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

Yoshitoku Watabe; Sakurako Kobayashi; Takahiro Nakayama; Satoru Takahashi; Masaharu Yoshihara

doi:10.12688/f1000research.159630.5

. 2026 Aug 21;14:31. Originally published 2025 Jan 6. [Version 5] doi: 10.12688/f1000research.159630.5

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

Yoshitoku Watabe ^1,², Sakurako Kobayashi ^1,², Takahiro Nakayama ^1,², Satoru Takahashi ^1,³, Masaharu Yoshihara ^1,^4,^a

PMCID: PMC13637476 PMID: 42839969

Version Changes

Revised. Amendments from Version 4

Thank you so much for reading our paper. We have made significant improvements as suggested by the reviewers. We have made significant revisions, especially in the annotation of the single-cell RNA/ATAC-seq data (GSE183684) by Thomas et al., using already annotated single-cell RNA-seq data by Kriukov et al. Screening of the four Notch receptor genes in the two single-cell RNA-seq datasets (the reference data by Kriukov et al. and the query data by Thomas et al.) showed reproducible, prominent NOTCH1 expression in the undifferentiated cells (Retinal progenitors and Transitory cell clusters), prompting us to focus on NOTCH1. In addition, the new annotation strategy led to the identification of a RAX binding site in the dynamically remodeled region in the NOTCH1 locus. Through this revision, we believe the manuscript is more concise and provides a possible example of chromatin-level regulation of Notch receptor genes.

Abstract

Background

Notch signaling, especially via the Notch1 receptor, is involved in normal eye development by maintaining retinal progenitor cell state before their differentiation, although the regulatory mechanism of fine-tuned Notch1 expression is unclear.

Methods

We re-analyzed publicly available human fetal retina single-cell RNA/ATAC-seq data at days 59, 74, and 78, using Seurat/Signac pipelines.

Results

We observed NOTCH1 expression in retinal progenitor cells, which diminished at later stages of differentiation. Integration of single-cell RNA-seq and ATAC-seq revealed a decrease in chromatin accessibility at the NOTCH1 locus, accompanied by a decrease in its mRNA expression during differentiation into bipolar cells, horizontal cells, or retinal ganglion cells. Importantly, those chromatin remodeling regions contained the binding motifs of the RAX transcription factor, which is essential for retinal progenitor cells, Muller glia, and cones. This result suggested that the regulation of NOTCH1 expression might be achieved by chromatin remodeling at the NOTCH1 locus, although single-cell RNA-seq analysis suggested that RAX expression regulation might also contribute to NOTCH1 expression.

Conclusions

By analyzing retinal cell differentiation, this study identified a putative RAX binding site in the NOTCH1 locus and raised the possibility that NOTCH1 expression could be regulated by chromatin remodeling.

Keywords: differentiation, epigenetics, eye development, single-cell ATAC-seq, single-cell RNA-seq

Introduction

Signal transduction via the Notch signaling pathway depends on the expression of receptors regulated at multiple levels. In addition to intracellular machineries such as glycosylation, ¹ ubiquitination, ² and phosphorylation, ³ these regulations include chromatin remodeling and DNA-binding proteins, such as transcription factors and transcriptional repressors, which could regulate the levels of Notch ligands and receptors. Since 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.

The retina is a good model for investigating cellular differentiation since cell fate determination is well documented. The developed retina is composed of multiple cell types, including retinal ganglion cells, amacrine cells, bipolar cells, horizontal cells, rods, cones, and glial cells (Muller glia), all of which originate from retinal progenitor cells (retinal progenitors). In addition, astrocytes and microglia exist during retinal development. Recently, a detailed molecular characterization at single-cell resolution of the developing retina was reported. ⁴ In addition, retinal progenitors are also known to express several transcription factors such as Lhx2, ⁵ Pax6, ⁶ Rax, ⁷ and Vsx2. ⁸

Notch signaling involves four Notch receptors that are expressed differently in space. In rat embryonic eyes, Notch1 was expressed on the posterior side of the retina at embryonic days (E) 13.5 and E14.5; Notch2 was expressed in the retinal pigment epithelium throughout E12.5 and E14.5; and Notch3 was expressed throughout the retina from E12.5 to E14.5, with strong expression on the anterior side at E14.5. ⁹ ^, ¹⁰ Further analyses on Notch2 using a knock-in mouse suggested that Notch2 was expressed at a slightly detectable level from the E9.5 optic vesicle stage in the retinal neuroepithelium that contained retinal progenitors. ¹¹ In addition, deletion of Notch2 in outer ciliary epithelium disrupted ciliary body formation, ¹² suggesting that this gene acts in the temporal side of the eye. Indeed, the spatial information in the mouse retina at E13.5 determined upstream regulators involved in Hes1 induction, which acts as a hub for Notch-dependent and -independent inputs in retinal progenitors. ¹³ Notably, Notch4 was expressed in arterial endothelial cells rather than retinal progenitors and their descendants in the retina. ¹⁴

Notch signaling is also involved in the complex differentiation of retinal progenitors into differentiated cells. Loss-of-function of Notch common co-actors Rbpj ¹⁵ and Maml ¹⁶ increased Crx-expressing photoreceptor precursor cells and cone/horizontal cell-restricted retinal progenitors, respectively. More specifically, Notch1 has been shown to inhibit the rod fate and maintain pluripotency of retinal progenitors. ¹⁷ ⁻ ¹⁹ This notion is also supported by a single-cell ATAC-seq and disease-associated 5q14.3 enhancer knockout study. ²⁰ In addition, Notch3, as well as Notch1, was shown to be expressed as early as the optic vesicle stage. ²¹ Indeed, Notch3 knockout mice showed a reduced number of retinal ganglion cells, ²² although zebrafish Notch3 was also essential for Muller glial cells. ²³ These results suggest that Notch1 and Notch3 act in retinal progenitors to regulate their differentiation, although it is unclear how retinal progenitor-dominant expressions of Notch1 and Notch3 are regulated during retinal progenitor differentiation.

A murine ophthalmological study examined global epigenetic changes and interacting transcription factors during retinal cell differentiation. ²⁴ By chromVAR analysis in the Signac pipeline and CUT&Tag analysis, this study revealed that the epigenetic landscape of cell type-specific enhancers shifted during retinal cell differentiation and identified several transcription factors (e.g., Otx2, Atoh7, Pou4f2, and Isl1) that potentially work in each differentiation stage. ²⁴ ChromVAR analysis identified five Atoh7-binding motifs in the Notch1 locus that were specific to naïve retinal progenitors, although CUT&Tag analysis did not result in positive findings for any of these motifs. In contrast, this report also identified chromatin remodeling at retinal progenitor-specific enhancers for key transcription factors, including Vsx2, and showed binding of transcription factors and their binding sites for key transcription factors, including Rax, Lhx2, and Pax6, in naïve and transitional retinal progenitors, although specific genomic regions for these transcription factors are still lacking, as chromVAR was used. Another study also reported chromatin remodeling in the NOTCH1 locus in humans, ²⁰ although a comprehensive analysis focusing on possible epigenetic regulation of the four Notch receptor gene expressions in humans, including the cis-regulatory elements, is still lacking, as this study focused on retinal cell differentiation instead of Notch signaling.

The Notch loci are subject to epigenetic regulation under both normal ²⁵ ^– ³⁰ and pathological ³¹ ^– ³⁹ conditions in a context-dependent manner. Although a single-cell RNA/ATAC-seq dataset from developing retinas, in which differentiation trajectories were well characterized, is available, and the accessibility of the NOTCH1 locus is known to decrease over differentiation, ²⁰ how this chromatin remodeling affects NOTCH1 expression is still unknown. To gain insight into chromatin remodeling and possible cis-regulatory elements responsible for regulation of the Notch receptor gene expression, we re-analyzed a publicly available multi-omics dataset of single-cell RNA-seq and single-cell ATAC-seq of human fetal retinas. ²⁰

Methods

To perform fine-grained annotation of the single-cell RNA-seq and ATAC-seq data, we downloaded an h5ad file, ⁴ which includes cell annotations from detailed molecular characteristics analysis of embryonic retinal cells from the CZ CELLxGENE Discover platform ( https://cellxgene.cziscience.com/collections/c11009b8-b113-4a99-9890-78b2f9df9d79). The h5ad file was converted into an RDS file and then analyzed in the Seurat pipeline. ⁴⁰

A single-cell RNA/ATAC-seq dataset (GSE183684) ²⁰ was downloaded from the Gene Expression Omnibus ( https://www.ncbi.nlm.nih.gov/geo/). Data from the earliest three days (day 59, 74, and 78) in this dataset were used to balance computational burden and the number of retinal progenitors. The data were processed in the pipelines using Seurat and Signac. ⁴¹ Detailed session information for each step is provided in the archived code (See Data availability section). Integration of the single-cell RNA-seq data and the single-cell ATAC-seq data was conducted using the “FindTransferAnchors” function of Signac. We used the FIMO tool ⁴² in the MEME Suite, ⁴³ referencing the JASPAR motif database ( https://jaspar.elixir.no/) and human genome assembly hg38 in the University of California, Santa Cruz genome browser ( https://genome.ucsc.edu/) to search transcription factor binding motifs.

Results

We started by analyzing the single-cell RNA-seq data ⁴ that we will use for annotation in the other single-cell RNA/ATAC-seq data. ²⁰ Uniform manifold approximation and projection (UMAP) analysis showed complexly ramified cell clusters ( Figure 1A), and in concordance with the original report, ⁴ we identified SOX2 and MKI67-double-positive Retinal progenitors ( Figure 1B-C). The cell number in each cluster is available in Additional Table 1. We performed dot plot analysis of the marker genes ( SOX2 and MKI67 for Retinal progenitors; RLBP1 for Muller glia; PAX6 for Retinal ganglion cell/Amacrine cell/Horizontal cell fate; RCVRN, OTX2, PDE6H, NRL, and RHO for Rods and Cones; VSX2 for Bipolar cells; TFAP2A, TFAP2B for Amacrine cells; ONECUT2 for Horizontal cells; POU4F2 and RBPMS for Retinal ganglion cell) according to the original report ⁴ to provide the average expression levels and proportion of these marker-expressing cells to guarantee the validity of annotation ( Figure 1D). Importantly, some of the markers were expressed in multiple cell clusters. We then screened the four Notch receptor genes and found that NOTCH1 was expressed in the Retinal progenitors (log2 fold change to background: 1.09, adjusted p-value: 0), Transitory (log2 fold change to background: 1.32, adjusted p-value: 0), and Muller glia (log2 fold change to background: 1.43, adjusted p-value: 0) cell clusters. The other Notch receptor genes were also expressed in the Retinal progenitors, although the expression was sparse, prompting us to focus on NOTCH1. We also examined the expression of RAX, PAX6, LHX2, and VSX2. RAX was enriched in the Retinal progenitors (log2 fold change to background: 0.62, adjusted p-value: 0), Transitory (log2 fold change to background: 0.87, adjusted p-value: 0), Muller glia (log2 fold change to background: 0.72, adjusted p-value: 1.66 x 10 ⁻¹⁴⁹), Cone precursors (log2 fold change to background: 0.59, adjusted p-value: 7.02 x 10 ⁻³⁵), Cones (log2 fold change to background: 1.31, adjusted p-value: 0), and Astrocytes (log2 fold change to background: 0.48, adjusted p-value: 5.36 x 10 ⁻³) cell clusters. PAX6 was also enriched in Retinal progenitors (log2 fold change to background: 0.15, adjusted p-value: 1.84 x 10 ⁻⁵⁴), Transitory (log2 fold change to background: 0.23, adjusted p-value: 2.34 x 10 ⁻¹⁶³), Amacrine cells (log2 fold change to background: 1.20, adjusted p-value: 0), Horizontal cells (log2 fold change to background: 1.13, adjusted p-value: 0), Retinal ganglion cells (log2 fold change to background: 0.35, adjusted p-value: 1.15 x 10 ⁻¹⁶⁰), and Muller glia (log2 fold change to background: 0.18, adjusted p-value: 6.83 x 10 ⁻³⁹) cell clusters. LHX2 was enriched in Retinal progenitors (log2 fold change to background: 0.95, adjusted p-value: 0), Transitory (log2 fold change to background: 1.43, adjusted p-value: 0), Muller glia (log2 fold change to background: 0.94, adjusted p-value: 1.59 x 10 ⁻¹⁶³), Astrocytes (log2 fold change to background: 1.15, adjusted p-value: 5.20 x 10 ⁻²⁰) cell clusters. VSX2 was enriched in Retinal progenitors (log2 fold change to background: 0.42, adjusted p-value: 2.12 x 10 ⁻¹⁷⁵), Transitory (log2 fold change to background: 1.07, adjusted p-value: 0), Bipolar cells (log2 fold change to background: 2.07, adjusted p-value: 0), Muller glia (log2 fold change to background: 0.60, adjusted p-value: 1.60 x 10 ⁻⁹¹) cell clusters. All the enriched gene information is available in Additional Table 2. For the following single-cell ATAC-seq analysis, we provide UMAP results highlighting Retinal progenitors ( Figure 1E) and Transitory ( Figure 1F) cell clusters, and feature plots for NOTCH1 ( Figure 1G) and RAX ( Figure 1H). We note that Retinal progenitors and Transitory cell clusters showed similar distributions in the UMAP, in which NOTCH1 and RAX were expressed.

Figure 1. — (A) UMAP analysis of the single-cell RNA-seq data ⁴ for annotation of the other single-cell RNA/ATAC-seq dataset. ²⁰ (B) Feature plot for *SOX2* expression. (C) A feature plot for *MKI67* expression. (D) A dot plot of marker gene expressions along with Notch genes and Retinal progenitors-associated TFs ( *RAX*, *PAX6*, *LHX2*, and *VSX2*). (E) Clear representation of Retinal progenitors in the UMAP result. (F) Clear representation of Transitory cell clusters in the UMAP result. (G) *NOTCH1* expression in the UMAP result. (H) *RAX* expression in the UMAP result.

Next, we examined single-cell RNA-seq data from human fetal retinas at day 59, 74, and 78 from GSE183684. ²⁰ The cell number in each cluster is available in Additional Table 3. After using the “RunHarmony” function ⁴⁴ with the Seurat pipeline to reduce batch effects, we annotated the cells according to the earlier single-cell RNA-seq data ⁴ using the “FindTransferAnchor” function with the Seurat pipeline. This data also contained all types of the aforementioned cell clusters ( Figure 2A). Indeed, we observed SOX2/ MKI67-double-positive Retinal progenitors ( Figure 2B-C). We also provided a dot plot for characteristic gene expression for each cell cluster ( Figure 2D). As expected, screening of the Notch receptor genes suggested prominent NOTCH1 expression in the Retinal progenitors (log2 fold change to background: 1.26, adjusted p-value: 0), Transitory (log2 fold change to background: 1.03, adjusted p-value: 1.88 x 10 ⁻²³¹), and Muller glia (log2 fold change to background: 1.45, adjusted p-value: 1.50 x 10 ⁻¹⁰⁶) cell clusters. In addition, RAX was enriched in Retinal progenitors (log2 fold change to background: 0.64, adjusted p-value: 1.32 x 10 ⁻¹³¹), Transitory (log2 fold change to background: 0.81, adjusted p-value: 3.88 x 10 ⁻¹⁵³), Muller glia (log2 fold change to background: 0.38, adjusted p-value: 2.03 x 10 ⁻¹⁰), Cone precursors (log2 fold change to background: 0.95, adjusted p-value: 6.79 x 10 ⁻⁸²), Cones (log2 fold change to background: 1.43, adjusted p-value: 3.67 x 10 ⁻¹⁰⁷), and Rods (log2 fold change to background: 0.96, adjusted p-value: 8.94 x 10 ⁻¹⁶) cell clusters. PAX6 was enriched in Horizontal cells (log2 fold change to background: 0.84, adjusted p-value: 2.53 x 10 ⁻¹²²) and Retinal ganglion cells (log2 fold change to background: 0.21, adjusted p-value: 1.98 x 10 ⁻⁴³) cell clusters but not in Retinal progenitors or Transitory cell clusters, possibly owing to its wide expression across the cell clusters. LHX2 was enriched in Retinal progenitors (log2 fold change to background: 0.87, adjusted p-value: 4.90 x 10 ⁻¹⁶⁹), Transitory (log2 fold change to background: 1.03, adjusted p-value: 1.62 x 10 ⁻¹⁵³), Muller glia (log2 fold change to background: 0.21, adjusted p-value: 1.31 x 10 ⁻³), Amacrine cells (log2 fold change to background: 0.49, adjusted p-value: 8.75 x 10 ⁻²), and Astrocytes (log2 fold change to background: 1.29, adjusted p-value: 1.16 x 10 ⁻⁴) cell clusters. VSX2 was enriched in Retinal progenitors (log2 fold change to background: 0.67, adjusted p-value: 3.82 x 10 ⁻¹⁵²), Transitory (log2 fold change to background: 1.14, adjusted p-value: 6.82 x 10 ⁻²⁹⁰), Muller glia (log2 fold change to background: 0.75, adjusted p-value: 2.38 x 10 ⁻⁴⁴), Amacrine cells (log2 fold change to background: 0.41, adjusted p-value: 1.50 x 10 ⁻⁸), Bipolar cells (log2 fold change to background: 2.64, adjusted p-value: 7.01 x 10 ⁻¹⁵²) cell clusters. All the enriched gene information is available in Additional Table 4. For the following single-cell ATAC-seq analysis, we provide UMAP results highlighting Retinal progenitors ( Figure 2E) and Transitory ( Figure 2F) cell clusters, and feature plots for NOTCH1 ( Figure 2G) and RAX ( Figure 2H). Similar to the previous data ( Figure 1E-H), feature plot analysis suggested that NOTCH1 and RAX were expressed in Retinal progenitors and Transitory cell clusters.

Figure 2. — (A) UMAP analysis of the single-cell RNA-seq data of a single-cell RNA-seq dataset. ²⁰ (B) A feature plot for *SOX2* expression. (C) A feature plot for *MKI67* expression. (D) A dot plot of marker gene expression along with Notch genes and retinal progenitor-associated TFs ( *RAX*, *PAX6*, *LHX2*, and *VSX2*). (E) Clear representation of Retinal progenitors in the UMAP result. (F) Clear representation of Transitory cell clusters in the UMAP result. (G) *NOTCH1* expression in the UMAP result. (H) *RAX* expression in the UMAP result.

To examine epigenetic changes underlying NOTCH1 expression dynamics, we examined single-cell ATAC-seq data from human fetal eyes at day 59, 74, and 78 from GSE183684. ²⁰ UMAP analysis confirmed that this data contains Retinal progenitors and Transitory cell clusters, along with other differentiated cell clusters, including Cones ( Figure 3A-C). The cell number in each cluster is available in Additional Table 5. We examined Tn5 insertion frequency in the genomic NOTCH1 region, including 100 kb upstream and downstream, with the Signac pipeline ( Figure 3D). To identify the statistically significant Tn5 insertion sites, we used the “Links” function in the Signac pipeline to obtain z-scores and p-values (Additional Table 6). To examine the transcription factor binding sites of RAX, PAX6, LHX2, and VSX2 in those Tn5 insertion sites, we used the FIMO tool and identified potential transcription factor binding sites (Additional Table 7). By comparing the Tn5 insertion sites and the potential transcription factor binding sites, we found a statistically significant Tn5 insertion site in the genomic NOTCH1 region (chr9: 136542126–136542839) containing a transcription factor binding site (chr9: 136542636–136542645) for RAX, but not for PAX6, LHX2, and VSX2. This region is indicated with an arrowhead in the whole genomic NOTCH1 region ( Figure 3D) and the focused region ( Figure 3E). We also performed footprinting analysis and found that RAX-binding is frequently observed in Retinal progenitors, Transitory, and Muller glia cell clusters ( Figure 3F), although this analysis is genome-wide and not specific to the genomic NOTCH1 region. Importantly, the Tn5 insertion event is frequent in Retinal progenitors, Transitory, Amacrine cells, Cone precursors, Cones, Rods, and Muller glia compared with Bipolar cells, Horizontal cells, and Retinal ganglion cells. As RAX is not a classic pioneer factor, in contrast to SOX2 and PAX6, this observation raises the possibility that fine-tuned RAX and NOTCH1 action in Retinal progenitors, Transitory, Amacrine cells, Cone precursors, Cones, Rods, and Muller glia is supported by chromatin remodeling, along with regulated RAX expression.

Figure 3. — (A) UMAP analysis of the single-cell ATAC-seq data. ²⁰ (B) Clear representation of Retinal progenitors in the UMAP result . (C) Clear representation of Transitory cell cluster in the UMAP result. (D) A peak plot for the *NOTCH1* locus with the 100 kb upstream and downstream. The arrowhead shows the chromatin remodeling region in the *NOTCH1* locus, which contains a RAX-binding site and is highlighted in (E). (E) A peak plot for the *NOTCH1* locus focusing on the chromatin remodeling region containing a *RAX*-binding site (arrowhead). (F) Footprint analysis revealed frequent RAX binding in Retinal progenitors, Transitory, and Muller glia.

Discussion

The involvement of Notch signaling in cell fate choices is well documented, including in Drosophila neurogenesis ⁴⁵ and mammalian biliary development. ⁴⁶ A computer simulation study suggested that the regulation of Notch receptor expression is necessary for proper signal transduction in the developing tissue. ⁴⁷ To the best of our knowledge, few studies have used genome-wide investigations for regulatory mechanisms of Notch receptor gene expression. Although the accessibility of the NOTCH1 locus is known to decrease during retinal cell differentiation, ²⁰ how this chromatin remodeling affects NOTCH1 expression, along with candidate cis-regulatory elements, is still unknown. In this study, we hypothesized that chromatin remodeling and transcription factor levels are involved and sought possible examples using a public single-cell RNA/ATAC-seq dataset. ²⁰ We used an already processed dataset from another study that intensively examined molecular characteristics of developing retinal cells ⁴ to enable reliable annotation of the cells in the single-cell RNA/ATAC-seq dataset. ²⁰ We observed prominent NOTCH1 expression both in the reference and query single-cell RNA-seq datasets, although the involvement of other Notch receptors in the developing retina was also reported. ⁹ ^– ¹⁴ Therefore, in the single-cell ATAC-seq analysis, we focused on the NOTCH1 locus, including 100 kb upstream and downstream of its coding region. We observed distinct Tn5 insertion frequencies depending on differentiation status and, importantly, identified one transcription factor binding site for RAX among four transcription factors implicated in retinal progenitors ( RAX, PAX6, LHX2, and VSX2). This RAX-binding site is open in Retinal progenitors, Transitory, Amacrine cells, Cone precursors, Cones, Rods, and Muller glia compared with Bipolar cells, Horizontal cells, and Retinal ganglion cells, which is in line with previous studies. ⁷ ^, ⁴⁸

One of the transcription factors linked to NOTCH1 is PAX6. PAX6 haploinsufficiency has been linked to aniridia, ⁴⁹ an ophthalmological disease often accompanied by fovea hypoplasia. AAV-mediated PAX6 overexpression in the Small eye ( Sey) mouse (a mouse model of aniridia) ⁵⁰ ^, ⁵¹ rescued ganglion cell layer thickness, which was preceded by increased Notch1 expression. ⁵² However, the timing of this overexpression (postnatal day 21) mimicked the average age of diagnosis in human patients (22.1 months) ⁵³ and was much later than the timing of the fetal samples re-analyzed in this study. It is known that PAX6’s DNA-binding affinity is so delicate that disease-causing PAX6 mutations alter rather than ablate this protein-DNA binding ⁵⁴ or require additional factors other than alterations in their DNA-binding affinities for their roles in pathogenesis. ⁵⁵ We observed PAX6 expression in a wide variety of cell clusters, including Retinal progenitors and Transitory cell clusters, both in the reference and query single-cell RNA-seq datasets. In embryonic stem cells, SOX2 (one of the Retinal progenitors markers) ⁴ binds to “hidden” chromatin regions in the nucleosome, and then PAX6 binds to the SOX2-bound “unwrapped” genomic regions to facilitate neural differentiation. ⁵⁶ Expression of these pioneer factors ⁵⁷ was indeed observed in the Retinal progenitors and Transitory cell clusters in the reference and query single-cell RNA-seq datasets. Given that rax upregulated notch1 transcription, ⁷ it is possible that PAX6 and SOX2 unwrap multiple genomic regions before RAX binding to the NOTCH1 locus; however, we were not able to identify PAX6-binding sites in the NOTCH1 locus, although the delicate nature of PAX6 binding might be involved. Although this point needs further investigation, it does not jeopardize the finding that chromatin remodeling occurs at the RAX binding site in the NOTCH1 locus. As RAX expression is observed in Retinal progenitors, Transitory, Muller glia, Cone precursors, and Cones cell clusters in the two single-cell RNA-seq datasets, it is likely that NOTCH1 expression in Retinal progenitors and Transitory cell clusters is in part regulated at the RAX protein level. However, it is also possible that the chromatin remodeling that we observed in this study could help fine-tune regulation of NOTCH1 expression. In conclusion, we provide a chromatin remodeling region in the NOTCH1 locus that contains a RAX binding site, raising the possibility of regulation of NOTCH1 expression at the transcription factor protein and chromatin levels.

Ethical considerations

This study does not generate new data from humans. 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.

Funding Statement

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.

[version 5; peer review: 1 approved

Data availability

A single-cell multiomics dataset (GSE183684) ²⁰ was downloaded from the Gene Expression Omnibus database ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183684).

An h5ad file, ⁴ which includes cell annotation from detailed molecular characteristics analysis of embryonic retinal cells, was downloaded from the CZ CELLxGENE Discover platform ( https://cellxgene.cziscience.com/collections/c11009b8-b113-4a99-9890-78b2f9df9d79).

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).

Archived Additional Tables available from: https://doi.org/10.5281/zenodo.21755650 ⁵⁸

License: OSI-approved open-license software is under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Universal).

The project contains the following underlying data:

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Additional_Table_1.csv.
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Additional_Table_2.csv.
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Additional_Table_3.csv.
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Additional_Table_4.csv.
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Additional_Table_5.csv.
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Additional_Table_6.csv.
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Additional_Table_7.csv.

Software availability

Archived source R code available from: https://doi.org/10.5281/zenodo.21755678 ⁵⁹

License: OSI-approved open-license software is under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Universal).

The project contains the following underlying data:

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260320_retina_chromatin_d59_before_merge.R (code No.1)
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260319_retina_chromatin_d59_d74_d78_merged_No0.R (code No.2)
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260319_retina_chromatin_d59_d74_d78_merged_No1.R (code No.3)
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260319_retina_chromatin_d59_d74_d78_merged_No2.R (code No.4)
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260319_retina_chromatin_d59_d74_d78_merged_No3.R (code No.5)
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Watabe_F1000_ver5_new_No1.R (code No.6)
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Watabe_F1000_ver5_new_No2.R (code No.7)
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Watabe_F1000_ver5_new_No3.R (code No.8)
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Watabe_F1000_ver5_new_No4.R (code No.9)
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Watabe_F1000_ver5_new_No5.R (code No.10)

Acknowledgements

The authors thank Dr. Shunya Sadaki (University of Tsukuba) for his constructive advice on FIMO and Dr. Petr Baranov and Dr. Emil Kriukov (University of Pittsburgh) for kindly providing already annotated single-cell RNA-seq data. ⁴

References

1. Pandey A, Niknejad N, Jafar-Nejad H: Multifaceted regulation of Notch signaling by glycosylation. Glycobiology. 2021;31(1):8–28. 10.1093/glycob/cwaa049 [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Bray SJ: Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol. 2006;7(9):678–689. 10.1038/nrm2009 [DOI] [PubMed] [Google Scholar]
3. Fryer CJ, White JB, Jones KA: Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol Cell. 2004;16(4):509–520. 10.1016/j.molcel.2004.10.014 [DOI] [PubMed] [Google Scholar]
4. Kriukov E, Soucy JR, Labrecque E, et al. : Unraveling the developmental heterogeneity of human retinal ganglion cells within the developing retina to study to the continuity of maturation. Dev Biol. 2025;528:91–104. 10.1016/j.ydbio.2025.08.025 [DOI] [PubMed] [Google Scholar]
5. Gordon PJ, Yun S, Clark AM, et al. : Lhx2 balances progenitor maintenance with neurogenic output and promotes competence state progression in the developing retina. J Neurosci. 2013;33(30):12197–12207. 10.1523/JNEUROSCI.1494-13.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Marquardt T, Ashery-Padan R, Andrejewski N, et al. : Pax6 is required for the multipotent state of retinal progenitor cells. Cell. 2001;105(1):43–55. 10.1016/s0092-8674(01)00295-1 [DOI] [PubMed] [Google Scholar]
7. Furukawa T, Mukherjee S, Bao ZZ, et al. : rax, Hes1, and notch1 promote the formation of Müller glia by postnatal retinal progenitor cells. Neuron. 2000;26(2):383–394. 10.1016/s0896-6273(00)81171-x [DOI] [PubMed] [Google Scholar]
8. Zou C, Levine EM: Vsx2 controls eye organogenesis and retinal progenitor identity via homeodomain and non-homeodomain residues required for high affinity DNA binding. PLoS Genet. 2012;8(9):e1002924. 10.1371/journal.pgen.1002924 [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Lindsell CE, Boulter J, diSibio G, et al. : Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 genes identify ligand-receptor pairs that may function in neural development. Mol Cell Neurosci. 1996;8(1):14–27. 10.1006/mcne.1996.0040 [DOI] [PubMed] [Google Scholar]
10. Bao ZZ, Cepko CL: The expression and function of Notch pathway genes in the developing rat eye. J Neurosci. 1997;17(4):1425–1434. 10.1523/JNEUROSCI.17-04-01425.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Zhu MY, Gasperowicz M, Chow RL: The expression of NOTCH2, HES1 and SOX9 during mouse retinal development. Gene Expr Patterns. 2013;13:78–83. 10.1016/j.gep.2012.12.001 [DOI] [PubMed] [Google Scholar]
12. Zhou Y, Tanzie C, Yan Z, et al. : Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye. Proc Natl Acad Sci U S A. 2013;110(22):8966–8971. 10.1073/pnas.1218145110 [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Bosze B, Suarez-Navarro J, Cajias I, et al. : Notch pathway mutants do not equivalently perturb mouse embryonic retinal development. PLoS Genet. 2023;19(9):e1010928. 10.1371/journal.pgen.1010928 [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Claxton S, Fruttiger M: Periodic Delta-like 4 expression in developing retinal arteries. Gene Expr Patterns. 2004;5(1):123–127. 10.1016/j.modgep.2004.05.004 [DOI] [PubMed] [Google Scholar]
15. Riesenberg AN, Liu Z, Kopan R, et al. : Rbpj cell autonomous regulation of retinal ganglion cell and cone photoreceptor fates in the mouse retina. J Neurosci. 2009;29(41):12865–12877. 10.1523/JNEUROSCI.3382-09.2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Chen X, Emerson MM: Notch signaling represses cone photoreceptor formation through the regulation of retinal progenitor cell states. Sci Rep. 2021;11(1):14525. 10.1038/s41598-021-93692-w [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Jadhav AP, Cho SH, Cepko CL: Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property. Proc Natl Acad Sci U S A. 2006;103(50):18998–19003. 10.1073/pnas.0608155103 [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Jadhav AP, Mason HA, Cepko CL: Notch 1 inhibits photoreceptor production in the developing mammalian retina. Development. 2006;133(5):913–923. 10.1242/dev.02245 [DOI] [PubMed] [Google Scholar]
19. Mizeracka K, DeMaso CR, Cepko CL: Notch1 is required in newly postmitotic cells to inhibit the rod photoreceptor fate. Development. 2013;140(15):3188–3197. 10.1242/dev.090696 [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Thomas ED, Timms AE, Giles S, et al. : Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids. Dev Cell. 2022;57(6):820–836.e6. 10.1016/j.devcel.2022.02.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Watabe Y, Kobayashi S, Takahashi S, et al. : Computer identification of Notch3 in the neurogenic progenitor cells of mammalian early optic vesicles. BMC Res Notes. 2025;18(1):367. 10.1186/s13104-025-07440-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Dvoriantchikova G, Perea-Martinez I, Pappas S, et al. : Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina. PLoS One. 2015;10:26091508. 10.1371/journal.pone.0131054 [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Jin M, Zhang H, Xu B, et al. : Jag2b-Notch3/1b-mediated neuron-to-glia crosstalk controls retinal gliogenesis. EMBO Rep. 2022;23(10):e54922. 10.15252/embr.202254922 [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Ge Y, Chen X, Nan N, et al. : Key transcription factors influence the epigenetic landscape to regulate retinal cell differentiation. Nucleic Acids Res. 2023;51(5):2151–2176. 10.1093/nar/gkad026 [DOI] [PMC free article] [PubMed] [Google Scholar]
25. He T, Fan Y, Du J, et al. : Stuxnet fine-tunes Notch dose during development using a functional Polycomb response element. Development. 2023;150. 10.1242/dev.201297 [DOI] [PubMed] [Google Scholar]
26. Schlereth K, Weichenhan D, Bauer T, et al. : The transcriptomic and epigenetic map of vascular quiescence in the continuous lung endothelium. Elife. 2018;7:e34423. 10.7554/eLife.34423 [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Lussi YC, Mariani L, Friis C, et al. : Impaired removal of H3K4 methylation affects cell fate determination and gene transcription. Development. 2016;143(20):3751–3762. 10.1242/dev.139139 [DOI] [PubMed] [Google Scholar]
28. Marttila S, Kananen L, Jylhävä J, et al. : Length of paternal lifespan is manifested in the DNA methylome of their nonagenarian progeny. Oncotarget. 2015;6(31):30557–30567. 10.18632/oncotarget.5905 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Gu B, Watanabe K, Sun P, et al. : Chromatin effector Pygo2 mediates Wnt-notch crosstalk to suppress luminal/alveolar potential of mammary stem and basal cells. Cell Stem Cell. 2013;13(1):48–61. 10.1016/j.stem.2013.04.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Rincon-Arano H, Halow J, Delrow JJ, et al. : UpSET recruits HDAC complexes and restricts chromatin accessibility and acetylation at promoter regions. Cell. 2012;151(6):1214–1228. 10.1016/j.cell.2012.11.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Hussan SS, Ali MS, Fatima M, et al. : Epigenetically dysregulated NOTCH-Delta-HES signaling cascade can serve as a subtype classifier for acute lymphoblastic leukemia. Ann Hematol. 2024;103(2):511–523. 10.1007/s00277-023-05515-9 [DOI] [PubMed] [Google Scholar]
32. Zhu Y, Ye M, Xu H, et al. : Methylation status of CpG sites in the NOTCH4 promoter region regulates NOTCH4 expression in patients with tetralogy of Fallot. Mol Med Rep. 2020;22(5):4412–4422. 10.3892/mmr.2020.11535 [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Jeong GY, Park MK, Choi HJ, et al. : NSD3-induced methylation of H3K36 activates NOTCH signaling to drive breast tumor initiation and metastatic progression. Cancer Res. 2021;81(1):77–90. 10.1158/0008-5472.CAN-20-0360 [DOI] [PubMed] [Google Scholar]
34. Huang YC, Lin SJ, Shih HY, et al. : Epigenetic regulation of NOTCH1 and NOTCH3 by KMT2A inhibits glioma proliferation. Oncotarget. 2017;8(38):63110–63120. 10.18632/oncotarget.18668 [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Liu M, Liang K, Zhen J, et al. : Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling. Nat Commun. 2017;8. 10.1038/s41467-017-00498-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Liu S, Liu D, Chen C, et al. : MSC transplantation improves osteopenia via epigenetic regulation of notch signaling in lupus. Cell Metab. 2015;22(4):606–618. 10.1016/j.cmet.2015.08.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Kuang SQ, Fang Z, Zweidler-McKay PA, et al. : Epigenetic inactivation of Notch-Hes pathway in human B-cell acute lymphoblastic leukemia. PLoS One. 2013;8. 10.1371/journal.pone.0061807 [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Jithesh PV, Risk JM, Schache AG, et al. : The epigenetic landscape of oral squamous cell carcinoma. Br J Cancer. 2013;108(2):370–379. 10.1038/bjc.2012.568 [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Jeannet R, Mastio J, Macias-Garcia A, et al. : Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL. Blood. 2010;116(25):5443–5454. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Hao Y, Stuart T, Kowalski MH, et al. : Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat Biotechnol. 2024;42(2):293–304. 10.1038/s41587-023-01767-y [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Stuart T, Srivastava A, Madad S, et al. : Single-cell chromatin state analysis with Signac. Nat Methods. 2021;18(11):1333–1341. 10.1038/s41592-021-01282-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Grant CE, Bailey TL, Noble WS: FIMO: scanning for occurrences of a given motif. Bioinformatics. 2011;27(7):1017–1018. 10.1093/bioinformatics/btr064 [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Bailey TL, Johnson J, Grant CE, et al. : The MEME Suite. Nucleic Acids Res. 2015;43(W1):W39–W49. 10.1093/nar/gkv416 [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Korsunsky I, Millard N, Fan J, et al. : Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods. 2019;16(12):1289–1296. 10.1038/s41592-019-0619-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Heitzler P, Simpson P: The choice of cell fate in the epidermis of Drosophila. Cell. 1991;64(6):1083–1092. 10.1016/0092-8674(91)90263-x [DOI] [PubMed] [Google Scholar]
46. Tanimizu N, Miyajima A: Notch signaling controls hepatoblast differentiation by altering the expression of liver-enriched transcription factors. J Cell Sci. 2004;117(Pt 15):3165–3174. 10.1242/jcs.01169 [DOI] [PubMed] [Google Scholar]
47. Yoshihara M, Nishino T, Yadav MK, et al. : Mathematical analysis of the effect of portal vein cells on biliary epithelial cell differentiation through the Delta-Notch signaling pathway. BMC Res Notes. 2021;14 10.1186/s13104-021-05656-y [DOI] [PMC free article] [PubMed] [Google Scholar]
48. Irie S, Sanuki R, Muranishi Y, et al. : Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina. Mol Cell Biol. 2015;35(15):2583–2596. 10.1128/MCB.00048-15 [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Jordan T, Hanson I, Zaletayev D, et al. : The human PAX6 gene is mutated in two patients with aniridia. Nat Genet. 1992;1(5):328–332. 10.1038/ng0892-328 [DOI] [PubMed] [Google Scholar]
50. Glaser T, Lane J, Housman D: A mouse model of the aniridia-Wilms tumor deletion syndrome. Science. 1990;250(4982):823–827. 10.1126/science.2173141 [DOI] [PubMed] [Google Scholar]
51. Hill RE, Favor J, Hogan BL, et al. : Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature.1991;354(6354):522–525. 10.1038/354522a0 [DOI] [PubMed]
52. Djaksigulova D, Kaad SG, Korecki AJ, et al. : First pilot study of intravenous rAAV-PAX6 gene therapy increases retinal-ganglion-cell-layer thickness and Notch1 transcription in a mouse model of aniridia. Gene Ther. 2026; 10.1038/s41434-026-00605-5 [DOI] [PubMed] [Google Scholar]
53. Netland PA, Scott ML, Boyle JW 4th, et al. : Ocular and systemic findings in a survey of aniridia subjects. J AAPOS. 2011;15(6):562–566. 10.1016/j.jaapos.2011.07.009 [DOI] [PubMed] [Google Scholar]
54. Williamson KA, Hall HN, Owen LJ, et al. : Recurrent heterozygous PAX6 missense variants cause severe bilateral microphthalmia via predictable effects on DNA-protein interaction. Genet Med. 2020;22(3):598–609. 10.1038/s41436-019-0685-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
55. Lee S, Lee SH, Heo H, et al. : Impaired DNA-binding affinity of novel PAX6 mutations. Sci Rep. 2020;10. 10.1038/s41598-020-60017-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
56. Zhang S, Bell E, Zhi H, et al. : OCT4 and PAX6 determine the dual function of SOX2 in human ESCs as a key pluripotent or neural factor. Stem Cell Res Ther. 2019;10. 10.1186/s13287-019-1228-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
57. Barral A, Zaret KS: Pioneer factors: roles and their regulation in development. Trends Genet. 2024;40(2):134–148. 10.1016/j.tig.2023.10.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
58. Watabe Y, Yoshihara M: Additional_Tables. 2026-08-02. [CSV files]. 10.5281/zenodo.21755650 [DOI]
59. Watabe Y, Yoshihara M: R_code_files. 2026-08-02. [R code files]. 10.5281/zenodo.21755678 [DOI]

F1000Res. 2026 Sep 28. doi: 10.5256/f1000research.208123.r513381

Reviewer response for version 5

Tomohiro Masuda ¹

I have no further major comments on the latest version of the manuscript. Although some questions remain to be answered to fully validate the authors' hypothesis, addressing them may be technically challenging as long as the study relies solely on in silico analysis. However, please double-check whether the progenitor clusters on the UMAP plots in Figs. 1 and 2 are appropriate and reanalyze them if necessary, as they appear to be somewhat dispersed. Nevertheless, the authors seem to have sincerely addressed my previous comments on Version 4.

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?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

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

Partly

Reviewer Expertise:

Ophthalmology, Developmental biology, gene regulation

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

F1000Res. 2026 Sep 3. doi: 10.5256/f1000research.208123.r513384

Reviewer response for version 5

Benedikt Obermayer ¹

in the new version, the authors have addressed previous reviewers' comments to some degree, but I don't feel this has substantially improved the paper.

- transferring cell type labels from Kriukov et al. is a reasonable approach, but unfortunately it doesn't help solve the annotation issue that I've raised multiple times. The learned labels don't agree with the dataset-intrinsic cluster structure seen in the UMAP: many clusters, in particular retinal progenitor and transitory cells are spread out and overlapping, which suggests a label transfer artifact, batch integration issues, substructure in the Thomas et al. data, or an incompatibility of query and reference datasets, maybe because of different sequencing technology. This inconsistency should be resolved.

- the RAX binding site claimed as novel discovery is already contained in the JASPAR tracks as well, just with a score (225) that's below the default, so it amounts to a lower-confidence result. If the authors' own analysis indicates better statistical support, this should be stated explicitly and the differences investigated.

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.

F1000Res. 2026 Jul 14. doi: 10.5256/f1000research.198475.r498343

Reviewer response for version 4

Tomohiro Masuda ¹

In this report, the authors re-analyzed single-cell RNA-seq and single-cell ATAC-seq data of the human fetal retina at days 59, 74, and 78, obtained from a paper published by Thomas et al. The authors found that the NOTCH1, NOTCH2, and NOTCH3 genes were preferentially expressed in what they defined as "Late RPCs," where typical RPC TFs (such as VSX2, LHX2, RAX, and PAX6) were also expressed. They also found that PAX6 TFBS partially overlapped with the open chromatin regions of the NOTCH1 and NOTCH3 loci, although foot printing analysis failed to identify PAX6 binding to the Notch loci. Although NOTCH signaling is important for the maintenance of retinal progenitors, and hence the identification of factors that regulate NOTCH gene expression is an interesting topic, this re-analysis, in its current state, does not seem to go into enough depth to advance our understanding of retinal development.

1. I understand the technical difficulties associated with single-cell data analysis, but your results suggest that PAX6 does not sufficiently explain the regulation of NOTCH gene expression. Have you searched for factors other than VSX2, LHX2, RAX, and PAX6 that could more clearly explain NOTCH regulation? Searching beyond these known factors might lead to the discovery of novel factors, thereby increasing the value of this report.

2. You mentioned Blimp1 as a developing cone photoreceptor marker gene, but it is not a cone-specific marker; rather, it is a general photoreceptor marker. THRB, as shown in Figure 1D, is a more appropriate marker.

3. Is your cluster definition of the "PR/BC precursor" correct? You defined GADD45G as the marker, but in Figure 1D, the expression levels of ONECUT2 and PAX6 genes are much higher than that of GADD45G in this cluster. These two genes are not markers for PRs and BCs.

4. Is your cluster definition of the "early RPC" correct? The Monocle3 pseudotime analysis shown in Figure 1C suggests that the early RPCs developed later than RPC1–4 ("Later RPCs" in your definition).

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?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

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

Partly

Reviewer Expertise:

Ophthalmology, Developmental biology, gene regulation

F1000Res. 2026 Aug 6.

Masaharu Yoshihara ¹

Dear Dr. Tomohiro Masuda,

Thank you very much for your time and effort in reviewing our manuscript. We appreciate your constructive comments and have significantly revised the manuscript to address all points, using the annotated single-cell RNA-seq data from Kriukov et al. in Developmental Biology (2025). We are submitting a revised version of this manuscript with this response letter.

Comments from Dr. Tomohiro Masuda:

We corrected cell annotation and found that, in line with previous literature by Furukawa et al. in Neuron (2000) and Irie et al. in Molecular and Cellular Biology (2015), the NOTCH1 locus contains a RAX binding site that has frequent Tn5 insertions. We revised the Discussion section to fully explain PAX6 expression and this RAX binding site.

We adopted cell annotation from Kriukov et al. in Developmental Biology (2025) and transferred the cell labeling to make it more concise.

3. Is your cluster definition of the "PR/BC precursor" correct? You defined GADD45G as the marker, but in Figure 1D, the expression levels of ONECUT2 and PAX6genes are much higher than that of GADD45G in this cluster. These two genes are not markers for PRs and BCs.

We adopted cell annotation from Kriukov et al. in Developmental Biology (2025) and transferred the cell labeling to make it more concise.

Again, thank you very much for your constructive comments. We look forward to hearing from you soon.

Sincerely,

Masaharu Yoshihara, M.D., Ph.D.

Specially Appointed Assistant Professor

University of Tsukuba, Japan

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

F1000Res. 2026 May 14. doi: 10.5256/f1000research.198475.r475951

Reviewer response for version 4

Benedikt Obermayer ¹

in the new version, the authors have re-done the analysis on data integrated over timepoints, which makes cell type labels easier to compare, even though an actual label transfer from Thomas et al. or Zuo et al. would have helped put their results into the context of the literature. Thomas et al. is now cited, but the findings of that paper are not discussed. In particular, I'm still missing a motivation for their re-analysis of that data, or where this re-analysis leads to new or different results: Notch accessibility and expression was already discussed in the supplementary figures of Thomas et al; the identification of PAX6 binding sites at the NOTCH1/NOTCH3 loci is presented as a key novel finding, but these sites are readily visible in the UCSC browser using the JASPAR predicted TF binding track, and the authors' own 'confirmation' via the browser tracks from Thomas et al. is therefore somewhat circular.

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

F1000Res. 2026 Aug 6.

Masaharu Yoshihara ¹

Dear Dr. Benedikt Obermayer,

Comments from Dr. Benedikt Obermayer:

In the new version, the authors have re-done the analysis on data integrated over timepoints, which makes cell type labels easier to compare, even though an actual label transfer from Thomas et al. or Zuo et al. would have helped put their results into the context of the literature. Thomas et al. is now cited, but the findings of that paper are not discussed. In particular, I'm still missing a motivation for their re-analysis of that data, or where this re-analysis leads to new or different results: Notch accessibility and expression was already discussed in the supplementary figures of Thomas et al; the identification of PAX6 binding sites at the NOTCH1/NOTCH3 loci is presented as a key novel finding, but these sites are readily visible in the UCSC browser using the JASPAR predicted TF binding track, and the authors' own 'confirmation' via the browser tracks from Thomas et al. is therefore somewhat circular.

We agree with you that cell annotation is important and transferred the cell label from a recent paper by Kriukov et al. in Developmental Biology (2025), in which the authors extensively examined molecular characteristics of developing retinal cells. This label transfer made it more concise and led to the identification of a new RAX transcription factor binding site instead of PAX6. We believe it is important to report putative CREs alongside transcription factor expression levels, even if chromatin remodeling is visible in the UCSC browser. In addition, our research focuses on the regulation of Notch signaling, which is why we examined the eye, in which cell differentiation is well characterized. We revised the manuscript so that our motivation in Notch signaling biology is clear, and the link to the ophthalmological finding by Thomas et al. is also clear. For example, we have added the following sentences in the Discussion section:

Although the accessibility of the NOTCH1 locus is known to decrease during retinal cell differentiation (Thomas et al.), how this chromatin remodeling affects NOTCH1 expression, along with candidate cis-regulatory elements, is still unknown. In this study, we hypothesized that chromatin remodeling and transcription factor levels are involved and sought possible examples using this dataset.

Again, thank you very much for your constructive comments. We look forward to hearing from you soon.

Sincerely,

Masaharu Yoshihara, M.D., Ph.D.

Specially Appointed Assistant Professor

University of Tsukuba, Japan

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

F1000Res. 2026 Feb 26. doi: 10.5256/f1000research.190734.r460837

Reviewer response for version 3

Benedikt Obermayer ¹

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

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 [DOI] [PMC free article] [PubMed] [Google Scholar]
2. : 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.2022.02.018820-836.e6 10.1016/j.devcel.2022.02.018 [DOI] [PMC free article] [PubMed] [Google Scholar]

F1000Res. 2025 Sep 30. doi: 10.5256/f1000research.188207.r416419

Reviewer response for version 2

Zhongjie Tang ¹

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

F1000Res. 2025 Nov 1.

Masaharu Yoshihara ¹

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.

F1000Res. 2025 Aug 22. doi: 10.5256/f1000research.175389.r398458

Reviewer response for version 1

Zhongjie Tang ¹

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

F1000Res. 2025 Sep 16.

Masaharu Yoshihara ¹

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

F1000Res. 2025 Feb 5. doi: 10.5256/f1000research.175389.r359501

Reviewer response for version 1

Mariko Kashiwagi ¹

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

F1000Res. 2025 Sep 16.

Masaharu Yoshihara ¹

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:

The authors stated in the text that "NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes." However, this claim is not fully supported by the data (Fig. 1D, Additional File 1B; Fig. 2D, Additional File 2B; Fig. 3D, Additional File 3B). A comprehensive interpretation of the three developmental stages suggests that NOTCH1 was the predominant isoform expressed in early RPC clusters, although expression levels varied across clusters and stages. At day 59, the expression hierarchy was NOTCH2 > NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully.

Response: Thank you for your constructive comments. We agree to your comment and clearly have indicated that the early RPCs expressed NOTCH1, NOTCH2 and NOTCH3 for each sample in the Results section. In particular, NOTCH1 showed the highest expressions throughout the three samples and we have clearly mentioned that we have focused in NOTCH1 and NOTCH3 in this study since 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

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

A single-cell multiomics dataset (GSE183684) ²⁰ was downloaded from the Gene Expression Omnibus database ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183684).

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).

Archived Additional Tables available from: https://doi.org/10.5281/zenodo.21755650 ⁵⁸

License: OSI-approved open-license software is under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Universal).

The project contains the following underlying data:

•
Additional_Table_1.csv.
•
Additional_Table_2.csv.
•
Additional_Table_3.csv.
•
Additional_Table_4.csv.
•
Additional_Table_5.csv.
•
Additional_Table_6.csv.
•
Additional_Table_7.csv.

[ref1] 1. Pandey A, Niknejad N, Jafar-Nejad H: Multifaceted regulation of Notch signaling by glycosylation. Glycobiology. 2021;31(1):8–28. 10.1093/glycob/cwaa049 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref2] 2. Bray SJ: Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol. 2006;7(9):678–689. 10.1038/nrm2009 [DOI] [PubMed] [Google Scholar]

[ref3] 3. Fryer CJ, White JB, Jones KA: Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol Cell. 2004;16(4):509–520. 10.1016/j.molcel.2004.10.014 [DOI] [PubMed] [Google Scholar]

[ref4] 4. Kriukov E, Soucy JR, Labrecque E, et al. : Unraveling the developmental heterogeneity of human retinal ganglion cells within the developing retina to study to the continuity of maturation. Dev Biol. 2025;528:91–104. 10.1016/j.ydbio.2025.08.025 [DOI] [PubMed] [Google Scholar]

[ref5] 5. Gordon PJ, Yun S, Clark AM, et al. : Lhx2 balances progenitor maintenance with neurogenic output and promotes competence state progression in the developing retina. J Neurosci. 2013;33(30):12197–12207. 10.1523/JNEUROSCI.1494-13.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref6] 6. Marquardt T, Ashery-Padan R, Andrejewski N, et al. : Pax6 is required for the multipotent state of retinal progenitor cells. Cell. 2001;105(1):43–55. 10.1016/s0092-8674(01)00295-1 [DOI] [PubMed] [Google Scholar]

[ref7] 7. Furukawa T, Mukherjee S, Bao ZZ, et al. : rax, Hes1, and notch1 promote the formation of Müller glia by postnatal retinal progenitor cells. Neuron. 2000;26(2):383–394. 10.1016/s0896-6273(00)81171-x [DOI] [PubMed] [Google Scholar]

[ref8] 8. Zou C, Levine EM: Vsx2 controls eye organogenesis and retinal progenitor identity via homeodomain and non-homeodomain residues required for high affinity DNA binding. PLoS Genet. 2012;8(9):e1002924. 10.1371/journal.pgen.1002924 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref9] 9. Lindsell CE, Boulter J, diSibio G, et al. : Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 genes identify ligand-receptor pairs that may function in neural development. Mol Cell Neurosci. 1996;8(1):14–27. 10.1006/mcne.1996.0040 [DOI] [PubMed] [Google Scholar]

[ref10] 10. Bao ZZ, Cepko CL: The expression and function of Notch pathway genes in the developing rat eye. J Neurosci. 1997;17(4):1425–1434. 10.1523/JNEUROSCI.17-04-01425.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref11] 11. Zhu MY, Gasperowicz M, Chow RL: The expression of NOTCH2, HES1 and SOX9 during mouse retinal development. Gene Expr Patterns. 2013;13:78–83. 10.1016/j.gep.2012.12.001 [DOI] [PubMed] [Google Scholar]

[ref12] 12. Zhou Y, Tanzie C, Yan Z, et al. : Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye. Proc Natl Acad Sci U S A. 2013;110(22):8966–8971. 10.1073/pnas.1218145110 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref13] 13. Bosze B, Suarez-Navarro J, Cajias I, et al. : Notch pathway mutants do not equivalently perturb mouse embryonic retinal development. PLoS Genet. 2023;19(9):e1010928. 10.1371/journal.pgen.1010928 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref14] 14. Claxton S, Fruttiger M: Periodic Delta-like 4 expression in developing retinal arteries. Gene Expr Patterns. 2004;5(1):123–127. 10.1016/j.modgep.2004.05.004 [DOI] [PubMed] [Google Scholar]

[ref15] 15. Riesenberg AN, Liu Z, Kopan R, et al. : Rbpj cell autonomous regulation of retinal ganglion cell and cone photoreceptor fates in the mouse retina. J Neurosci. 2009;29(41):12865–12877. 10.1523/JNEUROSCI.3382-09.2009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref16] 16. Chen X, Emerson MM: Notch signaling represses cone photoreceptor formation through the regulation of retinal progenitor cell states. Sci Rep. 2021;11(1):14525. 10.1038/s41598-021-93692-w [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref17] 17. Jadhav AP, Cho SH, Cepko CL: Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property. Proc Natl Acad Sci U S A. 2006;103(50):18998–19003. 10.1073/pnas.0608155103 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref18] 18. Jadhav AP, Mason HA, Cepko CL: Notch 1 inhibits photoreceptor production in the developing mammalian retina. Development. 2006;133(5):913–923. 10.1242/dev.02245 [DOI] [PubMed] [Google Scholar]

[ref19] 19. Mizeracka K, DeMaso CR, Cepko CL: Notch1 is required in newly postmitotic cells to inhibit the rod photoreceptor fate. Development. 2013;140(15):3188–3197. 10.1242/dev.090696 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref20] 20. Thomas ED, Timms AE, Giles S, et al. : Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids. Dev Cell. 2022;57(6):820–836.e6. 10.1016/j.devcel.2022.02.018 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref21] 21. Watabe Y, Kobayashi S, Takahashi S, et al. : Computer identification of Notch3 in the neurogenic progenitor cells of mammalian early optic vesicles. BMC Res Notes. 2025;18(1):367. 10.1186/s13104-025-07440-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref22] 22. Dvoriantchikova G, Perea-Martinez I, Pappas S, et al. : Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina. PLoS One. 2015;10:26091508. 10.1371/journal.pone.0131054 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref23] 23. Jin M, Zhang H, Xu B, et al. : Jag2b-Notch3/1b-mediated neuron-to-glia crosstalk controls retinal gliogenesis. EMBO Rep. 2022;23(10):e54922. 10.15252/embr.202254922 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref24] 24. Ge Y, Chen X, Nan N, et al. : Key transcription factors influence the epigenetic landscape to regulate retinal cell differentiation. Nucleic Acids Res. 2023;51(5):2151–2176. 10.1093/nar/gkad026 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref25] 25. He T, Fan Y, Du J, et al. : Stuxnet fine-tunes Notch dose during development using a functional Polycomb response element. Development. 2023;150. 10.1242/dev.201297 [DOI] [PubMed] [Google Scholar]

[ref26] 26. Schlereth K, Weichenhan D, Bauer T, et al. : The transcriptomic and epigenetic map of vascular quiescence in the continuous lung endothelium. Elife. 2018;7:e34423. 10.7554/eLife.34423 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref27] 27. Lussi YC, Mariani L, Friis C, et al. : Impaired removal of H3K4 methylation affects cell fate determination and gene transcription. Development. 2016;143(20):3751–3762. 10.1242/dev.139139 [DOI] [PubMed] [Google Scholar]

[ref28] 28. Marttila S, Kananen L, Jylhävä J, et al. : Length of paternal lifespan is manifested in the DNA methylome of their nonagenarian progeny. Oncotarget. 2015;6(31):30557–30567. 10.18632/oncotarget.5905 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref29] 29. Gu B, Watanabe K, Sun P, et al. : Chromatin effector Pygo2 mediates Wnt-notch crosstalk to suppress luminal/alveolar potential of mammary stem and basal cells. Cell Stem Cell. 2013;13(1):48–61. 10.1016/j.stem.2013.04.012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref30] 30. Rincon-Arano H, Halow J, Delrow JJ, et al. : UpSET recruits HDAC complexes and restricts chromatin accessibility and acetylation at promoter regions. Cell. 2012;151(6):1214–1228. 10.1016/j.cell.2012.11.009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref31] 31. Hussan SS, Ali MS, Fatima M, et al. : Epigenetically dysregulated NOTCH-Delta-HES signaling cascade can serve as a subtype classifier for acute lymphoblastic leukemia. Ann Hematol. 2024;103(2):511–523. 10.1007/s00277-023-05515-9 [DOI] [PubMed] [Google Scholar]

[ref32] 32. Zhu Y, Ye M, Xu H, et al. : Methylation status of CpG sites in the NOTCH4 promoter region regulates NOTCH4 expression in patients with tetralogy of Fallot. Mol Med Rep. 2020;22(5):4412–4422. 10.3892/mmr.2020.11535 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref33] 33. Jeong GY, Park MK, Choi HJ, et al. : NSD3-induced methylation of H3K36 activates NOTCH signaling to drive breast tumor initiation and metastatic progression. Cancer Res. 2021;81(1):77–90. 10.1158/0008-5472.CAN-20-0360 [DOI] [PubMed] [Google Scholar]

[ref34] 34. Huang YC, Lin SJ, Shih HY, et al. : Epigenetic regulation of NOTCH1 and NOTCH3 by KMT2A inhibits glioma proliferation. Oncotarget. 2017;8(38):63110–63120. 10.18632/oncotarget.18668 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref35] 35. Liu M, Liang K, Zhen J, et al. : Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling. Nat Commun. 2017;8. 10.1038/s41467-017-00498-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref36] 36. Liu S, Liu D, Chen C, et al. : MSC transplantation improves osteopenia via epigenetic regulation of notch signaling in lupus. Cell Metab. 2015;22(4):606–618. 10.1016/j.cmet.2015.08.018 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref37] 37. Kuang SQ, Fang Z, Zweidler-McKay PA, et al. : Epigenetic inactivation of Notch-Hes pathway in human B-cell acute lymphoblastic leukemia. PLoS One. 2013;8. 10.1371/journal.pone.0061807 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref38] 38. Jithesh PV, Risk JM, Schache AG, et al. : The epigenetic landscape of oral squamous cell carcinoma. Br J Cancer. 2013;108(2):370–379. 10.1038/bjc.2012.568 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref39] 39. Jeannet R, Mastio J, Macias-Garcia A, et al. : Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL. Blood. 2010;116(25):5443–5454. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref40] 40. Hao Y, Stuart T, Kowalski MH, et al. : Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat Biotechnol. 2024;42(2):293–304. 10.1038/s41587-023-01767-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref41] 41. Stuart T, Srivastava A, Madad S, et al. : Single-cell chromatin state analysis with Signac. Nat Methods. 2021;18(11):1333–1341. 10.1038/s41592-021-01282-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref42] 42. Grant CE, Bailey TL, Noble WS: FIMO: scanning for occurrences of a given motif. Bioinformatics. 2011;27(7):1017–1018. 10.1093/bioinformatics/btr064 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref43] 43. Bailey TL, Johnson J, Grant CE, et al. : The MEME Suite. Nucleic Acids Res. 2015;43(W1):W39–W49. 10.1093/nar/gkv416 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref44] 44. Korsunsky I, Millard N, Fan J, et al. : Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods. 2019;16(12):1289–1296. 10.1038/s41592-019-0619-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref45] 45. Heitzler P, Simpson P: The choice of cell fate in the epidermis of Drosophila. Cell. 1991;64(6):1083–1092. 10.1016/0092-8674(91)90263-x [DOI] [PubMed] [Google Scholar]

[ref46] 46. Tanimizu N, Miyajima A: Notch signaling controls hepatoblast differentiation by altering the expression of liver-enriched transcription factors. J Cell Sci. 2004;117(Pt 15):3165–3174. 10.1242/jcs.01169 [DOI] [PubMed] [Google Scholar]

[ref47] 47. Yoshihara M, Nishino T, Yadav MK, et al. : Mathematical analysis of the effect of portal vein cells on biliary epithelial cell differentiation through the Delta-Notch signaling pathway. BMC Res Notes. 2021;14 10.1186/s13104-021-05656-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref48] 48. Irie S, Sanuki R, Muranishi Y, et al. : Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina. Mol Cell Biol. 2015;35(15):2583–2596. 10.1128/MCB.00048-15 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref49] 49. Jordan T, Hanson I, Zaletayev D, et al. : The human PAX6 gene is mutated in two patients with aniridia. Nat Genet. 1992;1(5):328–332. 10.1038/ng0892-328 [DOI] [PubMed] [Google Scholar]

[ref50] 50. Glaser T, Lane J, Housman D: A mouse model of the aniridia-Wilms tumor deletion syndrome. Science. 1990;250(4982):823–827. 10.1126/science.2173141 [DOI] [PubMed] [Google Scholar]

[ref51] 51. Hill RE, Favor J, Hogan BL, et al. : Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature.1991;354(6354):522–525. 10.1038/354522a0 [DOI] [PubMed]

[ref52] 52. Djaksigulova D, Kaad SG, Korecki AJ, et al. : First pilot study of intravenous rAAV-PAX6 gene therapy increases retinal-ganglion-cell-layer thickness and Notch1 transcription in a mouse model of aniridia. Gene Ther. 2026; 10.1038/s41434-026-00605-5 [DOI] [PubMed] [Google Scholar]

[ref53] 53. Netland PA, Scott ML, Boyle JW 4th, et al. : Ocular and systemic findings in a survey of aniridia subjects. J AAPOS. 2011;15(6):562–566. 10.1016/j.jaapos.2011.07.009 [DOI] [PubMed] [Google Scholar]

[ref54] 54. Williamson KA, Hall HN, Owen LJ, et al. : Recurrent heterozygous PAX6 missense variants cause severe bilateral microphthalmia via predictable effects on DNA-protein interaction. Genet Med. 2020;22(3):598–609. 10.1038/s41436-019-0685-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref55] 55. Lee S, Lee SH, Heo H, et al. : Impaired DNA-binding affinity of novel PAX6 mutations. Sci Rep. 2020;10. 10.1038/s41598-020-60017-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref56] 56. Zhang S, Bell E, Zhi H, et al. : OCT4 and PAX6 determine the dual function of SOX2 in human ESCs as a key pluripotent or neural factor. Stem Cell Res Ther. 2019;10. 10.1186/s13287-019-1228-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref57] 57. Barral A, Zaret KS: Pioneer factors: roles and their regulation in development. Trends Genet. 2024;40(2):134–148. 10.1016/j.tig.2023.10.007 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref58] 58. Watabe Y, Yoshihara M: Additional_Tables. 2026-08-02. [CSV files]. 10.5281/zenodo.21755650 [DOI]

[ref59] 59. Watabe Y, Yoshihara M: R_code_files. 2026-08-02. [R code files]. 10.5281/zenodo.21755678 [DOI]

PERMALINK

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

Yoshitoku Watabe

Sakurako Kobayashi

Takahiro Nakayama

Satoru Takahashi

Masaharu Yoshihara

Roles

Version Changes

Revised. Amendments from Version 4

Abstract

Background

Methods

Results

Conclusions

Introduction

Methods

Results

Figure 1. Single-cell RNA-seq analysis of a dataset for annotation.

Figure 2. Single-cell RNA-seq analysis of human fetal retina at day 59, 74, and 78.

Figure 3. Single-cell ATAC-seq analysis of human fetal retina at day 59, 74, and 78.

Discussion

Ethical considerations

Consent for publication

Authors’ contributions

Funding Statement

Data availability

Software availability

Acknowledgements

References

Reviewer response for version 5

Tomohiro Masuda

Roles

Reviewer response for version 5

Benedikt Obermayer

Roles

Reviewer response for version 4

Tomohiro Masuda

Roles

Masaharu Yoshihara

Reviewer response for version 4

Benedikt Obermayer

Roles

Masaharu Yoshihara

Reviewer response for version 3

Benedikt Obermayer

Roles

References

Reviewer response for version 2

Zhongjie Tang

Roles

Masaharu Yoshihara

Reviewer response for version 1

Zhongjie Tang

Roles

Masaharu Yoshihara

Reviewer response for version 1

Mariko Kashiwagi

Roles

Masaharu Yoshihara

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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