{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T12:02:11Z","timestamp":1791892931573,"version":"4.3.4"},"update-to":[{"DOI":"10.12688\/f1000research.159630.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000}},{"DOI":"10.12688\/f1000research.159630.2","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000}},{"DOI":"10.12688\/f1000research.159630.3","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000}},{"DOI":"10.12688\/f1000research.159630.4","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000}}],"reference-count":57,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP23K14429"],"award-info":[{"award-number":["JP23K14429"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p>\n                    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\n                    <ns3:italic>NOTCH1<\/ns3:italic>\n                    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\n                    <ns3:italic>NOTCH1<\/ns3:italic>\n                    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\n                    <ns3:italic>RAX<\/ns3:italic>\n                    transcription factor, which is essential for retinal progenitor cells, Muller glia, and cones. This result suggested that the regulation of\n                    <ns3:italic>NOTCH1<\/ns3:italic>\n                    expression might be achieved by chromatin remodeling at the\n                    <ns3:italic>NOTCH1<\/ns3:italic>\n                    locus, although single-cell RNA-seq analysis suggested that\n                    <ns3:italic>RAX<\/ns3:italic>\n                    expression regulation might also contribute to\n                    <ns3:italic>NOTCH1<\/ns3:italic>\n                    expression. Conclusions By analyzing retinal cell differentiation, this study identified a putative\n                    <ns3:italic>RAX<\/ns3:italic>\n                    binding site in the\n                    <ns3:italic>NOTCH1<\/ns3:italic>\n                    locus and raised the possibility that\n                    <ns3:italic>NOTCH1<\/ns3:italic>\n                    expression could be regulated by chromatin remodeling.\n                  <\/ns3:p>","DOI":"10.12688\/f1000research.159630.5","type":"journal-article","created":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T10:29:10Z","timestamp":1787308150000},"page":"31","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Expression status transition of\u00a0NOTCH1\u00a0accompanies chromatin remodeling in human early retinal progenitor cells"],"prefix":"10.12688","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-4611-8193","authenticated-orcid":false,"given":"Yoshitoku","family":"Watabe","sequence":"first","affiliation":[{"name":"College of Medicine, School of Medicine and Health Sciences Tsukuba Ibaraki Prefecture University of Tsukuba"},{"name":"Department of Anatomy and Embryology, Institute of Medicine Tsukuba University of Tsukuba"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-1242-2097","authenticated-orcid":false,"given":"Sakurako","family":"Kobayashi","sequence":"additional","affiliation":[{"name":"College of Medicine, School of Medicine and Health Sciences Tsukuba Ibaraki Prefecture University of Tsukuba"},{"name":"Department of Anatomy and Embryology, Institute of Medicine Tsukuba University of Tsukuba"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4023-4560","authenticated-orcid":false,"given":"Takahiro","family":"Nakayama","sequence":"additional","affiliation":[{"name":"College of Medicine, School of Medicine and Health Sciences Tsukuba Ibaraki Prefecture University of Tsukuba"},{"name":"Department of Anatomy and Embryology, Institute of Medicine Tsukuba University of Tsukuba"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8540-7760","authenticated-orcid":false,"given":"Satoru","family":"Takahashi","sequence":"additional","affiliation":[{"name":"College of Medicine, School of Medicine and Health Sciences Tsukuba Ibaraki Prefecture University of Tsukuba"},{"name":"Transborder Medical Center, Institute of Medicine Tsukuba University of Tsukuba"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0212-0909","authenticated-orcid":false,"given":"Masaharu","family":"Yoshihara","sequence":"additional","affiliation":[{"name":"College of Medicine, School of Medicine and Health Sciences Tsukuba Ibaraki Prefecture University of Tsukuba"},{"name":"Department of Primary Care and Medical Education, Institute of Medicine Tsukuba University of Tsukuba"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"8-28","DOI":"10.1093\/glycob\/cwaa049","article-title":"Multifaceted regulation of Notch signaling by glycosylation.","volume":"31","author":"A Pandey","year":"2021","journal-title":"Glycobiology."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"678-689","DOI":"10.1038\/nrm2009","article-title":"Notch signalling: a simple pathway becomes 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status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.175389.r359501, Mariko Kashiwagi, Massachusetts General Hospital, Charlestown, 05 Feb 2025, version 1, 2 approved with reservations","URL":"https:\/\/f1000research.com\/articles\/14-31\/v1#referee-response-359501","order":0,"name":"referee-response-359501","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Masaharu Yoshihara<\/b>; \n<i>Posted: 16 Sep 2025<\/i>; Dear Dr. Mariko Kashiwagi,  Thank you so much for your time and efforts in reviewing our manuscript titled \u201cExpression status transition of \n<i>NOTCH1<\/i> accompanies chromatin remodeling in human early retinal progenitor cells\u201d. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter. We would like to respond to your comments. Your comments are sequentially numbered and followed by our response in a point-by-point manner.  \n<b>Comments:<\/b> In this manuscript, the authors investigated whether NOTCH family genes (NOTCH1\u20134) 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. \n<b>Major Concerns and Comments:<\/b> \n<b>(1) Chromatin Accessibility Analysis:<\/b> 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. \n<b>Response:<\/b> 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 (\n<i>NOTCH1<\/i> and \n<i>NOTCH3<\/i> for the three samples) and Additional_file_1, 2 and 3 (\n<i>NOTCH2<\/i> and \n<i>NOTCH4&nbsp;<\/i>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., \u201cATAC_peaks_NOTCH1_d59.csv\u201d) in our GitHub repository (https:\/\/doi.org\/10.5281\/zenodo.17084024).  \n<b>(2) Interpretation of the Results:<\/b> The authors stated in the text that &quot;NOTCH1-3 were expressed primarily in early RPCs, with NOTCH1 and NOTCH3 being the most prominent genes.&quot; 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 &gt; NOTCH3, whereas at days 74 and 78, NOTCH2 and NOTCH3 exhibited similar expression levels. The authors are advised to interpret these data more carefully. \n<b>Response: <\/b>Thank you for your constructive comments. We agree to your comment and clearly have indicated that the early RPCs expressed \n<i>NOTCH1<\/i>, \n<i>NOTCH2<\/i> and \n<i>NOTCH3<\/i> for each sample in the Results section. In particular, \n<i>NOTCH1<\/i> showed the highest expressions throughout the three samples and we have clearly mentioned that we have focused in \n<i>NOTCH1<\/i> and \n<i>NOTCH3<\/i> in this study since \n<i>NOTCH3<\/i>has been suggested for early RPC differentiation (ref. 25) in the Results section for day 59 sample. Since the early RPCs expressed \n<i>NOTCH2<\/i> in all the three samples, we have also analyzed chromatin accessibility for \n<i>NOTCH2<\/i> in addition to \n<i>NOTCH1<\/i> and \n<i>NOTCH3<\/i>although we are unable to provide solid conclusions regarding this gene owing to varying findings on its chromatin accessibility, which is clearly mentioned in the Discussion section. Our findings and conclusion that \n<i>NOTCH1<\/i> mRNA expression and chromatin accessibility concomitantly changed during RPC differentiation, however, are solid in front of the unexplained results on \n<i>NOTCH2<\/i>.  \n<b>(3)<\/b> 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. \n<b>Response: <\/b>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 \n<i>NOTCH2<\/i> mRNA expression and chromatin accessibility, we are confident to the main conclusion regarding \n<i>NOTCH1<\/i>.  Sincerely, Masaharu Yoshihara, M.D., Ph.D. Specially Appointed Assistant Professor University of Tsukuba, Japan","URL":"https:\/\/f1000research.com\/articles\/14-31\/v5#referee-comment-14603","order":1,"name":"referee-comment-14603","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.188207.r416419, Zhongjie Tang, University of Southern California, Los Angeles, Southern California, 30 Sep 2025, version 2, 2 approved with reservations","URL":"https:\/\/f1000research.com\/articles\/14-31\/v2#referee-response-416419","order":2,"name":"referee-response-416419","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Masaharu Yoshihara<\/b>; \n<i>Posted: 01 Nov 2025<\/i>; Dear Dr. Zhongjie Tang  Thank you so much for your time and efforts in reviewing our manuscript titled \u201cExpression status transition of \n<i>NOTCH1&nbsp;<\/i>accompanies chromatin remodeling in human early retinal progenitor cells\u201d. We appreciate your constructive comments and have tried to include all the points. Here, we would like to submit a revised version of this manuscript with this response letter.  The authors have made some revisions to clarify the NOTCH1\u20133 expression patterns, expand the ATAC-seq analysis window, and add discussion on the NOTCH2 mRNA\u2013chromatin 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\u2014or explicitly noting what remains unknown\u2014about NOTCH3 in retinal or neural development would help justify its inclusion beyond descriptive expression levels.  Response: Thank you for your constructive comments. We agree with your comments and have included brief biological context for \n<i>NOTCH3<\/i> in the Introduction section. \n<i>NOTCH3<\/i> 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 \n<i>NOTCH3<\/i> 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) \n<i>Notch3<\/i> is well known for its involvement in the endothelial cells and vascular development as suggested by causing a cerebral vascular disease, CADASIL. \n<sup>26, 27 <\/sup>In addition to involvement in endothelium development, this gene is also reported to be associated with differentiation of glial cells in zebrafish retinas. \n<sup>28 <\/sup>However, it is unclear when and how these Notch receptor expressions are switched on and off in RPCs during early differentiation to impact on overall Notch signaling amount and oscillation.  2.Quantify motif evidence  The identification of LHX2\/PAX6\/RAX\/VSX2 motifs in variable peaks near NOTCH1 is currently presented qualitatively. Including simple summary statistics, such as enrichment p-values or correlations between TF expression and accessibility, would provide basic quantitative support for these observations without expanding the study\u2019s scope.  Response: Thank you for your insightful comments. We agree with your comments. To enhance quantitative aspects, we have added JASPAR motif search in the transcription factor binding motif enrichment analysis using the Signac pipeline, and observed that the motifs for LHX2, RAX and VSX2 were significantly enriched in the early RPCs while that for PAX6 showed relatively less enrichment. In addition, to support biological significance of these motif enrichments, we also carried out footprinting analysis and showed that actual bindings of these three transcription factors (LHX2, RAX and VSX2) in the early retinal progenitor cells. Unfortunately, we were technically unable to conduct the \n<i>NOTCH1<\/i> 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 \n<i>NOTCH1<\/i> 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 \n<i>NOTCH1<\/i> locus were presented in the Method section and \u201cSupplementary_table_motif.xlsx\u201d, respectively, with partial overlap with JASPAR motif database. The motifplot for the transcription factors was presented in \u201cJASPAR_motif.png.\u201d), chromatin remodeling of these regions possibly regulates \n<i>NOTCH1<\/i> expression transition during RPC differentiation although the expression transitions of \n<i>LHX2<\/i>, \n<i>PAX6<\/i>, \n<i>RAX<\/i> and \n<i>VSX2<\/i> may also support the \n<i>NOTCH1<\/i> 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) (\u201cMotif_enrichment_d59.csv\u201d). 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.","URL":"https:\/\/f1000research.com\/articles\/14-31\/v5#referee-comment-14887","order":3,"name":"referee-comment-14887","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.208123.r513384, Benedikt Obermayer, Berlin Institute of Health at Charit\u00e9 \u2013 Universit\u00e4tsmedizin Berlin, Berlin, 03 Sep 2026, version 5, indexed","URL":"https:\/\/f1000research.com\/articles\/14-31\/v5#referee-response-513384","order":4,"name":"referee-response-513384","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Masaharu Yoshihara<\/b>; \n<i>Posted: 03 Oct 2026<\/i>; Dear Dr. Benedikt Obermayer  Thank you very much for your time and effort in reviewing our manuscript. We appreciate your constructive comments and have added a paragraph in the Discussion section to address all points.  Added paragraph The chromatin remodeling region (chr9: 136542126\u2013136542839) containing a RAX-binding site (chr9: 136542636\u2013136542645) partly overlaps with a candidate CRE (chr9: 136542638\u2013136542845, Accession #: EH38E3919891) provided by SCREEN v4 (Zhiping Weng Lab, UMass Chan Medical School, https:\/\/screen.wenglab.org, Access date: Sep.25, 2026). \n<sup>49<\/sup> However, one limitation is that the score of this RAX-binding site in the JASPAR track of the UCSC Genome Browser is low (226, corresponding to a p-value of 10\n<sup>\u2212<\/sup>\n<sup>2.26<\/sup>), below the default display threshold of 400. Therefore, the RAX-binding site identified by our integrated analysis of transcription factor expression and binding motifs should be considered a candidate requiring future experimental validation. Another limitation is that the single-cell RNA\/ATAC-seq dataset would significantly affect the results. We used the three early time points (days 59, 74, and 78) in a single-cell RNA\/ATAC-seq dataset (GSE183684) \n<sup>20<\/sup> to balance computational burden and the number of retinal progenitors, although performing investigations using the full dataset could lead to different results.&nbsp;Indeed, the Retinal progenitors and Transitory cell clusters are widely distributed and partly overlap in the UMAP results (Figure 1E-F, Figure 2E-F, and Figure 3B-C). This is mainly because these cell clusters show similar gene expression before terminal differentiation, as shown in the original paper for this annotation, \n<sup>4<\/sup> and, in the query dataset, partly because we used only the three early time points, which may have excluded more differentiated cells. This limitation also supports the need for future experimental validation.  \n<b>Comment 1 (Dr. Benedikt Obermayer)<\/b> - 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.  \n<b>Response 1<\/b> We agree with you that retinal progenitor and transitory cells are widespread, with some overlap. We also see a similar trend in Figure 1A of the original paper used as the annotation reference by Kriukov et al. (PMID: 40912448), and these two cell clusters also showed similar gene expression (Figure 1B). However, because annotation is critical, as understood by the fact that your previous comment has already improved our manuscript, we added a discussion on the widespread and overlapping retinal progenitor and transitory cells and a limitation on using the three early samples.  \n<b>Comment 2 (Dr. Benedikt Obermayer)<\/b> - 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.  \n<b>Response 2<\/b> Thank you for your critical comment. It made us carefully consider the biological significance of our finding. We found that the remodeled region containing a RAX-binding site partly overlaps a candidate cis-regulatory element suggested in the recently published database (SCREEN-v4) by Zhiping Weng Lab (UMass Chan Medical School, https:\/\/screen.wenglab.org) (PMID: 41501460), supporting the potential significance of this region. However, we also agree that the JASPAR transcription factor score is important; we added a discussion of this point.  Thank you again for your constructive comments.","URL":"https:\/\/f1000research.com\/articles\/14-31\/v5#referee-comment-17333","order":5,"name":"referee-comment-17333","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.208123.r513381, Tomohiro Masuda, Kobe City Eye Hospital, Kobe, 28 Sep 2026, version 5, indexed","URL":"https:\/\/f1000research.com\/articles\/14-31\/v5#referee-response-513381","order":6,"name":"referee-response-513381","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Masaharu Yoshihara<\/b>; \n<i>Posted: 03 Oct 2026<\/i>; Dear Dr. Tomohiro Masuda,  Thank you very much for your time and effort in reviewing our manuscript. We appreciate your constructive comments.  \n<b>Comment 1 (Dr. Tomohiro Masuda)<\/b> 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&nbsp;\n<i>in silico<\/i>&nbsp;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.  \n<b>Response&nbsp;<\/b> Thank you for your comment. We checked and confirmed that the UMAP plots are correctly labeled. As we mentioned in Response 1, we believe the dispersed plots reflect similar gene expression between retinal progenitor and transitory cells, as well as our choice of three early samples from the GSE183684 dataset. We clearly mentioned this limitation in the newly added paragraph.  Thank you again for your constructive comments.","URL":"https:\/\/f1000research.com\/articles\/14-31\/v5#referee-comment-17334","order":7,"name":"referee-comment-17334","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"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.","order":8,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}