Background

F1000Research

2046-1402

F1000 Research Limited

London, UK

10.12688/f1000research.159630.3

Brief Report

Articles

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

[version 3; peer review: 3 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 1 3 Yoshihara

Masaharu

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

14 11 2025

2025

5 11 2025

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. Compared to the knowledge on this signaling pathway in the differentiation of retinal ganglion cells, less is known about its involvement in earlier stages of retinal progenitor cell differentiation and its regulation, although NOTCH1, and probably NOTCH3, are involved in earlier stage 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 and NOTCH3 mRNA expressions 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 and NOTCH3 loci were accompanied by transitions in their mRNA expressions. Importantly, the NOTCH1 locus, which showed chromatin remodeling, contained multiple binding motifs for transcription factors important in early retinal progenitor cell differentiation. Since genome-wide investigation of transcription factor binding motifs showed enrichment of such motifs for the early retinal progenitor cell-associated transcription factors (LHX2, RAX and VSX2), the motifs in the NOTCH1 locus might be of biological significance rather than observed by chance. Indeed, footprinting analysis suggested actual binding of those transcription factors (LHX2, RAX and VSX2) and their motifs in the whole genome, strengthening potential involvement of these transcription factor activities in early retinal progenitor cell differentiation.

Conclusions

These results suggest that chromatin remodeling may be involved in the differential expression of NOTCH1, although another type of Notch mRNA expression regulations may also 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.

Revised Amendments from Version 2

We have made additional improvements as suggested by the reviewer. Concretely, we have elaborated the Introduction section to include the biological significance of NOTCH3investigation and additionally conducted motif enrichment and footprinting analyses to quantitatively examine involvement of the transcription factors specific to the early retinal progenitor cells (RPCs) (LHX2, PAX6, RAX and VSX2). The details of the changes in each section of the revised manuscript are described below. In Abstract: We have mentioned that the findings from motif enrichment supported by footprinting analysis suggested the involvement of LHX2, RAX and VSX2 in early retinal progenitor cell differentiation. In Introduction: We have included the biological context of NOTCH3 with emphasis on retinal development to justify its inclusion in the present study. In Methods: We have added JASPAR motif enrichment and footprinting analyses. In Results: For each dataset (day 59, 74 and 78), we have conducted the motif enrichment and footprinting analyses (e.g. “Motif_enrichment_d59.csv”, “JASPAR_motif.png” and “Additional_file_1.tif” for day 59 sample) In Discussion: We have added the results of JASPAR motif enrichment and footprinting analyses on a genome-wide scale (but not in a NOTCH1 locus-specific manner owing to technical limitations) and discussed their interpretation and limitation on the NOTCH1expression regulation in early RPCs. In summary, newly added quantitative analyses have collectively strengthened chromatin remodeling associated with LHX2, RAX and VSX2 during RPC differentiation although the NOTCH1 locus-specific investigation would further validate our conclusion.

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 for understanding the regulation of cell differentiation and subsequent tissue development.

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 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. ²⁵ 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. Here, we re-analyzed a public multi-omics dataset of single-cell RNA-seq and single-cell ATAC-seq from three human fetal retinas ²⁹ 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) ²⁹ 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 ³⁰ and Signac version 1.14.0 and 1.15.0 ³¹ 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 were conducted by using the “FindTransferAnchors” function of Monocle3 version 1.3.7 ³² and Signac, respectively. We searched the transcription factor binding motifs for LHX2 (TAATTA), ³³ PAX6 (CGCCTGA), ³⁴ ^, ³⁵ RAX ([C/T]AATTA) ³⁶ ^, ³⁷ and VSX2 (TAATT [A/G]) ³⁸ (“Supplementary_ table_motif.xlsx”) manually by referencing human genome assembly hg38 in the University of California Santa Cruz genome browser ( https://genome.ucsc.edu/). We also added DNA sequence motif enrichment analysis in Signac pipeline, using JASPAR motif database ( https://jaspar.elixir.no/). This database stores information of transcription factor binding motifs as position frequency matrices (PFMs), which consider biological significance of each base for possible transcription factor binding motifs in a quantitative manner. 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 14 clusters ( Figure 1A), which were further characterized by marker gene expressions ( Figure 1B). This sample primarily contained RPCs with various differentiation statuses except for PAX2-expressing glial cells. We defined LHX2, PAX6, RAX and VSX2 as early markers. We considered that early markers-expressing RPCs and MKI67-expressing RPCs constitute early RPCs. In addition, pseudotime analysis suggested that RPCs decreased expressions of LHX2, PAX6, RAX and VSX2 during differentiation into either ONECUT1/ 2-, ELAVL2/ 4- or EEF1A1-expressing RPCs ( Figure 1C). The details of each cluster are available in “Supplementary_table_count.xlsx” in our GitHub repository. Next, we examined Notch mRNA expressions and found that NOTCH1, NOTCH2 and NOTCH3 were expressed primarily in early RPCs with NOTCH1 showing the highest expression, which prompted us to focus on NOTCH1 and NOTCH3, ²⁵ whereas NOTCH4 expression was barely detectable ( Figure 1D). Then, we re-analyzed single-cell ATAC-seq data, which was mathematically integrated with its single-cell RNA-seq data by using the “FindTransferAnchors” function of Signac ( Figure 1E). In the early RPCs (Early markers-expressing RPC1- 3_d59 and MKI67-expressing RPC1- 2_d59), we observed multiple peaks in the 100 kb upstream and downstream chromosome regions of NOTCH1, some of which were diminished in the other RPC clusters such as ELAVL2/ 4- or EEF1A1-expressing RPCs ( Figure 2A, arrowheads). 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). Since NOTCH3 has also been suggested for early RPC differentiation, ²⁵ we also conducted chromatin accessibility analysis of NOTCH3 and observed less prominent chromatin remodeling in the 100 kb upstream and downstream regions spanning the NOTCH3 locus and the mRNA diminishment supports the previous report ²⁵ ( Figure 2B). The tables of the chromosome regions of each variable peak in NOTCH1 and NOTCH3 are provided in our GitHub repository as “ATAC_peaks_NOTCH1_d59.csv” and “ATAC_peaks_NOTCH3_d59.csv”, respectively. Although NOTCH2 and NOTCH4 have been less documented for RPC differentiation in the literature, we have provided the dot plots of the Notch expressions and the coverage plots of NOTCH2 and NOTCH4 in “Additional_file_2.tif” in our GitHub repository. In contrast to NOTCH2 mRNA expression in the early RPCs ( Figure 1D), we observed several peaks in the chromosome region spanning the NOTCH2 locus although it was difficult to point out the peak regions specific for the early RPCs (Additional_file_2C). Conversely, in contrast to little NOTCH4 mRNA expression ( Figure 1D), we found that multiple peaks were constantly open in the NOTCH4 locus during RPC differentiation (Additional_file_2D, arrowheads), some of which contained multiple binding motifs for LHX2, PAX6, RAX and VSX2 (Additional_file_2D, arrowheads numbered 1-2) (“Supplementary_table_motif.xlsx”). The table of the chromosome regions of each variable peak in NOTCH4 is provided in our GitHub repository as “ATAC_peaks_NOTCH4_d59.csv.”

Figure 1. Notch mRNA expression decrease in the day 59 sample.

UMAP analysis of the single-cell RNA-seq data identified 14 clusters. (B) Dot plot of marker genes. (C) Monocle3 pseudotime analysis for clarifying the differentiation status. (D) Feature plot of NOTCH1- 4. Note that NOTCH1, NOTCH2 and NOTCH3 expressions 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.

Figure 2. Concomitant chromatin remodeling of <italic toggle="yes">NOTCH1</italic> and <italic toggle="yes">NOTCH3</italic> in the day 59 sample.

The coverage plot of the 100 kb upstream and downstream chromosome regions of NOTCH1. The upstream of the genes is on the right. Variable peak regions are indicated by arrowheads, with the regions containing transcription binding motifs numbered as 1-3. (B) The coverage plot of the 100 kb upstream and downstream chromosome regions of NOTCH3. The upstream of the genes is on the right. A variable peak region which contains transcription binding motifs is indicated by an arrowhead numbered as 1.

Re-analysis day 74 human fetal retina

To validate the changes in Notch mRNA expressions and chromatin accessibility in the Notch loci during RPC differentiation, we investigated another early gestational stage sample (day 74). UMAP analysis identified 15 clusters ( Figure 3A) that were further characterized by marker gene expressions ( Figure 3B). This sample contained RPCs with various differentiation statuses. Similar to day 59 sample, pseudotime analysis suggested that the early RPCs differentiated into either ELAVL2/ 4, ONECUT1/ MEIS2 or ONECUT1/2-expressing RPCs ( Figure 3C). ONECUT1/ MEIS2-expressing RPCs differentiated into RPCs that markedly expressed VSX2. Next, we examined Notch mRNA expressions and found that NOTCH1, NOTCH2 and NOTCH3 were expressed primarily in early RPCs with NOTCH1 showing the highest expression whereas NOTCH4 expression was barely detectable ( Figure 3D), similar to the day 59 sample. We then re-analyzed the single-cell ATAC-seq data, which were integrated with the single-cell RNA-seq data ( Figure 3E). In the early RPCs (Early markers-expressing RPC1- 4_d74 and MKI67-expressing RPC_d74), we observed multiple peaks in the 100 kb upstream and downstream chromosome regions of NOTCH1, some of which were diminished in the other RPC clusters, such as ELAVL2/ 4- or ONECUT1/2-expressing RPCs ( Figure 4A, arrowheads). Since these regions contained multiple binding motifs for LHX2, PAX6, RAX and VSX2 ( Figure 4A, arrowheads numbered 1-4) (The frequencies of each transcription factor binding motif in the NOTCH1 locus was presented in “Supplementary_table_motif.xlsx”), 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 4A). 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 2.21, 2.14 and 2.10, respectively) although that for PAX6 showed relatively slight increase in fold enrichment (1.55) (“Motif_enrichment_d74.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_3A, 3B, 3C and 3D). Since NOTCH3 has also been suggested for early RPC differentiation, ²⁵ we also conducted chromatin accessibility analysis of NOTCH3 and observed less prominent chromatin remodeling in the 100 kb upstream and downstream regions spanning the NOTCH3 locus ( Figure 4B). The tables of the chromosome regions of each variable peak in NOTCH1 and NOTCH3 are provided in our GitHub repository as “ATAC_peaks_NOTCH1_d74.csv” and “ATAC_peaks_NOTCH3_d74.csv”, respectively. In summary, similar to the day 59 sample, in the day 74 sample, a concomitant mRNA decrease and chromatin remodeling in the NOTCH1 locus were observed. Although NOTCH2 and NOTCH4 have been less documented for RPC differentiation in the literature, we have provided the dot plots of the Notch expressions and the coverage plots of NOTCH2 and NOTCH4 in “Additional_file_4.tif” in our GitHub repository. In contrast to NOTCH2 mRNA expression ( Figure 4D), we observed a single peak in the NOTCH2 locus for EEF1A1-expressing RPC_d74, and therefore, no peak regions in the NOTCH2 locus specific for the early RPCs (Additional_file_4C). Conversely, in contrast to little NOTCH4 mRNA expression ( Figure 4D), we found that multiple peaks were constantly open in the NOTCH4 locus during RPC differentiation (Additional_file_4D, arrowheads), some of which contained multiple binding motifs for LHX2, PAX6, RAX and VSX2 (Additional_file_4D, arrowheads numbered 1-3) (“Supplementary_table_motif.xlsx”). The table of the chromosome regions of each variable peak in NOTCH4 is provided in our GitHub repository as “ATAC_peaks_NOTCH4_d74.csv.”

Figure 3. Notch mRNA expression decrease in the day 74 sample.

(A) UMAP analysis of the single-cell RNA-seq data identified 15 clusters. (B) Dot plot of marker genes. (C) Monocle3 pseudotime analysis. (D) Feature plot of NOTCH1- 4. Note that NOTCH1, NOTCH2 and NOTCH3 expressions 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.

Figure 4. Concomitant chromatin remodeling of <italic toggle="yes">NOTCH1</italic> and <italic toggle="yes">NOTCH3</italic> in the day 74 sample.

(A) The coverage plot of the 100 kb upstream and downstream chromosome regions of NOTCH1. The upstream of the gene is on the right. Variable peak regions are indicated by arrowheads, with the regions containing transcription binding motifs numbered as 1-4. (B) The coverage plot of the 100 kb upstream and downstream chromosome regions of NOTCH3. The upstream of the gene is on the right. Variable peak regions are indicated by arrowheads, with the regions containing transcription binding motifs numbered as 1-2.

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 added one more early gestational stage sample (day 78). UMAP analysis identified 18 clusters ( Figure 5A) that were further characterized by marker gene expressions ( Figure 5B). The sample contained RPCs with various differentiation statuses. Pseudotime analysis suggested that the early RPCs differentiated into either ONECUT1/ 2-expressing RPCs, which later differentiated into PTF1A and LHX1-expressing RPCs, ONECUT1/ MEIS2-expressing RPCs, or ELAVL2/4-expressing RPCs ( Figure 5C). Next, we examined Notch mRNA expressions and found that NOTCH1, NOTCH2 and NOTCH3 were expressed primarily in early RPCs with NOTCH1 showing the highest expression whereas NOTCH4 expression was barely detectable ( Figure 5D), similar to the day 59 and 74 samples. We then re-analyzed the single-cell ATAC-seq data, which was integrated with the single-cell RNA-seq data ( Figure 5E). In the early RPCs (Early markers-expressing RPC1-3_d78 and MKI67-expressing RPC1-2_78), we observed multiple peaks in the 100 kb upstream and downstream chromosome regions of NOTCH1, some of which were diminished in the other RPC clusters ( Figure 6A, arrowheads). Since these regions contained multiple binding motifs for LHX2, PAX6, RAX and VSX2 ( Figure 6A, arrowheads numbered 1-4) (The frequencies of each transcription factor binding motif in the NOTCH1 locus was presented in “Supplementary_table_motif.xlsx”), 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 6A). 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 2.47, 2.30 and 2.47, respectively) although that for PAX6 showed relatively slight increase in fold enrichment (1.67) (“Motif_enrichment_d78.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_5A, 5B, 5C and 5D). Since NOTCH3 has also been suggested for early RPC differentiation, ²⁵ we also conducted chromatin accessibility analysis of NOTCH3 and observed less prominent chromatin remodeling in the 100 kb upstream and downstream regions spanning the NOTCH3 locus ( Figure 6B). The tables of the chromosome regions of each variable peak in NOTCH1 and NOTCH3 are provided in our GitHub repository as “ATAC_peaks_NOTCH1_d78.csv” and “ATAC_peaks_NOTCH3_d78.csv”, respectively. In summary, examinations of all the three independent samples suggested that NOTCH1 mRNA expression decreased as RPC differentiation progresses, which was concomitant with chromatin remodeling in the NOTCH1 locus. Importantly, chromatin remodeling regions contained multiple transcription factor binding motifs. Although NOTCH2 and NOTCH4 have been less documented for RPC differentiation in the literature, we have provided the dot plots of the Notch expressions and the coverage plots of NOTCH2 and NOTCH4 in “Additional_file_6.tif” in our GitHub repository. In contrast to NOTCH2 mRNA expression ( Figure 5D), we observed several peaks in the chromosome regions spanning the NOTCH2 locus although it was difficult to point out peak regions specific for the early RPCs (Additional_file_6C). Conversely, in contrast to little NOTCH4 mRNA expression ( Figure 5D), we found that multiple peaks were constantly open in the NOTCH4 locus during RPC differentiation (Additional_file_6D, arrowheads), some of which contained multiple binding motifs for LHX2, PAX6, RAX and VSX2 (Additional_file_6D, arrowheads numbered 1-3) (“Supplementary_table_motif.xlsx”). The table of the chromosome regions of each variable peak in NOTCH4 is provided in our GitHub repository as “ATAC_peaks_NOTCH4_d78.csv.”

Figure 5. Notch mRNA expression decrease in the day 78 sample.

(A) UMAP analysis of the single-cell RNA-seq data identified 18 clusters. (B) Dot plot of marker genes. (C) Monocle3 pseudotime analysis. (D) Feature plot of NOTCH1- 4. Note that NOTCH1, NOTCH2 and NOTCH3 expressions 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.

Figure 6. Concomitant chromatin remodeling of <italic toggle="yes">NOTCH1</italic> and <italic toggle="yes">NOTCH3</italic> in the day 78 sample.

(A) The coverage plot of the 100 kb upstream and downstream chromosome regions of NOTCH1. The upstream of the gene is on the right. Variable peak regions are indicated by arrowheads, with the regions containing transcription binding motifs numbered as 1-4. (B) The coverage plot of the 100 kb upstream and downstream chromosome regions of NOTCH3. The upstream of the gene is on the right. Variable peak regions are indicated by arrowheads, with the regions with transcription binding motifs are numbered as 1-2.

Discussion

The involvement of Notch signaling in cell fate choices is well documented, including in Drosophila neurogenesis ³⁹ and mammalian biliary development. ⁴⁰ 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 the NOTCH1 locus, which contains multiple binding motifs for transcription factors, concomitant with changes in its mRNA expression during RPC differentiation. On genome-wide scale, we also found that the binding motifs for LHX2, RAX and VSX2 were significantly enriched in the early RPCs, and that the footprinting analysis supported actual bindings of these transcription factors to those motifs. These findings supported global interactions between the transcription factors (LHX2, RAX and VSX2) and their binding motifs, suggesting that the early RPC differentiation accompanied chromatin remodeling in association with multiple transcription factor bindings although the NOTCH1 locus-specific investigation was not achieved owing to technical limitations. In summary, the present study highlights that the fine-tuned regulation of Notch receptor expressions occurs at an epigenetic level.

NOTCH1 and NOTCH3 were well documented in the early RPC differentiation compared to NOTCH2 and NOTCH4. Indeed, we observed NOTCH1 and NOTCH3 mRNA expression in contrast to little NOTCH4 mRNA expression throughout the three samples. We observed comparable NOTCH2 mRNA expression in the early RPCs although we are unable to fully explain the discrepancy between mRNA expression and chromatin accessibility regarding this gene owing to lack of significant peak regions in the NOTCH2 locus (Additional_file_1C, 2C and 3C). Some possible causes such as regulation via distal enhancer and technical limitations of ATAC-seq may exist. When NOTCH2 mRNA is transcribed in support with those mechanisms, the decrease of NOTCH2 mRNA expression during RPC differentiation may be influenced by a post-transcriptional mechanism since a small non-coding regulatory RNA (CAT1) reportedly promotes stabilization of Notch2 mRNA. ⁴¹ The roles and regulations of NOTCH2 are, however, beyond the scope of this study. Conversely, we observed little NOTCH4 mRNA expression and its high chromatin accessibility with multiple binding motifs for LHX2, PAX6, RAX and VSX2. This observation might be owing to the absence of other transcription factors for NOTCH4 mRNA expression since this gene expression has been suggested to highly depend on the cell type and its expressing transcription factors. For example, Notch4 is limitedly expressed in endothelial cells, compared to Notch1, under regulation of AP-1 transcription factor. ⁴² Although we are unable to provide solid conclusions on the expression and regulation of NOTCH2 and NOTCH4 in the early RPCs, we believe that this does not impede our findings that NOTCH1 mRNA expression transition during RPC differentiation accompanied chromatin remodeling.

An ophthalmological study revealed that the epigenetic landscape of cell type-specific enhancers shifted during differentiation of RPCs. ⁴³ 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) ²⁹ 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/eyeATAC_supplementary_data/tree/main. ⁴⁴

Archived software available from: https://doi.org/10.5281/zenodo.17463838.

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: •

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JASPAR_motif.png

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ATAC_peaks_NOTCH1_d59.csv

•

ATAC_peaks_NOTCH3_d59.csv

•

ATAC_peaks_NOTCH4_d59.csv

•

ATAC_peaks_NOTCH1_d74.csv

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ATAC_peaks_NOTCH3_d74.csv

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ATAC_peaks_NOTCH4_d74.csv

•

ATAC_peaks_NOTCH1_d78.csv

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ATAC_peaks_NOTCH3_d78.csv

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ATAC_peaks_NOTCH4_d78.csv

•

Motif_enrichment_d59.csv

•

Motif_enrichment_d74.csv

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Motif_enrichment_d78.csv

Software availability

Source code available from: https://github.com/Yoshitokky/eyeATAC_software/tree/main. ⁴⁵

Archived software available from: https://doi.org/10.5281/zenodo.17461764.

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: •

251016_retina_chromatin_d59_No0.R (R code script for d59 data followed by No1).

•

251016_retina_chromatin_d59_No1.R (R code script for d59 data followed by No2).

•

251016_retina_chromatin_d59_No2.R (R code script for d59 data followed by No3).

•

251016_retina_chromatin_d59_No3.R (R code script for d59 data followed by No4).

•

251016_retina_chromatin_d59_No4.R (R code script for d59 data followed by No5).

•

251016_retina_chromatin_d59_No5.R.

•

251016_retina_chromatin_d74_No0.R (R code script for d74 data followed by No1).

•

251016_retina_chromatin_d74_No1.R (R code script for d74 data followed by No2).

•

251016_retina_chromatin_d74_No2.R (R code script for d74 data followed by No3).

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251016_retina_chromatin_d74_No3.R (R code script for d74 data followed by No4).

•

251016_retina_chromatin_d74_No4.R (R code script for d74 data followed by No5).

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251016_retina_chromatin_d74_No5.R.

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251016_retina_chromatin_d78_No0.R (R code script for d78 data followed by No1).

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251016_retina_chromatin_d78_No1.R (R code script for d78 data followed by No2).

•

251016_retina_chromatin_d78_No2.R (R code script for d78 data followed by No3).

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251016_retina_chromatin_d78_No3.R (R code script for d78 data followed by No4).

•

251016_retina_chromatin_d78_No4.R (R code script for d78 data followed by No5).

•

251016_retina_chromatin_d78_No5.R.

Acknowledgements

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

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Reviewer response for version 3

Obermayer

Benedikt

1 Referee https://orcid.org/0000-0002-9116-630X 1Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany

Competing interests: No competing interests were disclosed.

26 2 2026

2026

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

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?

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

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:

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.

References 1

: Single cell dual-omic atlas of the human developing retina. Nature Communications .2024;15(1) : 10.1038/s41467-024-50853-5

10.1038/s41467-024-50853-5

: Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids. Developmental Cell .2022;57(6) : 10.1016/j.devcel.2022.02.018 820-836.e6

10.1016/j.devcel.2022.02.018

10.5256/f1000research.188207.r416419

Reviewer response for version 2

Tang

Zhongjie

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

Competing interests: No competing interests were disclosed.

30 9 2025

2025

recommendation

approve-with-reservations

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.

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

Yoshihara

Masaharu

University of Tsukuba, Tsukuba, Ibaraki Prefecture, Japan

Competing interests: We have no competing interests to declare.

1 11 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.

Two points remain that could be addressed with minimal additional work:

1.Provide brief biological context for NOTCH3

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. ²⁸However, 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

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.

10.5256/f1000research.175389.r398458

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

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

Yoshihara

Masaharu

University of Tsukuba, Tsukuba, Ibaraki Prefecture, Japan

Competing interests: We have no competing interests.

16 9 2025

Dear Dr. Zhongjie Tang

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