Background

F1000Research

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F1000 Research Limited

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10.12688/f1000research.159630.1

Brief Report

Articles

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

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

Watabe

Yoshitoku

Data Curation Formal Analysis Software Visualization Writing – Original Draft Preparation https://orcid.org/0009-0007-4611-8193 1 2 Kobayashi

Sakurako

Writing – Original Draft Preparation https://orcid.org/0009-0001-1242-2097 1 2 Nakayama

Takahiro

Writing – Original Draft Preparation https://orcid.org/0000-0002-4023-4560 1 2 Takahashi

Satoru

Supervision Writing – Review & Editing https://orcid.org/0000-0002-8540-7760 2 3 Yoshihara

Masaharu

Conceptualization Formal Analysis Funding Acquisition Methodology Project Administration Software Writing – Original Draft Preparation https://orcid.org/0000-0002-0212-0909 a 2 4 1Department of Anatomy and Embryology, Institute of Medicine, University of Tsukuba, Tsukuba, Japan 2College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, Tsukuba, Ibaraki Prefecture, Japan 3Transborder Medical Center, Institute of Medicine, University of Tsukuba, Tsukuba, Japan 4Department of Primary Care and Medical Education, Institute of Medicine, University of Tsukuba, Tsukuba, Japan

a yoshihara.masahar.ly@alumni.tsukuba.ac.jp

No competing interests were disclosed.

6 1 2025

2025

20 12 2024

2025

This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

differentiation epigenetics eye development single-cell ATAC-seq single-cell RNA-seq

Japan Society for the Promotion of Science

JP23K14429

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.

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 ( Elavl2/4), 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. <italic toggle="yes">NOTCH1</italic> 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. <italic toggle="yes">NOTCH1</italic> 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. <italic toggle="yes">NOTCH1</italic> 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).

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Additional_file_2.jpg (Extended data for d74 data).

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Additional_file_3.jpg (Extended data for d78 data).

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Figure 1A. png

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Figure 1B. png

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Figure 1C. png

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Figure 1D_NOTCH1.png

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Figure 1D_NOTCH2.png

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Figure 1D_NOTCH3.png

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Figure 1D_NOTCH4.png

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Figure 1E. png

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Figure 1F. png

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Figure 1G. png

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Figure 2A. png

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Figure 2B. png

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Figure 2C. png

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Figure 2D_NOTCH1.png

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Figure 2D_NOTCH2.png

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Figure 2D_NOTCH3.png

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Figure 2D_NOTCH4.png

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Figure 2E. png

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Figure 2F. png

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Figure 2G. png

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Figure 3A. png

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Figure 3B. png

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Figure 3C. png

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Figure 3D_NOTCH1.png

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Figure 3D_NOTCH2.png

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Figure 3D_NOTCH3.png

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Figure 3D_NOTCH4.png

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Figure 3E. png

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Figure 3F. png

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Figure 3G. png

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Additional_file_1_A.png

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Additional_file_1_B.png

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Additional_file_1_C.png

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Additional_file_1_D.png

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Additional_file_2_A.png

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Additional_file_2_B.png

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Additional_file_2_C.png

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Additional_file_2_D.png

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Additional_file_3_A.png

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Additional_file_3_B.png

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Additional_file_3_C.png

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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).

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241108_retina_chromatin_d59_No1.R (R code script for d59 data followed by No2).

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241108_retina_chromatin_d59_No2.R (R code script for d59 data followed by No3).

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241108_retina_chromatin_d59_No3.R (R code script for d59 data followed by No4).

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241108_retina_chromatin_d59_No4.R (R code script for d59 data).

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241108_retina_chromatin_d74_No0.R (R code script for d74 data followed by No1).

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241108_retina_chromatin_d74_No1.R (R code script for d74 data followed by No2).

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241108_retina_chromatin_d74_No2.R (R code script for d74 data followed by No3).

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241108_retina_chromatin_d74_No3.R (R code script for d74 data followed by No4).

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241108_retina_chromatin_d74_No4.R (R code script for d74 data).

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241108_retina_chromatin_d78_No0.R (R code script for d78 data followed by No1).

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241108_retina_chromatin_d78_No1.R (R code script for d78 data followed by No2).

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241108_retina_chromatin_d78_No2.R (R code script for d78 data followed by No3).

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241108_retina_chromatin_d78_No3.R (R code script for d78 data followed by No4).

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241108_retina_chromatin_d78_No4.R (R code script for d78 data).

Acknowledgements

The authors thank Editage ( https://www.editage.jp) for their support with English language editing.

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Reviewer response for version 1

Tang

Zhongjie

1 Referee 1University of Southern California, Los Angeles, Southern California, USA

Competing interests: No competing interests were disclosed.

22 8 2025

2025

This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

recommendation

approve-with-reservations

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

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

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

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.

Yoshihara

Masaharu

University of Tsukuba, Tsukuba, Ibaraki Prefecture, Japan

Competing interests: We have no competing interests.

16 9 2025

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

Essential Revisions

(1) Clarify the expression hierarchy of NOTCH1–3

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

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

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

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

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

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

yoshihara.masahar.ly@alumni.tsukuba.ac.jp

10.5256/f1000research.175389.r359501

Reviewer response for version 1

Kashiwagi

Mariko

1 Referee https://orcid.org/0000-0001-9625-9372 1Massachusetts General Hospital, Charlestown, USA

Competing interests: No competing interests were disclosed.

5 2 2025

2025

recommendation

approve-with-reservations

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

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

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Skin biology, Immunology, Gene regulation

Yoshihara

Masaharu

University of Tsukuba, Tsukuba, Ibaraki Prefecture, Japan

Competing interests: We have no competing interests.

16 9 2025

Dear Dr. Mariko Kashiwagi,

Comments:

Major Concerns and Comments:

(1) Chromatin Accessibility Analysis:

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:

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 NOTCH3has 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 NOTCH3although 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

yoshihara.masahar.ly@alumni.tsukuba.ac.jp