expressing Proliferating RPC1-2 cells) were characterized by LHX2 , PAX6 , RAX , and VSX2. In addition, pseudotime analysis suggested that these RPCs differentiated into ONECUT1 / 2 -expressing RPCs, which later differentiated into PTF1A and LHX1 -expressing RPCs, MEIS2 -expressing RPCs, or ELAVL2/4 -expressing RPCs ( Figure 3C ). Next, we examined Notch mRNA expression and found that NOTCH1 - 3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes ( Figure 3D ). We then re-analyzed the single-cell ATAC-seq data for the day 78 sample, which was integrated with the single-cell RNA-seq data ( Figure 3E ). In the early RPCs (RPC1-3), we observed peaks between Chr9 136510000-136520000 of the NOTCH1 gene, which diminished in the other RPC clusters ( Figure 3F ). However, we noted that the chromatin accessibility of the upstream region of the NOTCH1 locus remained high in ONECUT -expressing RPCs. In contrast to these populations, the chromatin accessibility of the NOTCH1 locus in ELAVL2/4 -expressing RPCs was very low, which was consistent with the low mRNA expression in these RPC populations. In addition, the chromatin accessibility of the NOTCH3 locus remained unchanged during RPC differentiation ( Figure 3G ). In summary, examination of all three independent samples suggested that NOTCH1 mRNA expression decreased, which was concomitant with chromatin remodeling in Chr9 136510000-136520000 of the NOTCH1 locus. The feature plots of the marker genes and the dot plots of the NOTCH genes from single-cell RNA-seq along with coverage plots of NOTCH2 and NOTCH4 from single-cell ATAC-seq are available in “Additional_file_3” in our GitHub repository. Figure 3. NOTCH1 mRNA expression decrease and concomitant chromatin remodeling of the day 78 sample. (A) UMAP analysis of the single-cell RNA-seq data identified 17 clusters. (B) Dot plot of marker genes. Note that early RPC clusters expressed LHX2 , PAX6 , RAX , and VSX2. (C) Monocle3 pseudotime analysis. (D) Feature plot of NOTCH1 - 4. Note that NOTCH1 and NOTCH3 expression were prominent in early RPC clusters. (E) UMAP analysis of the single-cell ATAC-seq data which was integrated with the single-cell RNA-seq data. (F) The coverage plot of the NOTCH1 locus. (G) The coverage plot of the NOTCH3 locus. Discussion The involvement of Notch signaling in cell fate choices is well documented, including in Drosophila neurogenesis 29 and mammalian biliary development. 30 Although the regulation of Notch receptor expression is necessary for these processes, to the best of our knowledge, few studies have used genome-wide investigations of the underlying molecular mechanisms. To examine chromatin remodeling in such regulatory mechanisms, we re-analyzed a single-cell RNA-seq and ATAC-seq dataset from developing retinas in which differentiation trajectories were well characterized. By re-analyzing three independent samples, we observed chromatin remodeling in part of the NOTCH1 locus, concomitant with changes in its mRNA expression during RPC differentiation. Transcriptional regulation occurs at many levels, including DNA-binding proteins and miRNAs, as well as chromatin remodeling. Importantly, the chromatin accessibility of the upstream regions of the NOTCH1 locus was unaffected in ONECUT -expressing RPCs. Therefore, the observed mRNA changes might be driven by pathways other than chromatin remodeling. Indeed, NOTCH3 mRNA expression also diminished during differentiation, although we observed no chromatin remodeling in the NOTCH3 locus. Because chromatin accessibility in ELAVL2 / 4 -expressing RPCs was very low, this ensured low mRNA expression of NOTCH1. An ophthalmological study revealed that the epigenetic landscape of cell type-specific enhancers shifted during differentiation of RPCs. 31 For example, in the single-cell ATAC-seq data from embryonic day 14.5 mouse retina, motif enrichment for Lhx2 , Rax and Pax6 in the early RPCs were observed, and footprinting analysis validated binding of those transcription factors to their motifs. These high chromatin accessibilities decreased as they differentiated into retinal ganglion cells and non-retinal ganglion cells. Although that study is excellent in providing comprehensive and in-depth insights, ours is unique in focusing on the Notch loci for clarifying the regulatory mechanisms in view of Notch signaling biology. Finally, we note that further investigations, such as a large deletion of these regions, will be needed to evaluate the contribution of the identified chromatin remodeling to the differential expression of Notch receptors in RPC subsets. Ethical considerations This study does not generate new data from human. Therefore, we consider that there are no special requirements on recruitment and publication. In addition, this study does not involve analysis of animals and plants. Consent for publication Not applicable. Authors’ contributions Y. W.: Data Curation, Formal Analysis, Software, Visualization, Writing – Original Draft Preparation S.K.: Writing – Original Draft Preparation T.N.: Writing – Original Draft Preparation S.T.: Supervision, Writing – Review & Editing M.Y.: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Software, Writing – Original Draft Preparation Data availability A single-cell multiomics dataset (GSE183684) 18 was downloaded from the Gene Expression Omnibus database ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183684 ). The original data in the present study including additional data available from: https://github.com/Yoshitokky/Notch1_retina_multiomics_data/tree/main . 32 Archived software available from: https://doi.org/10.5281/zenodo.14507019 . 32 License: OSI approved open license software is under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). The project contains the following underlying data: • Additional_file_1.jpg (Extended data for d59 data). • Additional_file_2.jpg (Extended data for d74 data). • Additional_file_3.jpg (Extended data for d78 data). • Figure 1A. png • Figure 1B. png • Figure 1C. png • Figure 1D_NOTCH1.png • Figure 1D_NOTCH2.png • Figure 1D_NOTCH3.png • Figure 1D_NOTCH4.png • Figure 1E. png • Figure 1F. png • Figure 1G. png • Figure 2A. png • Figure 2B. png • Figure 2C. png • Figure 2D_NOTCH1.png • Figure 2D_NOTCH2.png • Figure 2D_NOTCH3.png • Figure 2D_NOTCH4.png • Figure 2E. png • Figure 2F. png • Figure 2G. png • Figure 3A. png • Figure 3B. png • Figure 3C. png • Figure 3D_NOTCH1.png • Figure 3D_NOTCH2.png • Figure 3D_NOTCH3.png • Figure 3D_NOTCH4.png • Figure 3E. png • Figure 3F. png • Figure 3G. png • Additional_file_1_A.png • Additional_file_1_B.png • Additional_file_1_C.png • Additional_file_1_D.png • Additional_file_2_A.png • Additional_file_2_B.png • Additional_file_2_C.png • Additional_file_2_D.png • Additional_file_3_A.png • Additional_file_3_B.png • Additional_file_3_C.png • Additional_file_3_D.png Software availability Source code available from: https://github.com/Yoshitokky/Notch1_retina_multiomics_software/tree/main . 33 Archived software available from: https://doi.org/10.5281/zenodo.14333877 . License: OSI approved open license software is under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). The project contains the following underlying data: • 241108_retina_chromatin_d59_No0.R (R code script for d59 data followed by No1). • 241108_retina_chromatin_d59_No1.R (R code script for d59 data followed by No2). • 241108_retina_chromatin_d59_No2.R (R code script for d59 data followed by No3). • 241108_retina_chromatin_d59_No3.R (R code script for d59 data followed by No4). • 241108_retina_chromatin_d59_No4.R (R code script for d59 data). • 241108_retina_chromatin_d74_No0.R (R code script for d74 data followed by No1). • 241108_retina_chromatin_d74_No1.R (R code script for d74 data followed by No2). • 241108_retina_chromatin_d74_No2.R (R code script for d74 data followed by No3). • 241108_retina_chromatin_d74_No3.R (R code script for d74 data followed by No4). • 241108_retina_chromatin_d74_No4.R (R code script for d74 data). • 241108_retina_chromatin_d78_No0.R (R code script for d78 data followed by No1). • 241108_retina_chromatin_d78_No1.R (R code script for d78 data followed by No2). • 241108_retina_chromatin_d78_No2.R (R code script for d78 data followed by No3). • 241108_retina_chromatin_d78_No3.R (R code script for d78 data followed by No4). • 241108_retina_chromatin_d78_No4.R (R code script for d78 data). 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Reference Source Comments on this article Comments (0) Version 4 VERSION 4 PUBLISHED 06 Jan 2025 ADD YOUR COMMENT Comment Author details Author details 1 Department of Anatomy and Embryology, Institute of Medicine, University of Tsukuba, Tsukuba, Japan 2 College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Ibaraki Prefecture, Japan 3 Transborder Medical Center, Institute of Medicine, University of Tsukuba, Tsukuba, Japan 4 Department of Primary Care and Medical Education, Institute of Medicine, University of Tsukuba, Tsukuba, Japan Yoshitoku Watabe Roles: Data Curation, Formal Analysis, Software, Visualization, Writing – Original Draft Preparation Sakurako Kobayashi Roles: Writing – Original Draft Preparation Takahiro Nakayama Roles: Writing – Original Draft Preparation Satoru Takahashi Roles: Supervision, Writing – Review & Editing Masaharu Yoshihara Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Software, Writing – Original Draft Preparation Competing interests No competing interests were disclosed. Grant information This study was supported by the JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (grant no. JP23K14429) to M.Y. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (4) version 4 Revised Published: 17 Apr 2026, 14:31 https://doi.org/10.12688/f1000research.159630.4 version 3 Revised Published: 14 Nov 2025, 14:31 https://doi.org/10.12688/f1000research.159630.3 version 2 Revised Published: 22 Sep 2025, 14:31 https://doi.org/10.12688/f1000research.159630.2 version 1 Published: 06 Jan 2025, 14:31 https://doi.org/10.12688/f1000research.159630.1 Copyright © 2025 Watabe Y 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. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Watabe Y, Kobayashi S, Nakayama T et al. Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.12688/f1000research.159630.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE 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 Version 1 VERSION 1 PUBLISHED 06 Jan 2025 Views 0 Cite How to cite this report: Tang Z. Reviewer Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.175389.r398458 ) The direct URL for this report is: https://f1000research.com/articles/14-31/v1#referee-response-398458 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 22 Aug 2025 Zhongjie Tang , University of Southern California, Los Angeles, Southern California, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.175389.r398458 Evaluation This study presents a reanalysis of publicly available single-cell multi-omics datasets to explore the relationship between NOTCH1 expression dynamics and chromatin accessibility during early stages of human retinal progenitor cell (RPC) differentiation. The authors apply standard single-cell RNA-seq ... Continue reading READ ALL Evaluation This study presents a reanalysis of publicly available single-cell multi-omics datasets to explore the relationship between NOTCH1 expression dynamics and chromatin accessibility during early stages of human retinal progenitor cell (RPC) differentiation. The authors apply standard single-cell RNA-seq and ATAC-seq integration methods to a biologically relevant question related to Notch signaling in retinal development. While the technical execution appears sound, the analysis remains largely descriptive and would benefit from further depth. Several issues—particularly concerning data interpretation and figure clarity—should be addressed to improve the overall quality of the manuscript. Essential Revisions 1. Clarify the expression hierarchy of NOTCH1–3 The manuscript currently implies that NOTCH3 is expressed at levels comparable to NOTCH1 across developmental stages. However, Figures 1D, 2D, and 3D clearly show that NOTCH1 is the dominant isoform, while NOTCH3 is expressed at notably lower levels. The text should be revised to accurately reflect these patterns and avoid overgeneralization. 2. Discuss the discrepancy between NOTCH2 expression and chromatin accessibility Although NOTCH2 is transcriptionally active throughout all three stages, there is little to no signal at its locus in the ATAC-seq data. This discrepancy warrants further discussion. Potential explanations might include technical limitations of ATAC-seq, regulation via distal enhancers, or post-transcriptional mechanisms. 3. Expand chromatin accessibility analysis beyond the immediate gene body The current analysis is restricted to a narrow window around the NOTCH1 locus (Chr9:136510000–136520000). Expanding the analysis region upstream and downstream (e.g., ±50–100 kb) may help identify putative enhancers or other regulatory elements contributing to the observed expression dynamics. 4. Enhance figure annotation and clarity The figures, particularly the UMAP and coverage plots, would benefit from clearer labeling. Specifically: Annotate key functional regions such as promoters or candidate enhancers in the ATAC-seq plots. Ensure cluster labels are consistent across UMAPs and provide legends that identify RPC subtypes. A supplementary table listing marker genes and cell counts per cluster would improve clarity for readers. Recommended Revisions Provide biological context for NOTCH3 Although NOTCH3 is included in the gene expression analysis, the manuscript does not explain its potential role in retinal development. Including a brief discussion of existing literature—or acknowledging the lack thereof—would better justify its inclusion in the study. Connect findings with known transcriptional regulators The manuscript mentions transcription factors such as LHX2, PAX6, and RAX, which are known regulators of retinal development. Further exploring how these factors may influence NOTCH1 expression or chromatin accessibility—either through motif analysis or referencing prior enhancer studies—would strengthen the biological interpretation. Summary Recommendation Major revision — The study addresses a relevant topic and is based on a solid dataset, but the analysis remains limited and the interpretation lacks depth. With substantial improvements in analytical scope, biological context, and figure presentation, the manuscript could make a valuable contribution to the field. 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? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Single-cell RNA-seq and ATAC-seq analysis, computational biology 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. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Tang Z. Reviewer Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.175389.r398458 ) The direct URL for this report is: https://f1000research.com/articles/14-31/v1#referee-response-398458 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 Nov 2025 Masaharu Yoshihara , College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Japan 10 Nov 2025 Author Response Dear Dr. Zhongjie Tang Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early ... Continue reading Dear Dr. Zhongjie Tang Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments from Dr. Zhongjie Tang This study presents a reanalysis of publicly available single-cell multi-omics datasets to explore the relationship between NOTCH1 expression dynamics and chromatin accessibility during early stages of human retinal progenitor cell (RPC) differentiation. The authors apply standard single-cell RNA-seq and ATAC-seq integration methods to a biologically relevant question related to Notch signaling in retinal development. While the technical execution appears sound, the analysis remains largely descriptive and would benefit from further depth. Several issues—particularly concerning data interpretation and figure clarity—should be addressed to improve the overall quality of the manuscript. Essential Revisions (1) Clarify the expression hierarchy of NOTCH1–3 The manuscript currently implies that NOTCH3 is expressed at levels comparable to NOTCH1 across developmental stages. However, Figures 1D, 2D, and 3D clearly show that NOTCH1 is the dominant isoform, while NOTCH3 is expressed at notably lower levels. The text should be revised to accurately reflect these patterns and avoid overgeneralization. Response: Thank you for your constructive comments. We agree to your comments and have carefully revised the manuscript and ensured that NOTCH1 showed the highest expression, followed by NOTCH2 or NOTCH3 . In the revised manuscript (the Results section for day 59 sample), we have emphasized that, considering the literature (ref. 25), we focused on NOTCH1 and NOTCH3 . To avoid overgeneralization, we have carried out analyses to the four NOTCH genes and reached to the conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation. (2) Discuss the discrepancy between NOTCH2 expression and chromatin accessibility Although NOTCH2 is transcriptionally active throughout all three stages, there is little to no signal at its locus in the ATAC-seq data. This discrepancy warrants further discussion. Potential explanations might include technical limitations of ATAC-seq, regulation via distal enhancers, or post-transcriptional mechanisms. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #2. (3) Expand chromatin accessibility analysis beyond the immediate gene body The current analysis is restricted to a narrow window around the NOTCH1 locus (Chr9:136510000–136520000). Expanding the analysis region upstream and downstream (e.g., ±50–100 kb) may help identify putative enhancers or other regulatory elements contributing to the observed expression dynamics. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #1. (4) Enhance figure annotation and clarity The figures, particularly the UMAP and coverage plots, would benefit from clearer labeling. Specifically: Annotate key functional regions such as promoters or candidate enhancers in the ATAC-seq plots. Response: Thank you for your constructive comment. We agree to your comment and have clearly indicated the peak regions with arrowheads in the ATAC-seq plots in Figure 2, 4 and 6, along with Additional_file_1, 2 and 3. In addition, the exact chromosome regions of each peak are now indicated in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Ensure cluster labels are consistent across UMAPs and provide legends that identify RPC subtypes. Response: Thank you for your constructive comment. We agree to your comment and have corrected all the cluster labels to ensure the consistency among the samples and to be reader friendly. A supplementary table listing marker genes and cell counts per cluster would improve clarity for readers. Response: Thank you for your constructive comment. We agree to your comment and have now provided a supplementary table listing marker genes and cell counts per cluster as “Supplementary_table_count.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Recommended Revisions (5) Provide biological context for NOTCH3 Although NOTCH3 is included in the gene expression analysis, the manuscript does not explain its potential role in retinal development. Including a brief discussion of existing literature—or acknowledging the lack thereof—would better justify its inclusion in the study. Response: Thank you for your constructive comment. We agree to your comment and have analyzed NOTCH3 as well as NOTCH1 since NOTCH3 has been suggested for retinal development (ref. 25). Therefore, we have added this reference paper in this revised manuscript. In addition, the biological significance of NOTCH3 is added in the Introduction section (“In addition to Notch1 , Notch3 expression has also been suggested in the early RPCs, which potentially affects the amount and oscillation of total Notch signaling via its susceptibility to the receptor cleavage and subsequent signal transduction). (6) Connect findings with known transcriptional regulators The manuscript mentions transcription factors such as LHX2, PAX6, and RAX, which are known regulators of retinal development. Further exploring how these factors may influence NOTCH1 expression or chromatin accessibility—either through motif analysis or referencing prior enhancer studies—would strengthen the biological interpretation. Response: Thank you for your constructive comment. We agree to your comment and have searched for binding motifs for those transcription factors in chromatin remodeling regions in the Notch loci. We have identified several binding motifs (indicated by arrowheads and numbers in Figure 2, 4 and 6 and Additional_file_1, 2 and 3, supporting the potential biological significance. The exact peak and binding motif information is provided in a supplementary file named “Supplementary_table_motif.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (7) Summary Recommendation Major revision — The study addresses a relevant topic and is based on a solid dataset, but the analysis remains limited and the interpretation lacks depth. With substantial improvements in analytical scope, biological context, and figure presentation, the manuscript could make a valuable contribution to the field. Response: We sincerely appreciate your thoughtful insights, all of which helped us improve the manuscript significantly. The R code used in this revised manuscript is available in our GitHub repository ( https://doi.org/10.5281/zenodo.17084033 ). Again, thank you so much for your constructive comments. We look forward to hearing from you. Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] Dear Dr. Zhongjie Tang Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments from Dr. Zhongjie Tang This study presents a reanalysis of publicly available single-cell multi-omics datasets to explore the relationship between NOTCH1 expression dynamics and chromatin accessibility during early stages of human retinal progenitor cell (RPC) differentiation. The authors apply standard single-cell RNA-seq and ATAC-seq integration methods to a biologically relevant question related to Notch signaling in retinal development. While the technical execution appears sound, the analysis remains largely descriptive and would benefit from further depth. Several issues—particularly concerning data interpretation and figure clarity—should be addressed to improve the overall quality of the manuscript. Essential Revisions (1) Clarify the expression hierarchy of NOTCH1–3 The manuscript currently implies that NOTCH3 is expressed at levels comparable to NOTCH1 across developmental stages. However, Figures 1D, 2D, and 3D clearly show that NOTCH1 is the dominant isoform, while NOTCH3 is expressed at notably lower levels. The text should be revised to accurately reflect these patterns and avoid overgeneralization. Response: Thank you for your constructive comments. We agree to your comments and have carefully revised the manuscript and ensured that NOTCH1 showed the highest expression, followed by NOTCH2 or NOTCH3 . In the revised manuscript (the Results section for day 59 sample), we have emphasized that, considering the literature (ref. 25), we focused on NOTCH1 and NOTCH3 . To avoid overgeneralization, we have carried out analyses to the four NOTCH genes and reached to the conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation. (2) Discuss the discrepancy between NOTCH2 expression and chromatin accessibility Although NOTCH2 is transcriptionally active throughout all three stages, there is little to no signal at its locus in the ATAC-seq data. This discrepancy warrants further discussion. Potential explanations might include technical limitations of ATAC-seq, regulation via distal enhancers, or post-transcriptional mechanisms. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #2. (3) Expand chromatin accessibility analysis beyond the immediate gene body The current analysis is restricted to a narrow window around the NOTCH1 locus (Chr9:136510000–136520000). Expanding the analysis region upstream and downstream (e.g., ±50–100 kb) may help identify putative enhancers or other regulatory elements contributing to the observed expression dynamics. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #1. (4) Enhance figure annotation and clarity The figures, particularly the UMAP and coverage plots, would benefit from clearer labeling. Specifically: Annotate key functional regions such as promoters or candidate enhancers in the ATAC-seq plots. Response: Thank you for your constructive comment. We agree to your comment and have clearly indicated the peak regions with arrowheads in the ATAC-seq plots in Figure 2, 4 and 6, along with Additional_file_1, 2 and 3. In addition, the exact chromosome regions of each peak are now indicated in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Ensure cluster labels are consistent across UMAPs and provide legends that identify RPC subtypes. Response: Thank you for your constructive comment. We agree to your comment and have corrected all the cluster labels to ensure the consistency among the samples and to be reader friendly. A supplementary table listing marker genes and cell counts per cluster would improve clarity for readers. Response: Thank you for your constructive comment. We agree to your comment and have now provided a supplementary table listing marker genes and cell counts per cluster as “Supplementary_table_count.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Recommended Revisions (5) Provide biological context for NOTCH3 Although NOTCH3 is included in the gene expression analysis, the manuscript does not explain its potential role in retinal development. Including a brief discussion of existing literature—or acknowledging the lack thereof—would better justify its inclusion in the study. Response: Thank you for your constructive comment. We agree to your comment and have analyzed NOTCH3 as well as NOTCH1 since NOTCH3 has been suggested for retinal development (ref. 25). Therefore, we have added this reference paper in this revised manuscript. In addition, the biological significance of NOTCH3 is added in the Introduction section (“In addition to Notch1 , Notch3 expression has also been suggested in the early RPCs, which potentially affects the amount and oscillation of total Notch signaling via its susceptibility to the receptor cleavage and subsequent signal transduction). (6) Connect findings with known transcriptional regulators The manuscript mentions transcription factors such as LHX2, PAX6, and RAX, which are known regulators of retinal development. Further exploring how these factors may influence NOTCH1 expression or chromatin accessibility—either through motif analysis or referencing prior enhancer studies—would strengthen the biological interpretation. Response: Thank you for your constructive comment. We agree to your comment and have searched for binding motifs for those transcription factors in chromatin remodeling regions in the Notch loci. We have identified several binding motifs (indicated by arrowheads and numbers in Figure 2, 4 and 6 and Additional_file_1, 2 and 3, supporting the potential biological significance. The exact peak and binding motif information is provided in a supplementary file named “Supplementary_table_motif.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (7) Summary Recommendation Major revision — The study addresses a relevant topic and is based on a solid dataset, but the analysis remains limited and the interpretation lacks depth. With substantial improvements in analytical scope, biological context, and figure presentation, the manuscript could make a valuable contribution to the field. Response: We sincerely appreciate your thoughtful insights, all of which helped us improve the manuscript significantly. The R code used in this revised manuscript is available in our GitHub repository ( https://doi.org/10.5281/zenodo.17084033 ). Again, thank you so much for your constructive comments. We look forward to hearing from you. Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] Competing Interests: We have no competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 Nov 2025 Masaharu Yoshihara , College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Japan 10 Nov 2025 Author Response Dear Dr. Zhongjie Tang Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early ... Continue reading Dear Dr. Zhongjie Tang Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments from Dr. Zhongjie Tang This study presents a reanalysis of publicly available single-cell multi-omics datasets to explore the relationship between NOTCH1 expression dynamics and chromatin accessibility during early stages of human retinal progenitor cell (RPC) differentiation. The authors apply standard single-cell RNA-seq and ATAC-seq integration methods to a biologically relevant question related to Notch signaling in retinal development. While the technical execution appears sound, the analysis remains largely descriptive and would benefit from further depth. Several issues—particularly concerning data interpretation and figure clarity—should be addressed to improve the overall quality of the manuscript. Essential Revisions (1) Clarify the expression hierarchy of NOTCH1–3 The manuscript currently implies that NOTCH3 is expressed at levels comparable to NOTCH1 across developmental stages. However, Figures 1D, 2D, and 3D clearly show that NOTCH1 is the dominant isoform, while NOTCH3 is expressed at notably lower levels. The text should be revised to accurately reflect these patterns and avoid overgeneralization. Response: Thank you for your constructive comments. We agree to your comments and have carefully revised the manuscript and ensured that NOTCH1 showed the highest expression, followed by NOTCH2 or NOTCH3 . In the revised manuscript (the Results section for day 59 sample), we have emphasized that, considering the literature (ref. 25), we focused on NOTCH1 and NOTCH3 . To avoid overgeneralization, we have carried out analyses to the four NOTCH genes and reached to the conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation. (2) Discuss the discrepancy between NOTCH2 expression and chromatin accessibility Although NOTCH2 is transcriptionally active throughout all three stages, there is little to no signal at its locus in the ATAC-seq data. This discrepancy warrants further discussion. Potential explanations might include technical limitations of ATAC-seq, regulation via distal enhancers, or post-transcriptional mechanisms. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #2. (3) Expand chromatin accessibility analysis beyond the immediate gene body The current analysis is restricted to a narrow window around the NOTCH1 locus (Chr9:136510000–136520000). Expanding the analysis region upstream and downstream (e.g., ±50–100 kb) may help identify putative enhancers or other regulatory elements contributing to the observed expression dynamics. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #1. (4) Enhance figure annotation and clarity The figures, particularly the UMAP and coverage plots, would benefit from clearer labeling. Specifically: Annotate key functional regions such as promoters or candidate enhancers in the ATAC-seq plots. Response: Thank you for your constructive comment. We agree to your comment and have clearly indicated the peak regions with arrowheads in the ATAC-seq plots in Figure 2, 4 and 6, along with Additional_file_1, 2 and 3. In addition, the exact chromosome regions of each peak are now indicated in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Ensure cluster labels are consistent across UMAPs and provide legends that identify RPC subtypes. Response: Thank you for your constructive comment. We agree to your comment and have corrected all the cluster labels to ensure the consistency among the samples and to be reader friendly. A supplementary table listing marker genes and cell counts per cluster would improve clarity for readers. Response: Thank you for your constructive comment. We agree to your comment and have now provided a supplementary table listing marker genes and cell counts per cluster as “Supplementary_table_count.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Recommended Revisions (5) Provide biological context for NOTCH3 Although NOTCH3 is included in the gene expression analysis, the manuscript does not explain its potential role in retinal development. Including a brief discussion of existing literature—or acknowledging the lack thereof—would better justify its inclusion in the study. Response: Thank you for your constructive comment. We agree to your comment and have analyzed NOTCH3 as well as NOTCH1 since NOTCH3 has been suggested for retinal development (ref. 25). Therefore, we have added this reference paper in this revised manuscript. In addition, the biological significance of NOTCH3 is added in the Introduction section (“In addition to Notch1 , Notch3 expression has also been suggested in the early RPCs, which potentially affects the amount and oscillation of total Notch signaling via its susceptibility to the receptor cleavage and subsequent signal transduction). (6) Connect findings with known transcriptional regulators The manuscript mentions transcription factors such as LHX2, PAX6, and RAX, which are known regulators of retinal development. Further exploring how these factors may influence NOTCH1 expression or chromatin accessibility—either through motif analysis or referencing prior enhancer studies—would strengthen the biological interpretation. Response: Thank you for your constructive comment. We agree to your comment and have searched for binding motifs for those transcription factors in chromatin remodeling regions in the Notch loci. We have identified several binding motifs (indicated by arrowheads and numbers in Figure 2, 4 and 6 and Additional_file_1, 2 and 3, supporting the potential biological significance. The exact peak and binding motif information is provided in a supplementary file named “Supplementary_table_motif.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (7) Summary Recommendation Major revision — The study addresses a relevant topic and is based on a solid dataset, but the analysis remains limited and the interpretation lacks depth. With substantial improvements in analytical scope, biological context, and figure presentation, the manuscript could make a valuable contribution to the field. Response: We sincerely appreciate your thoughtful insights, all of which helped us improve the manuscript significantly. The R code used in this revised manuscript is available in our GitHub repository ( https://doi.org/10.5281/zenodo.17084033 ). Again, thank you so much for your constructive comments. We look forward to hearing from you. Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] Dear Dr. Zhongjie Tang Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments from Dr. Zhongjie Tang This study presents a reanalysis of publicly available single-cell multi-omics datasets to explore the relationship between NOTCH1 expression dynamics and chromatin accessibility during early stages of human retinal progenitor cell (RPC) differentiation. The authors apply standard single-cell RNA-seq and ATAC-seq integration methods to a biologically relevant question related to Notch signaling in retinal development. While the technical execution appears sound, the analysis remains largely descriptive and would benefit from further depth. Several issues—particularly concerning data interpretation and figure clarity—should be addressed to improve the overall quality of the manuscript. Essential Revisions (1) Clarify the expression hierarchy of NOTCH1–3 The manuscript currently implies that NOTCH3 is expressed at levels comparable to NOTCH1 across developmental stages. However, Figures 1D, 2D, and 3D clearly show that NOTCH1 is the dominant isoform, while NOTCH3 is expressed at notably lower levels. The text should be revised to accurately reflect these patterns and avoid overgeneralization. Response: Thank you for your constructive comments. We agree to your comments and have carefully revised the manuscript and ensured that NOTCH1 showed the highest expression, followed by NOTCH2 or NOTCH3 . In the revised manuscript (the Results section for day 59 sample), we have emphasized that, considering the literature (ref. 25), we focused on NOTCH1 and NOTCH3 . To avoid overgeneralization, we have carried out analyses to the four NOTCH genes and reached to the conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation. (2) Discuss the discrepancy between NOTCH2 expression and chromatin accessibility Although NOTCH2 is transcriptionally active throughout all three stages, there is little to no signal at its locus in the ATAC-seq data. This discrepancy warrants further discussion. Potential explanations might include technical limitations of ATAC-seq, regulation via distal enhancers, or post-transcriptional mechanisms. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #2. (3) Expand chromatin accessibility analysis beyond the immediate gene body The current analysis is restricted to a narrow window around the NOTCH1 locus (Chr9:136510000–136520000). Expanding the analysis region upstream and downstream (e.g., ±50–100 kb) may help identify putative enhancers or other regulatory elements contributing to the observed expression dynamics. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #1. (4) Enhance figure annotation and clarity The figures, particularly the UMAP and coverage plots, would benefit from clearer labeling. Specifically: Annotate key functional regions such as promoters or candidate enhancers in the ATAC-seq plots. Response: Thank you for your constructive comment. We agree to your comment and have clearly indicated the peak regions with arrowheads in the ATAC-seq plots in Figure 2, 4 and 6, along with Additional_file_1, 2 and 3. In addition, the exact chromosome regions of each peak are now indicated in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Ensure cluster labels are consistent across UMAPs and provide legends that identify RPC subtypes. Response: Thank you for your constructive comment. We agree to your comment and have corrected all the cluster labels to ensure the consistency among the samples and to be reader friendly. A supplementary table listing marker genes and cell counts per cluster would improve clarity for readers. Response: Thank you for your constructive comment. We agree to your comment and have now provided a supplementary table listing marker genes and cell counts per cluster as “Supplementary_table_count.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Recommended Revisions (5) Provide biological context for NOTCH3 Although NOTCH3 is included in the gene expression analysis, the manuscript does not explain its potential role in retinal development. Including a brief discussion of existing literature—or acknowledging the lack thereof—would better justify its inclusion in the study. Response: Thank you for your constructive comment. We agree to your comment and have analyzed NOTCH3 as well as NOTCH1 since NOTCH3 has been suggested for retinal development (ref. 25). Therefore, we have added this reference paper in this revised manuscript. In addition, the biological significance of NOTCH3 is added in the Introduction section (“In addition to Notch1 , Notch3 expression has also been suggested in the early RPCs, which potentially affects the amount and oscillation of total Notch signaling via its susceptibility to the receptor cleavage and subsequent signal transduction). (6) Connect findings with known transcriptional regulators The manuscript mentions transcription factors such as LHX2, PAX6, and RAX, which are known regulators of retinal development. Further exploring how these factors may influence NOTCH1 expression or chromatin accessibility—either through motif analysis or referencing prior enhancer studies—would strengthen the biological interpretation. Response: Thank you for your constructive comment. We agree to your comment and have searched for binding motifs for those transcription factors in chromatin remodeling regions in the Notch loci. We have identified several binding motifs (indicated by arrowheads and numbers in Figure 2, 4 and 6 and Additional_file_1, 2 and 3, supporting the potential biological significance. The exact peak and binding motif information is provided in a supplementary file named “Supplementary_table_motif.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (7) Summary Recommendation Major revision — The study addresses a relevant topic and is based on a solid dataset, but the analysis remains limited and the interpretation lacks depth. With substantial improvements in analytical scope, biological context, and figure presentation, the manuscript could make a valuable contribution to the field. Response: We sincerely appreciate your thoughtful insights, all of which helped us improve the manuscript significantly. The R code used in this revised manuscript is available in our GitHub repository ( https://doi.org/10.5281/zenodo.17084033 ). Again, thank you so much for your constructive comments. We look forward to hearing from you. Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] Competing Interests: We have no competing interests. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Kashiwagi M. Reviewer Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.175389.r359501 ) The direct URL for this report is: https://f1000research.com/articles/14-31/v1#referee-response-359501 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 05 Feb 2025 Mariko Kashiwagi , Massachusetts General Hospital, Charlestown, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.175389.r359501 Remarks to the Author: In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1–4) were expressed in retinal progenitor cells (RPCs) and whether changes in chromatin accessibility at promoter regions contributed to their transcriptional activation. To ... Continue reading READ ALL Remarks to the Author: In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1–4) were expressed in retinal progenitor cells (RPCs) and whether changes in chromatin accessibility at promoter regions contributed to their transcriptional activation. To address these questions, they reanalyzed publicly available single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq datasets of the human fetal retina across three developmental stages (GSE183684). Overall, the analytical pipelines are valid and appropriately implemented. However, several concerns regarding the experimental design and the interpretation of the results require further consideration. Major Concerns and Comments: 1. Chromatin Accessibility Analysis: During development, enhancer regions play a crucial role in dynamic, lineage-specific gene regulation by modulating chromatin structure and recruiting transcription factors. In contrast, promoter regions generally maintain a more stable configuration following cellular differentiation into a specific lineage. With this in mind, I recommend that the authors expand their chromatin accessibility analysis beyond the promoter and gene body to include the 5' upstream and 3' downstream regions. This broader approach may provide a more comprehensive understanding of how various regulatory elements, including enhancers, contribute to the transcriptional regulation of NOTCH genes. 2. Interpretation of the Results: The authors stated in the text that "NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes." However, this claim is not fully supported by the data (Fig. 1D, Additional File 1B; Fig. 2D, Additional File 2B; Fig. 3D, Additional File 3B). A comprehensive interpretation of the three developmental stages suggests that NOTCH1 was the predominant isoform expressed in early RPC clusters, although expression levels varied across clusters and stages. At day 59, the expression hierarchy was NOTCH2 > NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully. Additionally, although NOTCH2 expression was consistently observed across all three stages (days 59, 74, and 78), the corresponding ATAC peaks were either absent or very low. The manuscript did not provide any discussion of this discrepancy. The authors should address this issue to clarify the relationship between chromatin accessibility and NOTCH2 expression, while also discussing whether this discrepancy may stem from technical limitations or reflect underlying biological factors. 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? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Skin biology, Immunology, Gene 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. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Kashiwagi M. Reviewer Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.175389.r359501 ) The direct URL for this report is: https://f1000research.com/articles/14-31/v1#referee-response-359501 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 Nov 2025 Masaharu Yoshihara , College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Japan 10 Nov 2025 Author Response Dear Dr. Mariko Kashiwagi, Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early ... Continue reading Dear Dr. Mariko Kashiwagi, Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments: In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1–4) were expressed in retinal progenitor cells (RPCs) and whether changes in chromatin accessibility at promoter regions contributed to their transcriptional activation. To address these questions, they reanalyzed publicly available single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq datasets of the human fetal retina across three developmental stages (GSE183684). Overall, the analytical pipelines are valid and appropriately implemented. However, several concerns regarding the experimental design and the interpretation of the results require further consideration. Major Concerns and Comments: (1) Chromatin Accessibility Analysis: During development, enhancer regions play a crucial role in dynamic, lineage-specific gene regulation by modulating chromatin structure and recruiting transcription factors. In contrast, promoter regions generally maintain a more stable configuration following cellular differentiation into a specific lineage. With this in mind, I recommend that the authors expand their chromatin accessibility analysis beyond the promoter and gene body to include the 5' upstream and 3' downstream regions. This broader approach may provide a more comprehensive understanding of how various regulatory elements, including enhancers, contribute to the transcriptional regulation of NOTCH genes. Response: Thank you for your insightful comments. We agree to your comment and have expanded the chromatin accessibility analysis to include 100 kb upstream and downstream of the Notch genes, and we have identified additional chromatin remodeling regions. The results are presented in Figure 2, 4 and 6 ( NOTCH1 and NOTCH3 for the three samples) and Additional_file_1, 2 and 3 ( NOTCH2 and NOTCH4 for the three samples). We have drastically revised the text for each sample in the Results section and provided the exact chromosome regions of each peak in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (2) Interpretation of the Results: The authors stated in the text that "NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes." However, this claim is not fully supported by the data (Fig. 1D, Additional File 1B; Fig. 2D, Additional File 2B; Fig. 3D, Additional File 3B). A comprehensive interpretation of the three developmental stages suggests that NOTCH1 was the predominant isoform expressed in early RPC clusters, although expression levels varied across clusters and stages. At day 59, the expression hierarchy was NOTCH2 > NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully. Response: Thank you for your constructive comments. We agree to your comment and clearly have indicated that the early RPCs expressed NOTCH1 , NOTCH2 and NOTCH3 for each sample in the Results section. In particular, NOTCH1 showed the highest expressions throughout the three samples and we have clearly mentioned that we have focused in NOTCH1 and NOTCH3 in this study since NOTCH3 has been suggested for early RPC differentiation (ref. 25) in the Results section for day 59 sample. Since the early RPCs expressed NOTCH2 in all the three samples, we have also analyzed chromatin accessibility for NOTCH2 in addition to NOTCH1 and NOTCH3 although we are unable to provide solid conclusions regarding this gene owing to varying findings on its chromatin accessibility, which is clearly mentioned in the Discussion section. Our findings and conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation, however, are solid in front of the unexplained results on NOTCH2 . (3) Additionally, although NOTCH2 expression was consistently observed across all three stages (days 59, 74, and 78), the corresponding ATAC peaks were either absent or very low. The manuscript did not provide any discussion of this discrepancy. The authors should address this issue to clarify the relationship between chromatin accessibility and NOTCH2 expression, while also discussing whether this discrepancy may stem from technical limitations or reflect underlying biological factors. Response: Thank you for your constructive comments. We agree to your comment and have revised our manuscript by adding several possible mechanisms such as distal enhancers and a post-transcriptional mechanism (small non-coding regulatory RNAs) (ref. 38) as well as technical limitations in the Discussion section. Although we are unable to fully explain the discrepancy of NOTCH2 mRNA expression and chromatin accessibility, we are confident to the main conclusion regarding NOTCH1 . Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] Dear Dr. Mariko Kashiwagi, Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments: In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1–4) were expressed in retinal progenitor cells (RPCs) and whether changes in chromatin accessibility at promoter regions contributed to their transcriptional activation. To address these questions, they reanalyzed publicly available single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq datasets of the human fetal retina across three developmental stages (GSE183684). Overall, the analytical pipelines are valid and appropriately implemented. However, several concerns regarding the experimental design and the interpretation of the results require further consideration. Major Concerns and Comments: (1) Chromatin Accessibility Analysis: During development, enhancer regions play a crucial role in dynamic, lineage-specific gene regulation by modulating chromatin structure and recruiting transcription factors. In contrast, promoter regions generally maintain a more stable configuration following cellular differentiation into a specific lineage. With this in mind, I recommend that the authors expand their chromatin accessibility analysis beyond the promoter and gene body to include the 5' upstream and 3' downstream regions. This broader approach may provide a more comprehensive understanding of how various regulatory elements, including enhancers, contribute to the transcriptional regulation of NOTCH genes. Response: Thank you for your insightful comments. We agree to your comment and have expanded the chromatin accessibility analysis to include 100 kb upstream and downstream of the Notch genes, and we have identified additional chromatin remodeling regions. The results are presented in Figure 2, 4 and 6 ( NOTCH1 and NOTCH3 for the three samples) and Additional_file_1, 2 and 3 ( NOTCH2 and NOTCH4 for the three samples). We have drastically revised the text for each sample in the Results section and provided the exact chromosome regions of each peak in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (2) Interpretation of the Results: The authors stated in the text that "NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes." However, this claim is not fully supported by the data (Fig. 1D, Additional File 1B; Fig. 2D, Additional File 2B; Fig. 3D, Additional File 3B). A comprehensive interpretation of the three developmental stages suggests that NOTCH1 was the predominant isoform expressed in early RPC clusters, although expression levels varied across clusters and stages. At day 59, the expression hierarchy was NOTCH2 > NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully. Response: Thank you for your constructive comments. We agree to your comment and clearly have indicated that the early RPCs expressed NOTCH1 , NOTCH2 and NOTCH3 for each sample in the Results section. In particular, NOTCH1 showed the highest expressions throughout the three samples and we have clearly mentioned that we have focused in NOTCH1 and NOTCH3 in this study since NOTCH3 has been suggested for early RPC differentiation (ref. 25) in the Results section for day 59 sample. Since the early RPCs expressed NOTCH2 in all the three samples, we have also analyzed chromatin accessibility for NOTCH2 in addition to NOTCH1 and NOTCH3 although we are unable to provide solid conclusions regarding this gene owing to varying findings on its chromatin accessibility, which is clearly mentioned in the Discussion section. Our findings and conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation, however, are solid in front of the unexplained results on NOTCH2 . (3) Additionally, although NOTCH2 expression was consistently observed across all three stages (days 59, 74, and 78), the corresponding ATAC peaks were either absent or very low. The manuscript did not provide any discussion of this discrepancy. The authors should address this issue to clarify the relationship between chromatin accessibility and NOTCH2 expression, while also discussing whether this discrepancy may stem from technical limitations or reflect underlying biological factors. Response: Thank you for your constructive comments. We agree to your comment and have revised our manuscript by adding several possible mechanisms such as distal enhancers and a post-transcriptional mechanism (small non-coding regulatory RNAs) (ref. 38) as well as technical limitations in the Discussion section. Although we are unable to fully explain the discrepancy of NOTCH2 mRNA expression and chromatin accessibility, we are confident to the main conclusion regarding NOTCH1 . Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] Competing Interests: We have no competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 Nov 2025 Masaharu Yoshihara , College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Japan 10 Nov 2025 Author Response Dear Dr. Mariko Kashiwagi, Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early ... Continue reading Dear Dr. Mariko Kashiwagi, Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments: In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1–4) were expressed in retinal progenitor cells (RPCs) and whether changes in chromatin accessibility at promoter regions contributed to their transcriptional activation. To address these questions, they reanalyzed publicly available single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq datasets of the human fetal retina across three developmental stages (GSE183684). Overall, the analytical pipelines are valid and appropriately implemented. However, several concerns regarding the experimental design and the interpretation of the results require further consideration. Major Concerns and Comments: (1) Chromatin Accessibility Analysis: During development, enhancer regions play a crucial role in dynamic, lineage-specific gene regulation by modulating chromatin structure and recruiting transcription factors. In contrast, promoter regions generally maintain a more stable configuration following cellular differentiation into a specific lineage. With this in mind, I recommend that the authors expand their chromatin accessibility analysis beyond the promoter and gene body to include the 5' upstream and 3' downstream regions. This broader approach may provide a more comprehensive understanding of how various regulatory elements, including enhancers, contribute to the transcriptional regulation of NOTCH genes. Response: Thank you for your insightful comments. We agree to your comment and have expanded the chromatin accessibility analysis to include 100 kb upstream and downstream of the Notch genes, and we have identified additional chromatin remodeling regions. The results are presented in Figure 2, 4 and 6 ( NOTCH1 and NOTCH3 for the three samples) and Additional_file_1, 2 and 3 ( NOTCH2 and NOTCH4 for the three samples). We have drastically revised the text for each sample in the Results section and provided the exact chromosome regions of each peak in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (2) Interpretation of the Results: The authors stated in the text that "NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes." However, this claim is not fully supported by the data (Fig. 1D, Additional File 1B; Fig. 2D, Additional File 2B; Fig. 3D, Additional File 3B). A comprehensive interpretation of the three developmental stages suggests that NOTCH1 was the predominant isoform expressed in early RPC clusters, although expression levels varied across clusters and stages. At day 59, the expression hierarchy was NOTCH2 > NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully. Response: Thank you for your constructive comments. We agree to your comment and clearly have indicated that the early RPCs expressed NOTCH1 , NOTCH2 and NOTCH3 for each sample in the Results section. In particular, NOTCH1 showed the highest expressions throughout the three samples and we have clearly mentioned that we have focused in NOTCH1 and NOTCH3 in this study since NOTCH3 has been suggested for early RPC differentiation (ref. 25) in the Results section for day 59 sample. Since the early RPCs expressed NOTCH2 in all the three samples, we have also analyzed chromatin accessibility for NOTCH2 in addition to NOTCH1 and NOTCH3 although we are unable to provide solid conclusions regarding this gene owing to varying findings on its chromatin accessibility, which is clearly mentioned in the Discussion section. Our findings and conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation, however, are solid in front of the unexplained results on NOTCH2 . (3) Additionally, although NOTCH2 expression was consistently observed across all three stages (days 59, 74, and 78), the corresponding ATAC peaks were either absent or very low. The manuscript did not provide any discussion of this discrepancy. The authors should address this issue to clarify the relationship between chromatin accessibility and NOTCH2 expression, while also discussing whether this discrepancy may stem from technical limitations or reflect underlying biological factors. Response: Thank you for your constructive comments. We agree to your comment and have revised our manuscript by adding several possible mechanisms such as distal enhancers and a post-transcriptional mechanism (small non-coding regulatory RNAs) (ref. 38) as well as technical limitations in the Discussion section. Although we are unable to fully explain the discrepancy of NOTCH2 mRNA expression and chromatin accessibility, we are confident to the main conclusion regarding NOTCH1 . Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] Dear Dr. Mariko Kashiwagi, Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments: In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1–4) were expressed in retinal progenitor cells (RPCs) and whether changes in chromatin accessibility at promoter regions contributed to their transcriptional activation. To address these questions, they reanalyzed publicly available single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq datasets of the human fetal retina across three developmental stages (GSE183684). Overall, the analytical pipelines are valid and appropriately implemented. However, several concerns regarding the experimental design and the interpretation of the results require further consideration. Major Concerns and Comments: (1) Chromatin Accessibility Analysis: During development, enhancer regions play a crucial role in dynamic, lineage-specific gene regulation by modulating chromatin structure and recruiting transcription factors. In contrast, promoter regions generally maintain a more stable configuration following cellular differentiation into a specific lineage. With this in mind, I recommend that the authors expand their chromatin accessibility analysis beyond the promoter and gene body to include the 5' upstream and 3' downstream regions. This broader approach may provide a more comprehensive understanding of how various regulatory elements, including enhancers, contribute to the transcriptional regulation of NOTCH genes. Response: Thank you for your insightful comments. We agree to your comment and have expanded the chromatin accessibility analysis to include 100 kb upstream and downstream of the Notch genes, and we have identified additional chromatin remodeling regions. The results are presented in Figure 2, 4 and 6 ( NOTCH1 and NOTCH3 for the three samples) and Additional_file_1, 2 and 3 ( NOTCH2 and NOTCH4 for the three samples). We have drastically revised the text for each sample in the Results section and provided the exact chromosome regions of each peak in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (2) Interpretation of the Results: The authors stated in the text that "NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes." However, this claim is not fully supported by the data (Fig. 1D, Additional File 1B; Fig. 2D, Additional File 2B; Fig. 3D, Additional File 3B). A comprehensive interpretation of the three developmental stages suggests that NOTCH1 was the predominant isoform expressed in early RPC clusters, although expression levels varied across clusters and stages. At day 59, the expression hierarchy was NOTCH2 > NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully. Response: Thank you for your constructive comments. We agree to your comment and clearly have indicated that the early RPCs expressed NOTCH1 , NOTCH2 and NOTCH3 for each sample in the Results section. In particular, NOTCH1 showed the highest expressions throughout the three samples and we have clearly mentioned that we have focused in NOTCH1 and NOTCH3 in this study since NOTCH3 has been suggested for early RPC differentiation (ref. 25) in the Results section for day 59 sample. Since the early RPCs expressed NOTCH2 in all the three samples, we have also analyzed chromatin accessibility for NOTCH2 in addition to NOTCH1 and NOTCH3 although we are unable to provide solid conclusions regarding this gene owing to varying findings on its chromatin accessibility, which is clearly mentioned in the Discussion section. Our findings and conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation, however, are solid in front of the unexplained results on NOTCH2 . (3) Additionally, although NOTCH2 expression was consistently observed across all three stages (days 59, 74, and 78), the corresponding ATAC peaks were either absent or very low. The manuscript did not provide any discussion of this discrepancy. The authors should address this issue to clarify the relationship between chromatin accessibility and NOTCH2 expression, while also discussing whether this discrepancy may stem from technical limitations or reflect underlying biological factors. Response: Thank you for your constructive comments. We agree to your comment and have revised our manuscript by adding several possible mechanisms such as distal enhancers and a post-transcriptional mechanism (small non-coding regulatory RNAs) (ref. 38) as well as technical limitations in the Discussion section. Although we are unable to fully explain the discrepancy of NOTCH2 mRNA expression and chromatin accessibility, we are confident to the main conclusion regarding NOTCH1 . Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] Competing Interests: We have no competing interests. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 4 VERSION 4 PUBLISHED 06 Jan 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline 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 Reviewer Reports Invited Reviewers 1 2 3 Version 4 (revision) 17 Apr 26 read Version 3 (revision) 14 Nov 25 read Version 2 (revision) 22 Sep 25 read Version 1 06 Jan 25 read read Mariko Kashiwagi , Massachusetts General Hospital, Charlestown, USA Zhongjie Tang , University of Southern California, Los Angeles, USA Benedikt Obermayer , Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Obermayer B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 14 May 2026 | for Version 4 Benedikt Obermayer , Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany 0 Views copyright © 2026 Obermayer B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline 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 in the new version, the authors have re-done the analysis on data integrated over timepoints, which makes cell type labels easier to compare, even though an actual label transfer from Thomas et al. or Zuo et al. would have helped put their results into the context of the literature. Thomas et al. is now cited, but the findings of that paper are not discussed. In particular, I'm still missing a motivation for their re-analysis of that data, or where this re-analysis leads to new or different results: Notch accessibility and expression was already discussed in the supplementary figures of Thomas et al; the identification of PAX6 binding sites at the NOTCH1/NOTCH3 loci is presented as a key novel finding, but these sites are readily visible in the UCSC browser using the JASPAR predicted TF binding track, and the authors' own 'confirmation' via the browser tracks from Thomas et al. is therefore somewhat circular. Competing Interests No competing interests were disclosed. Reviewer Expertise Bioinformatics, Functional and Single-Cell Genomics 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. reply Respond to this report Responses (0) Obermayer B. Peer Review Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.198475.r475951) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-31/v4#referee-response-475951 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Obermayer B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 26 Feb 2026 | for Version 3 Benedikt Obermayer , Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany 0 Views copyright © 2026 Obermayer B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline 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 This study re-analyzes selected timepoints from a comprehensive scRNAseq and scATACseq atlas of the developing human retina (Thomas et al., Dev Cell 2022, GSE183684), focusing on the NOTCH locus and potential chromatin remodeling during retina development. While I highly welcome re-analyses of publicly available datasets with different methods, different hypotheses or even just a somewhat narrow focus, I think this study does not properly acknowledge and engage with the prior work that produced that dataset. It doesn't even cite the original paper, and it also doesn't discuss any of the original findings. The motivation for that re-analysis is not clear - if they were interested in an exporatory hypothesis-generating analysis, couldn't they simply have used the extensive data provided in the supplement (including differentially accessible peaks) or the UCSC browser tracks ( http://genome.ucsc.edu/s/CherryLab/Nuclear_EyeBrowser_TrackHub) ? The authors come up with new cluster labels that don't clearly link to the cell types identified in the original paper, and because the different timepoints are analyzed separately instead of together, cluster labels are not consistent across developmental timepoints and comparison between them is much harder. While the original paper did not provide a ready-to-use Seurat object, they did publish their analysis code, so it should be possible to re-do their analysis (or simply ask them for their cell type annotation). Further, by now there is a more recent atlas of the developing human retina using (actual) multiOme libraries (https://www.nature.com/articles/s41467-024-50853-5), which does provide processed data that could be used for, e.g., cell type label transfer. Finally, I think it's hard to interpret accessibility changes in one specific locus without considering the wider genomic context. Are the observed changes comparable to those in other known regulators of retina development? are any of the CREs or enhancers identified by Thomas et al. or Zuo et al. re-identified in the current study? in summary: - the original paper should be cited and discussed - cell type labels should be harmonized across timepoints and with the original paper or retina data from Zuo et al. - changes in the NOTCH locus should be discussed in a genome-wide context Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly References 1. Zuo Z, Cheng X, Ferdous S, Shao J, et al.: Single cell dual-omic atlas of the human developing retina. Nature Communications . 2024; 15 (1). Publisher Full Text 2. Thomas E, Timms A, Giles S, Harkins-Perry S, et al.: Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids. Developmental Cell . 2022; 57 (6): 820-836.e6 Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Bioinformatics, Functional and Single-Cell Genomics 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. reply Respond to this report Responses (0) Obermayer B. Peer Review Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.190734.r460837) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-31/v3#referee-response-460837 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Tang Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 30 Sep 2025 | for Version 2 Zhongjie Tang , University of Southern California, Los Angeles, Southern California, USA 0 Views copyright © 2025 Tang Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline 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 The authors have made some revisions to clarify the NOTCH1–3 expression patterns, expand the ATAC-seq analysis window, and add discussion on the NOTCH2 mRNA–chromatin discrepancy. Figures and supplementary materials have been updated, which modestly improves the clarity of the study. Two points remain that could be addressed with minimal additional work: 1.Provide brief biological context for NOTCH3 A short discussion summarizing what is known—or explicitly noting what remains unknown—about NOTCH3 in retinal or neural development would help justify its inclusion beyond descriptive expression levels. 2.Quantify motif evidence The identification of LHX2/PAX6/RAX/VSX2 motifs in variable peaks near NOTCH1 is currently presented qualitatively. Including simple summary statistics, such as enrichment p-values or correlations between TF expression and accessibility, would provide basic quantitative support for these observations without expanding the study’s scope. Competing Interests No competing interests were disclosed. Reviewer Expertise Single-cell RNA-seq and ATAC-seq analysis, computational biology 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. reply Respond to this report Responses (1) Author Response 14 Nov 2025 Masaharu Yoshihara, College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Japan Dear Dr. Zhongjie Tang Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. The authors have made some revisions to clarify the NOTCH1–3 expression patterns, expand the ATAC-seq analysis window, and add discussion on the NOTCH2 mRNA–chromatin discrepancy. Figures and supplementary materials have been updated, which modestly improves the clarity of the study. Two points remain that could be addressed with minimal additional work: 1.Provide brief biological context for NOTCH3 A short discussion summarizing what is known—or explicitly noting what remains unknown—about NOTCH3 in retinal or neural development would help justify its inclusion beyond descriptive expression levels. Response: Thank you for your constructive comments. We agree with your comments and have included brief biological context for NOTCH3 in the Introduction section. NOTCH3 has been well documented in the endothelial cell and vascular development, as well as in a related disease (CADASIL) (references #26 and #27), and recent studies have added the involvement of NOTCH3 to the glial differentiation in retinal development (reference #28). The involvement of this gene in the neuronal cells, however, is still unclear. Since NOTCH3 has a unique biochemical property in ligand sensing and signal transduction (reference #25), revealing this gene expression and its regulation would add potential insights into the coordinated retinal neuronal cell lineages development. We have clarified this point by adding the following sentences in the Introduction section. (added sentences) Notch3 is well known for its involvement in the endothelial cells and vascular development as suggested by causing a cerebral vascular disease, CADASIL. 26, 27 In addition to involvement in endothelium development, this gene is also reported to be associated with differentiation of glial cells in zebrafish retinas. 28 However, it is unclear when and how these Notch receptor expressions are switched on and off in RPCs during early differentiation to impact on overall Notch signaling amount and oscillation. 2.Quantify motif evidence The identification of LHX2/PAX6/RAX/VSX2 motifs in variable peaks near NOTCH1 is currently presented qualitatively. Including simple summary statistics, such as enrichment p-values or correlations between TF expression and accessibility, would provide basic quantitative support for these observations without expanding the study’s scope. Response: Thank you for your insightful comments. We agree with your comments. To enhance quantitative aspects, we have added JASPAR motif search in the transcription factor binding motif enrichment analysis using the Signac pipeline, and observed that the motifs for LHX2, RAX and VSX2 were significantly enriched in the early RPCs while that for PAX6 showed relatively less enrichment. In addition, to support biological significance of these motif enrichments, we also carried out footprinting analysis and showed that actual bindings of these three transcription factors (LHX2, RAX and VSX2) in the early retinal progenitor cells. Unfortunately, we were technically unable to conduct the NOTCH1 locus-specific investigations nor calculating correlations between transcription factor expressions and their chromatin accessibility. Despite lack of direct evidence of transcription factor binding specifically in the NOTCH1 locus, we believe that the genome-wide quantitative analyses (JASPAR motif enrichment analysis and footprinting analysis) have enhanced the potential biological significance of chromatin remodeling in the early retinal progenitor cell differentiation. (added sentences in the Results section for day 59, for example) Since these regions contained multiple binding motifs for LHX2, PAX6, RAX and VSX2 ( Figure 2A, arrowheads numbered 1-3) (DNA sequences of each transcription factor binding motif and their frequencies in the NOTCH1 locus were presented in the Method section and “Supplementary_table_motif.xlsx”, respectively, with partial overlap with JASPAR motif database. The motifplot for the transcription factors was presented in “JASPAR_motif.png.”), chromatin remodeling of these regions possibly regulates NOTCH1 expression transition during RPC differentiation although the expression transitions of LHX2 , PAX6 , RAX and VSX2 may also support the NOTCH1 expression transition ( Figure 2A). DNA sequence motif enrichment analysis to the whole genomic regions revealed that these motifs were significantly enriched in the early RPCs (the fold changes for LHX2, RAX and VSX2 are 2.09, 2.30 and 2.26, respectively) although that for PAX6 showed relatively slight increase in fold enrichment (1.62) (“Motif_enrichment_d59.csv”). Moreover, transcription factor footprinting analysis suggested that LHX2, RAX and VSX2 bound to those motifs in the early RPCs while observing less PAX6 binding in those cells (Additional_file_1A, 1B, 1C and 1D). The updated R codes used in this revised manuscript and their result data are available in our GitHub repository ( https://doi.org/10.5281/zenodo.17461764 ) ( https://doi.org/10.5281/zenodo.17463838 ). Again, thank you so much for your constructive comments. We look forward to hearing from you soon. View more View less Competing Interests We have no competing interests to declare. reply Respond Report a concern Tang Z. Peer Review Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.188207.r416419) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-31/v2#referee-response-416419 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Tang Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Aug 2025 | for Version 1 Zhongjie Tang , University of Southern California, Los Angeles, Southern California, USA 0 Views copyright © 2025 Tang Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline 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 Evaluation This study presents a reanalysis of publicly available single-cell multi-omics datasets to explore the relationship between NOTCH1 expression dynamics and chromatin accessibility during early stages of human retinal progenitor cell (RPC) differentiation. The authors apply standard single-cell RNA-seq and ATAC-seq integration methods to a biologically relevant question related to Notch signaling in retinal development. While the technical execution appears sound, the analysis remains largely descriptive and would benefit from further depth. Several issues—particularly concerning data interpretation and figure clarity—should be addressed to improve the overall quality of the manuscript. Essential Revisions 1. Clarify the expression hierarchy of NOTCH1–3 The manuscript currently implies that NOTCH3 is expressed at levels comparable to NOTCH1 across developmental stages. However, Figures 1D, 2D, and 3D clearly show that NOTCH1 is the dominant isoform, while NOTCH3 is expressed at notably lower levels. The text should be revised to accurately reflect these patterns and avoid overgeneralization. 2. Discuss the discrepancy between NOTCH2 expression and chromatin accessibility Although NOTCH2 is transcriptionally active throughout all three stages, there is little to no signal at its locus in the ATAC-seq data. This discrepancy warrants further discussion. Potential explanations might include technical limitations of ATAC-seq, regulation via distal enhancers, or post-transcriptional mechanisms. 3. Expand chromatin accessibility analysis beyond the immediate gene body The current analysis is restricted to a narrow window around the NOTCH1 locus (Chr9:136510000–136520000). Expanding the analysis region upstream and downstream (e.g., ±50–100 kb) may help identify putative enhancers or other regulatory elements contributing to the observed expression dynamics. 4. Enhance figure annotation and clarity The figures, particularly the UMAP and coverage plots, would benefit from clearer labeling. Specifically: Annotate key functional regions such as promoters or candidate enhancers in the ATAC-seq plots. Ensure cluster labels are consistent across UMAPs and provide legends that identify RPC subtypes. A supplementary table listing marker genes and cell counts per cluster would improve clarity for readers. Recommended Revisions Provide biological context for NOTCH3 Although NOTCH3 is included in the gene expression analysis, the manuscript does not explain its potential role in retinal development. Including a brief discussion of existing literature—or acknowledging the lack thereof—would better justify its inclusion in the study. Connect findings with known transcriptional regulators The manuscript mentions transcription factors such as LHX2, PAX6, and RAX, which are known regulators of retinal development. Further exploring how these factors may influence NOTCH1 expression or chromatin accessibility—either through motif analysis or referencing prior enhancer studies—would strengthen the biological interpretation. Summary Recommendation Major revision — The study addresses a relevant topic and is based on a solid dataset, but the analysis remains limited and the interpretation lacks depth. With substantial improvements in analytical scope, biological context, and figure presentation, the manuscript could make a valuable contribution to the field. 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? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Single-cell RNA-seq and ATAC-seq analysis, computational biology 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. reply Respond to this report Responses (1) Author Response 10 Nov 2025 Masaharu Yoshihara, College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Japan Dear Dr. Zhongjie Tang Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments from Dr. Zhongjie Tang This study presents a reanalysis of publicly available single-cell multi-omics datasets to explore the relationship between NOTCH1 expression dynamics and chromatin accessibility during early stages of human retinal progenitor cell (RPC) differentiation. The authors apply standard single-cell RNA-seq and ATAC-seq integration methods to a biologically relevant question related to Notch signaling in retinal development. While the technical execution appears sound, the analysis remains largely descriptive and would benefit from further depth. Several issues—particularly concerning data interpretation and figure clarity—should be addressed to improve the overall quality of the manuscript. Essential Revisions (1) Clarify the expression hierarchy of NOTCH1–3 The manuscript currently implies that NOTCH3 is expressed at levels comparable to NOTCH1 across developmental stages. However, Figures 1D, 2D, and 3D clearly show that NOTCH1 is the dominant isoform, while NOTCH3 is expressed at notably lower levels. The text should be revised to accurately reflect these patterns and avoid overgeneralization. Response: Thank you for your constructive comments. We agree to your comments and have carefully revised the manuscript and ensured that NOTCH1 showed the highest expression, followed by NOTCH2 or NOTCH3 . In the revised manuscript (the Results section for day 59 sample), we have emphasized that, considering the literature (ref. 25), we focused on NOTCH1 and NOTCH3 . To avoid overgeneralization, we have carried out analyses to the four NOTCH genes and reached to the conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation. (2) Discuss the discrepancy between NOTCH2 expression and chromatin accessibility Although NOTCH2 is transcriptionally active throughout all three stages, there is little to no signal at its locus in the ATAC-seq data. This discrepancy warrants further discussion. Potential explanations might include technical limitations of ATAC-seq, regulation via distal enhancers, or post-transcriptional mechanisms. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #2. (3) Expand chromatin accessibility analysis beyond the immediate gene body The current analysis is restricted to a narrow window around the NOTCH1 locus (Chr9:136510000–136520000). Expanding the analysis region upstream and downstream (e.g., ±50–100 kb) may help identify putative enhancers or other regulatory elements contributing to the observed expression dynamics. Response: Thank you for your constructive comment. This point is also raised by Dr. Mariko Kashiwagi, and we have revised our manuscript as described in comment #1. (4) Enhance figure annotation and clarity The figures, particularly the UMAP and coverage plots, would benefit from clearer labeling. Specifically: Annotate key functional regions such as promoters or candidate enhancers in the ATAC-seq plots. Response: Thank you for your constructive comment. We agree to your comment and have clearly indicated the peak regions with arrowheads in the ATAC-seq plots in Figure 2, 4 and 6, along with Additional_file_1, 2 and 3. In addition, the exact chromosome regions of each peak are now indicated in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Ensure cluster labels are consistent across UMAPs and provide legends that identify RPC subtypes. Response: Thank you for your constructive comment. We agree to your comment and have corrected all the cluster labels to ensure the consistency among the samples and to be reader friendly. A supplementary table listing marker genes and cell counts per cluster would improve clarity for readers. Response: Thank you for your constructive comment. We agree to your comment and have now provided a supplementary table listing marker genes and cell counts per cluster as “Supplementary_table_count.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). Recommended Revisions (5) Provide biological context for NOTCH3 Although NOTCH3 is included in the gene expression analysis, the manuscript does not explain its potential role in retinal development. Including a brief discussion of existing literature—or acknowledging the lack thereof—would better justify its inclusion in the study. Response: Thank you for your constructive comment. We agree to your comment and have analyzed NOTCH3 as well as NOTCH1 since NOTCH3 has been suggested for retinal development (ref. 25). Therefore, we have added this reference paper in this revised manuscript. In addition, the biological significance of NOTCH3 is added in the Introduction section (“In addition to Notch1 , Notch3 expression has also been suggested in the early RPCs, which potentially affects the amount and oscillation of total Notch signaling via its susceptibility to the receptor cleavage and subsequent signal transduction). (6) Connect findings with known transcriptional regulators The manuscript mentions transcription factors such as LHX2, PAX6, and RAX, which are known regulators of retinal development. Further exploring how these factors may influence NOTCH1 expression or chromatin accessibility—either through motif analysis or referencing prior enhancer studies—would strengthen the biological interpretation. Response: Thank you for your constructive comment. We agree to your comment and have searched for binding motifs for those transcription factors in chromatin remodeling regions in the Notch loci. We have identified several binding motifs (indicated by arrowheads and numbers in Figure 2, 4 and 6 and Additional_file_1, 2 and 3, supporting the potential biological significance. The exact peak and binding motif information is provided in a supplementary file named “Supplementary_table_motif.xlsx” in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (7) Summary Recommendation Major revision — The study addresses a relevant topic and is based on a solid dataset, but the analysis remains limited and the interpretation lacks depth. With substantial improvements in analytical scope, biological context, and figure presentation, the manuscript could make a valuable contribution to the field. Response: We sincerely appreciate your thoughtful insights, all of which helped us improve the manuscript significantly. The R code used in this revised manuscript is available in our GitHub repository ( https://doi.org/10.5281/zenodo.17084033 ). Again, thank you so much for your constructive comments. We look forward to hearing from you. Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] View more View less Competing Interests We have no competing interests. reply Respond Report a concern Tang Z. Peer Review Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.175389.r398458) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-31/v1#referee-response-398458 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Kashiwagi M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 Feb 2025 | for Version 1 Mariko Kashiwagi , Massachusetts General Hospital, Charlestown, USA 0 Views copyright © 2025 Kashiwagi M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline 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 Remarks to the Author: In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1–4) were expressed in retinal progenitor cells (RPCs) and whether changes in chromatin accessibility at promoter regions contributed to their transcriptional activation. To address these questions, they reanalyzed publicly available single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq datasets of the human fetal retina across three developmental stages (GSE183684). Overall, the analytical pipelines are valid and appropriately implemented. However, several concerns regarding the experimental design and the interpretation of the results require further consideration. Major Concerns and Comments: 1. Chromatin Accessibility Analysis: During development, enhancer regions play a crucial role in dynamic, lineage-specific gene regulation by modulating chromatin structure and recruiting transcription factors. In contrast, promoter regions generally maintain a more stable configuration following cellular differentiation into a specific lineage. With this in mind, I recommend that the authors expand their chromatin accessibility analysis beyond the promoter and gene body to include the 5' upstream and 3' downstream regions. This broader approach may provide a more comprehensive understanding of how various regulatory elements, including enhancers, contribute to the transcriptional regulation of NOTCH genes. 2. Interpretation of the Results: The authors stated in the text that "NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes." However, this claim is not fully supported by the data (Fig. 1D, Additional File 1B; Fig. 2D, Additional File 2B; Fig. 3D, Additional File 3B). A comprehensive interpretation of the three developmental stages suggests that NOTCH1 was the predominant isoform expressed in early RPC clusters, although expression levels varied across clusters and stages. At day 59, the expression hierarchy was NOTCH2 > NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully. Additionally, although NOTCH2 expression was consistently observed across all three stages (days 59, 74, and 78), the corresponding ATAC peaks were either absent or very low. The manuscript did not provide any discussion of this discrepancy. The authors should address this issue to clarify the relationship between chromatin accessibility and NOTCH2 expression, while also discussing whether this discrepancy may stem from technical limitations or reflect underlying biological factors. 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? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Skin biology, Immunology, Gene 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. reply Respond to this report Responses (1) Author Response 10 Nov 2025 Masaharu Yoshihara, College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Japan Dear Dr. Mariko Kashiwagi, Thank you so much for your time and efforts in reviewing our manuscript titled “Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells”. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner. Comments: In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1–4) were expressed in retinal progenitor cells (RPCs) and whether changes in chromatin accessibility at promoter regions contributed to their transcriptional activation. To address these questions, they reanalyzed publicly available single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq datasets of the human fetal retina across three developmental stages (GSE183684). Overall, the analytical pipelines are valid and appropriately implemented. However, several concerns regarding the experimental design and the interpretation of the results require further consideration. Major Concerns and Comments: (1) Chromatin Accessibility Analysis: During development, enhancer regions play a crucial role in dynamic, lineage-specific gene regulation by modulating chromatin structure and recruiting transcription factors. In contrast, promoter regions generally maintain a more stable configuration following cellular differentiation into a specific lineage. With this in mind, I recommend that the authors expand their chromatin accessibility analysis beyond the promoter and gene body to include the 5' upstream and 3' downstream regions. This broader approach may provide a more comprehensive understanding of how various regulatory elements, including enhancers, contribute to the transcriptional regulation of NOTCH genes. Response: Thank you for your insightful comments. We agree to your comment and have expanded the chromatin accessibility analysis to include 100 kb upstream and downstream of the Notch genes, and we have identified additional chromatin remodeling regions. The results are presented in Figure 2, 4 and 6 ( NOTCH1 and NOTCH3 for the three samples) and Additional_file_1, 2 and 3 ( NOTCH2 and NOTCH4 for the three samples). We have drastically revised the text for each sample in the Results section and provided the exact chromosome regions of each peak in supplementary files (e.g., “ATAC_peaks_NOTCH1_d59.csv”) in our GitHub repository ( https://doi.org/10.5281/zenodo.17084024 ). (2) Interpretation of the Results: The authors stated in the text that "NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes." However, this claim is not fully supported by the data (Fig. 1D, Additional File 1B; Fig. 2D, Additional File 2B; Fig. 3D, Additional File 3B). A comprehensive interpretation of the three developmental stages suggests that NOTCH1 was the predominant isoform expressed in early RPC clusters, although expression levels varied across clusters and stages. At day 59, the expression hierarchy was NOTCH2 > NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully. Response: Thank you for your constructive comments. We agree to your comment and clearly have indicated that the early RPCs expressed NOTCH1 , NOTCH2 and NOTCH3 for each sample in the Results section. In particular, NOTCH1 showed the highest expressions throughout the three samples and we have clearly mentioned that we have focused in NOTCH1 and NOTCH3 in this study since NOTCH3 has been suggested for early RPC differentiation (ref. 25) in the Results section for day 59 sample. Since the early RPCs expressed NOTCH2 in all the three samples, we have also analyzed chromatin accessibility for NOTCH2 in addition to NOTCH1 and NOTCH3 although we are unable to provide solid conclusions regarding this gene owing to varying findings on its chromatin accessibility, which is clearly mentioned in the Discussion section. Our findings and conclusion that NOTCH1 mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation, however, are solid in front of the unexplained results on NOTCH2 . (3) Additionally, although NOTCH2 expression was consistently observed across all three stages (days 59, 74, and 78), the corresponding ATAC peaks were either absent or very low. The manuscript did not provide any discussion of this discrepancy. The authors should address this issue to clarify the relationship between chromatin accessibility and NOTCH2 expression, while also discussing whether this discrepancy may stem from technical limitations or reflect underlying biological factors. Response: Thank you for your constructive comments. We agree to your comment and have revised our manuscript by adding several possible mechanisms such as distal enhancers and a post-transcriptional mechanism (small non-coding regulatory RNAs) (ref. 38) as well as technical limitations in the Discussion section. Although we are unable to fully explain the discrepancy of NOTCH2 mRNA expression and chromatin accessibility, we are confident to the main conclusion regarding NOTCH1 . Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan [email protected] View more View less Competing Interests We have no competing interests. reply Respond Report a concern Kashiwagi M. Peer Review Report For: Expression status transition of NOTCH1 accompanies chromatin remodeling in human early retinal progenitor cells [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :31 ( https://doi.org/10.5256/f1000research.175389.r359501) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-31/v1#referee-response-359501 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. 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