A transcriptome atlas of the mouse iris at single-cell resolution defines cell types and the genomic response to pupil dilation
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
Using single nucleus RNA sequencing, the authors have characterized all major cell types in the mouse iris and ciliary body, defined new types of iris stromal and sphincter cells, and shown cell-specific transcriptome responses in the resting, constricted, and dilated states and identified and validated antibodies and in situ hybridization probes for visualization of major iris cell types. This work will be a valuable reference for investigations of iris development, disease, and pharmacology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (eLife)
Abstract
The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmologic diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting vs. dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre -expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre -expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.
Article activity feed
-
Evaluation Summary:
Using single nucleus RNA sequencing, the authors have characterized all major cell types in the mouse iris and ciliary body, defined new types of iris stromal and sphincter cells, and shown cell-specific transcriptome responses in the resting, constricted, and dilated states and identified and validated antibodies and in situ hybridization probes for visualization of major iris cell types. This work will be a valuable reference for investigations of iris development, disease, and pharmacology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
This study establishes fundamental information on the mouse iris and its function. Using single nucleus RNA sequencing, the authors have characterized all major cell types in the mouse iris and ciliary body, defined two types of iris stromal cells and two types of iris sphincter cells, and shown cell-specific transcriptome responses in the resting, constricted, and dilated states. They have identified and validated antibody and in situ hybridization probes for visualization of the major iris cell types. They have quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre expressing progenitors contribute only to stromal cells. This …
Reviewer #1 (Public Review):
This study establishes fundamental information on the mouse iris and its function. Using single nucleus RNA sequencing, the authors have characterized all major cell types in the mouse iris and ciliary body, defined two types of iris stromal cells and two types of iris sphincter cells, and shown cell-specific transcriptome responses in the resting, constricted, and dilated states. They have identified and validated antibody and in situ hybridization probes for visualization of the major iris cell types. They have quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre expressing progenitors contribute only to stromal cells. This work will be a valuable reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.
This paper was a pleasure to read. It is well written, thorough, and will provide tools to study the iris and ciliary body for the research community. I had no major concerns.
-
Reviewer #2 (Public Review):
Major strengths of the manuscript:
Using single nucleus RNA sequencing technology had several advantages over single cell RNA sequencing with minimum disturbance of the native transcriptional profiles.
This research revealed major cell types in the mouse iris and provided valuable and verifiable markers for each of the iris cell types. This research generated great resources for future studies on normal and diseased irises.
The study showed very interesting changes in the transcriptome and nuclear morphology associated with iris dilation, and the most upregulated genes identified could be great candidates for studying iris function and malfunction in diseases.
The study provided definitive experimental proof showing the neural crest contribution to the various iris cell types.
Overall, the study was well …
Reviewer #2 (Public Review):
Major strengths of the manuscript:
Using single nucleus RNA sequencing technology had several advantages over single cell RNA sequencing with minimum disturbance of the native transcriptional profiles.
This research revealed major cell types in the mouse iris and provided valuable and verifiable markers for each of the iris cell types. This research generated great resources for future studies on normal and diseased irises.
The study showed very interesting changes in the transcriptome and nuclear morphology associated with iris dilation, and the most upregulated genes identified could be great candidates for studying iris function and malfunction in diseases.
The study provided definitive experimental proof showing the neural crest contribution to the various iris cell types.
Overall, the study was well designed and precisely executed, the data analysis was clear and scientifically stringent, the results are comprehensive and revealing novel molecular correlates of cellular responses.
-
Reviewer #3 (Public Review):
This work defines the mouse iris transcriptomic atlas by single-nucleus RNA-seq (snRNA-seq), an approach that captures nuclear transcripts without enzymatic cell dissociation and processing. The major cell types defined/revealed are independently and rigorously validated by immunofluorescence and fluorescence in-situ hybridization. Immunofluorescence and fluorescence in-situ hybridization experiments further confirmed distinction between sphincter and dilator muscles and revealed distinct distribution of subtypes of sphincter and stromal cells. More importantly, the snRNA-seq approach they have undertaken, though only capturing the nuclear transcripts, is sufficient to profile the transcriptomic changes during constriction and dilation, and some of the expression changes were confirmed by immunofluorescence. …
Reviewer #3 (Public Review):
This work defines the mouse iris transcriptomic atlas by single-nucleus RNA-seq (snRNA-seq), an approach that captures nuclear transcripts without enzymatic cell dissociation and processing. The major cell types defined/revealed are independently and rigorously validated by immunofluorescence and fluorescence in-situ hybridization. Immunofluorescence and fluorescence in-situ hybridization experiments further confirmed distinction between sphincter and dilator muscles and revealed distinct distribution of subtypes of sphincter and stromal cells. More importantly, the snRNA-seq approach they have undertaken, though only capturing the nuclear transcripts, is sufficient to profile the transcriptomic changes during constriction and dilation, and some of the expression changes were confirmed by immunofluorescence. The identification of transcription factors associated with defined cell types also allows tests of an unexplored question- does nuclear morphology change along with known changes in the the cell plasma during dilation? The authors assessed the nuclear morphology of each cell type by immunofluorescence of cell-type specific transcription factors they identified from snRNA-seq in this study, and found cell-type specific changes of nuclear morphology during dilation. Finally, the authors revisited a partially conflicting result on the neural crest cells contribution to iris cell types, with characterized transcription factors in this study to increase resolution. Overall, this is a rigorous study and could have broad interests. This version of manuscript could benefit from more details in statistics and methodology in some analyses. Despite the insufficient technical/statistical details in some figures, the authors' major claims and the identified sub-celltypes are justified by their data.
-
-
