Identification and characterization of early human photoreceptor states and cell-state-specific retinoblastoma-related features
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Curated by eLife
eLife Assessment
In this paper, the authors use single-cell RNA sequencing to understand post-mitotic cone and rod developmental states and identify cone-specific features that contribute to retinoblastoma genesis. The work is important and the evidence is generally convincing. The findings of rod/cone fate determination at a very early stage are intriguing.
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Abstract
Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin, yet the developmental basis for their distinct behaviors is poorly understood. Here, we used deep full-length single-cell RNA-sequencing to distinguish post-mitotic cone and rod developmental states and identify cone-specific features that contribute to retinoblastomagenesis. The analyses revealed early post-mitotic cone- and rod-directed populations characterized by higher THRB or NRL regulon activities, an immature photoreceptor precursor population with concurrent cone and rod gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Unexpectedly, both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL and THRB isoforms and prematurely terminated THRB transcripts. Early L/M cone precursors exhibited successive expression of several lncRNAs along with MYCN , which composed the seventh most L/M-cone-specific regulon, and SYK , which contributed to the early cone precursors’ proliferative response to RB1 loss. These findings reveal previously unrecognized photoreceptor precursor states and a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation.
Impact Statement
Features acquired by early post-mitotic retinal cells underlie the distinct behaviors of rods and the cone cells of origin of retinoblastoma tumors.
Article activity feed
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
The authors have used full-length single-cell sequencing on a sorted population of human fetal retina to delineate expression patterns associated with the progression of progenitors to rod and cone photoreceptors. They find that rod and cone precursors contain a mix of rod/cone determinants, with a bias in both amounts and isoform balance likely deciding the ultimate cell fate. Markers of early rod/cone hybrids are clarified, and a gradient of lncRNAs is uncovered in maturing cones. Comparison of early rods and cones exposes an enriched MYCN regulon, as well as expression of SYK, which may contribute to tumor initiation in RB1 deficient cone precursors.
Strengths:
(1) The insight into how cone and rod transcripts are mixed together at first is important and …
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
The authors have used full-length single-cell sequencing on a sorted population of human fetal retina to delineate expression patterns associated with the progression of progenitors to rod and cone photoreceptors. They find that rod and cone precursors contain a mix of rod/cone determinants, with a bias in both amounts and isoform balance likely deciding the ultimate cell fate. Markers of early rod/cone hybrids are clarified, and a gradient of lncRNAs is uncovered in maturing cones. Comparison of early rods and cones exposes an enriched MYCN regulon, as well as expression of SYK, which may contribute to tumor initiation in RB1 deficient cone precursors.
Strengths:
(1) The insight into how cone and rod transcripts are mixed together at first is important and clarifies a long-standing notion in the field.
(2) The discovery of distinct active vs inactive mRNA isoforms for rod and cone determinants is crucial to understanding how cells make the decision to form one or the other cell type. This is only really possible with full-length scRNAseq analysis.
(3) New markers of subpopulations are also uncovered, such as CHRNA1 in rod/cone hybrids that seem to give rise to either rods or cones.
(4) Regulon analyses provide insight into key transcription factor programs linked to rod or cone fates.
(5) The gradient of lncRNAs in maturing cones is novel, and while the functional significance is unclear, it opens up a new line of questioning around photoreceptor maturation.
(6) The finding that SYK mRNA is naturally expressed in cone precursors is novel, as previously it was assumed that SYK expression required epigenetic rewiring in tumors.
Weaknesses:
(1) The writing is very difficult to follow. The nomenclature is confusing and there are contradictory statements that need to be clarified.
(2) The drug data is not enough to conclude that SYK inhibition is sufficient to prevent the division of RB1 null cone precursors. Drugs are never completely specific so validation is critical to make the conclusion drawn in the paper.
We thank the reviewer for describing the study’s strengths and weaknesses. In the upcoming revision, we will:
(1) improve the writing and clarify the nomenclature and contradictory statements, particularly those noted in the Reviewer’s Recommendations for Authors; and
(2) scale back the claims related to the role of SYK in the cone precursor response to RB1 loss; we agree that genetic perturbation of SYK is required to prove it’s role and will perform such analyses in a separate study.
Reviewer #2 (Public review):
Summary:
The authors used deep full-length single-cell sequencing to study human photoreceptor development, with a particular emphasis on the characteristics of photoreceptors that may contribute to retinoblastoma.
Strengths:
This single-cell study captures gene regulation in photoreceptors across different developmental stages, defining post-mitotic cone and rod populations by highlighting their unique gene expression profiles through analyses such as RNA velocity and SCENIC. By leveraging full-length sequencing data, the study identifies differentially expressed isoforms of NRL and THRB in L/M cone and rod precursors, illustrating the dynamic gene regulation involved in photoreceptor fate commitment. Additionally, the authors performed high-resolution clustering to explore markers defining developing photoreceptors across the fovea and peripheral retina, particularly characterizing SYK's role in the proliferative response of cones in the RB loss background. The study provides an in-depth analysis of developing human photoreceptors, with the authors conducting thorough analyses using full-length single-cell RNA sequencing. The strength of the study lies in its design, which integrates single-cell full-length RNA-seq, long-read RNA-seq, and follow-up histological and functional experiments to provide compelling evidence supporting their conclusions. The model of cell type-dependent splicing for NRL and THRB is particularly intriguing. Moreover, the potential involvement of the SYK and MYC pathways with RB in cone progenitor cells aligns with previous literature, offering additional insights into RB development.
Weaknesses:
The manuscript feels somewhat unfocused, with a lack of a strong connection between the analysis of developing photoreceptors, which constitutes the bulk of the manuscript, and the discussion on retinoblastoma. Additionally, given the recent publication of several single-cell studies on the developing human retina, it is important for the authors to cross-validate their findings and adjust their statements where appropriate.
We thank the reviewer for summarizing the main findings and for noting the compelling support for the conclusions, the intriguing cell type-dependent splicing of rod and cone lineage factors, and the insights into retinoblastoma development.
We concur that some studies of developing photoreceptors were not well connected to retinoblastoma, which diminished the focus. However, we suggest that it was valuable to highlight how deep, long read sequencing provided new insights into retinoblastoma. For example, our demonstration of similar rod- and cone-related gene expression in early cones and RB cells addressed concerns with the proposed cone cell-of-origin, adding disease relevance.
We will address the Reviewer’s request to cross-validate our findings with those of other single-cell studies of developing human retina and to adjust the related statements in our upcoming revision.
Reviewer #3 (Public review):
Summary:
The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.
Strengths:
The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.
Human developing fetal retinal tissue samples were collected between 13-19 gestational weeks and this provides a substantially higher depth of sequencing coverage, thereby identifying both rare transcripts and alternative splice forms, and thereby representing an important advance over previous droplet-based scRNA-Seq studies of human retinal development.
Weaknesses:
The weaknesses identified are relatively minor. This is a technically strong and thorough study, that is broadly useful to investigators studying retinal development and retinoblastoma.
We thank the reviewer for describing the strengths of the study. Our upcoming revision will address the minor concerns that were raised separately in the Reviewer’s Recommendations for Authors.
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eLife Assessment
In this paper, the authors use single-cell RNA sequencing to understand post-mitotic cone and rod developmental states and identify cone-specific features that contribute to retinoblastoma genesis. The work is important and the evidence is generally convincing. The findings of rod/cone fate determination at a very early stage are intriguing.
-
Reviewer #1 (Public review):
Summary:
The authors have used full-length single-cell sequencing on a sorted population of human fetal retina to delineate expression patterns associated with the progression of progenitors to rod and cone photoreceptors. They find that rod and cone precursors contain a mix of rod/cone determinants, with a bias in both amounts and isoform balance likely deciding the ultimate cell fate. Markers of early rod/cone hybrids are clarified, and a gradient of lncRNAs is uncovered in maturing cones. Comparison of early rods and cones exposes an enriched MYCN regulon, as well as expression of SYK, which may contribute to tumor initiation in RB1 deficient cone precursors.
Strengths:
(1) The insight into how cone and rod transcripts are mixed together at first is important and clarifies a long-standing notion in the …
Reviewer #1 (Public review):
Summary:
The authors have used full-length single-cell sequencing on a sorted population of human fetal retina to delineate expression patterns associated with the progression of progenitors to rod and cone photoreceptors. They find that rod and cone precursors contain a mix of rod/cone determinants, with a bias in both amounts and isoform balance likely deciding the ultimate cell fate. Markers of early rod/cone hybrids are clarified, and a gradient of lncRNAs is uncovered in maturing cones. Comparison of early rods and cones exposes an enriched MYCN regulon, as well as expression of SYK, which may contribute to tumor initiation in RB1 deficient cone precursors.
Strengths:
(1) The insight into how cone and rod transcripts are mixed together at first is important and clarifies a long-standing notion in the field.
(2) The discovery of distinct active vs inactive mRNA isoforms for rod and cone determinants is crucial to understanding how cells make the decision to form one or the other cell type. This is only really possible with full-length scRNAseq analysis.
(3) New markers of subpopulations are also uncovered, such as CHRNA1 in rod/cone hybrids that seem to give rise to either rods or cones.
(4) Regulon analyses provide insight into key transcription factor programs linked to rod or cone fates.
(5) The gradient of lncRNAs in maturing cones is novel, and while the functional significance is unclear, it opens up a new line of questioning around photoreceptor maturation.
(6) The finding that SYK mRNA is naturally expressed in cone precursors is novel, as previously it was assumed that SYK expression required epigenetic rewiring in tumors.
Weaknesses:
(1) The writing is very difficult to follow. The nomenclature is confusing and there are contradictory statements that need to be clarified.
(2) The drug data is not enough to conclude that SYK inhibition is sufficient to prevent the division of RB1 null cone precursors. Drugs are never completely specific so validation is critical to make the conclusion drawn in the paper.
-
Reviewer #2 (Public review):
Summary:
The authors used deep full-length single-cell sequencing to study human photoreceptor development, with a particular emphasis on the characteristics of photoreceptors that may contribute to retinoblastoma.
Strengths:
This single-cell study captures gene regulation in photoreceptors across different developmental stages, defining post-mitotic cone and rod populations by highlighting their unique gene expression profiles through analyses such as RNA velocity and SCENIC. By leveraging full-length sequencing data, the study identifies differentially expressed isoforms of NRL and THRB in L/M cone and rod precursors, illustrating the dynamic gene regulation involved in photoreceptor fate commitment. Additionally, the authors performed high-resolution clustering to explore markers defining developing …
Reviewer #2 (Public review):
Summary:
The authors used deep full-length single-cell sequencing to study human photoreceptor development, with a particular emphasis on the characteristics of photoreceptors that may contribute to retinoblastoma.
Strengths:
This single-cell study captures gene regulation in photoreceptors across different developmental stages, defining post-mitotic cone and rod populations by highlighting their unique gene expression profiles through analyses such as RNA velocity and SCENIC. By leveraging full-length sequencing data, the study identifies differentially expressed isoforms of NRL and THRB in L/M cone and rod precursors, illustrating the dynamic gene regulation involved in photoreceptor fate commitment. Additionally, the authors performed high-resolution clustering to explore markers defining developing photoreceptors across the fovea and peripheral retina, particularly characterizing SYK's role in the proliferative response of cones in the RB loss background. The study provides an in-depth analysis of developing human photoreceptors, with the authors conducting thorough analyses using full-length single-cell RNA sequencing. The strength of the study lies in its design, which integrates single-cell full-length RNA-seq, long-read RNA-seq, and follow-up histological and functional experiments to provide compelling evidence supporting their conclusions. The model of cell type-dependent splicing for NRL and THRB is particularly intriguing. Moreover, the potential involvement of the SYK and MYC pathways with RB in cone progenitor cells aligns with previous literature, offering additional insights into RB development.
Weaknesses:
The manuscript feels somewhat unfocused, with a lack of a strong connection between the analysis of developing photoreceptors, which constitutes the bulk of the manuscript, and the discussion on retinoblastoma. Additionally, given the recent publication of several single-cell studies on the developing human retina, it is important for the authors to cross-validate their findings and adjust their statements where appropriate.
-
Reviewer #3 (Public review):
Summary:
The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.
Strengths:
The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.
Human developing fetal retinal tissue samples were collected between 13-19 gestational weeks and this provides a substantially higher depth of sequencing coverage, thereby identifying both rare transcripts and alternative splice forms, and thereby representing an important advance over previous droplet-based scRNA-Seq …
Reviewer #3 (Public review):
Summary:
The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.
Strengths:
The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.
Human developing fetal retinal tissue samples were collected between 13-19 gestational weeks and this provides a substantially higher depth of sequencing coverage, thereby identifying both rare transcripts and alternative splice forms, and thereby representing an important advance over previous droplet-based scRNA-Seq studies of human retinal development.
Weaknesses:
The weaknesses identified are relatively minor. This is a technically strong and thorough study, that is broadly useful to investigators studying retinal development and retinoblastoma.
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