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Status |
Public on Apr 01, 2024 |
Title |
NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development |
Organism |
Homo sapiens |
Experiment type |
Expression profiling by high throughput sequencing Other
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Summary |
While dysfunction and/or death of light-detecting photoreceptor cells underlies most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids using induced pluripotent stem cells (iPSC) derived from a patient with genetic photoreceptor disease, an isogenic control, and an unrelated control. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation; up to 260 days of in vitro differentiation. Using single-cell transcriptomics data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint in development unique to the disease state that gives rise to a divergent rod photoreceptor cell population. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including expression of the light-sensitive protein rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes at both the transcript and protein level. Using joint multimodal single-cell sequencing on late-stage retinal organoids, we further identify the specific putative regulatory sites where rod-specific factors act to steer rod and cone photoreceptor cell development. Importantly, these findings are strikingly different than that observed in rodent models of disease. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation.
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Overall design |
Three iPSC lines (ND [no disease] Control, NR2E3-null, and Isogenic Control) were differentiated to retinal organoids for 260 days. Samples were assayed by 3' scRNAseq at D40, D80, D120, D160 and D260 from all three lines. In a separate differentiation batch, NR2E3-null and Isogenic Control organoids were assayed by joint (RNA & ATAC) single-nucleus multimodal sequencing at D160 and D280. Multimodal runs yield paired scRNAseq and scATACseq libraries. For AAV experiments, NR2E3-null organoids were cultured for 120 days. Organoids were treated with AAV carrying NR2E3-T2A-GFP or GFP or left untransduced as described. Organoids were harvested at D120 or following treatment at D160 and assayed by 3' scRNAseq. For ESCS Patient 2 experiment, an iPSC line from ESCS Patient 2 was differentiated to retinal organoids and assayed at D80 and D160 by 3' scRNAseq. >>>Raw data were not provided for ATAC-seq Samples due to patient privacy concerns<<<
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Contributor(s) |
Mullin NK, Bohrer LR, Tucker BA |
Citation(s) |
38652563 |
NIH grant(s) |
Grant ID |
Grant title |
Affiliation |
Name |
R01 EY033308 |
Molecular Studies of the Choriocapillaris in AMD |
THE UNIVERSITY OF IOWA |
Budd A Tucker |
R01 EY033331 |
A Next Generation Outer Retinal Microphysiological System |
THE UNIVERSITY OF IOWA |
Budd A Tucker |
T32 GM008629 |
Predoctoral Training Program in Genetics |
THE UNIVERSITY OF IOWA |
Daniel F Eberl |
T32 GM139776 |
Medical Scientist Training Program |
THE UNIVERSITY OF IOWA |
Steven R Lentz |
F30 EY034009 |
Pathogenesis of Mitochondrial Retina Disease |
THE UNIVERSITY OF IOWA |
Nathaniel Kevin Mullin |
F30 EY031923 |
The Membrane Attack Complex and the Choriocapillaris in Health and Age-Related Macular Degeneration |
THE UNIVERSITY OF IOWA |
Andrew P Voigt |
P30 EY025585 |
NEI Center Core Grant for Vision Research |
CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE - CWRU |
Bela Anand-Apte |
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Submission date |
Jun 29, 2023 |
Last update date |
Apr 25, 2024 |
Contact name |
Nathaniel Kevin Mullin |
Organization name |
University of Iowa
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Street address |
375 Newton Road
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City |
Iowa City |
State/province |
IA |
ZIP/Postal code |
52246 |
Country |
USA |
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Platforms (1) |
GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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Samples (29)
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GSM8181635 |
retinal organoid, NR2E3-null, D130, untransduced, scRNAseq |
GSM8181636 |
retinal organoid, NR2E3-null, D160, aav-gfp, scRNAseq |
GSM8181637 |
retinal organoid, NR2E3-null, D160, aav-nr2e3-gfp, scRNAseq |
GSM8181638 |
retinal organoid, NR2E3-null, D160, untransduced, scRNAseq |
GSM8181639 |
retinal organoid, escs_patient_2, D80, scRNAseq |
GSM8181640 |
retinal organoid, escs_patient_2, D160, scRNAseq |
GSM8181641 |
retinal organoid, ND Control, D40, scRNAseq |
GSM8181642 |
retinal organoid, NR2E3-null, D40, scRNAseq |
GSM8181643 |
retinal organoid, Isogenic Control, D40, scRNAseq |
GSM8181644 |
retinal organoid, ND Control, D80, scRNAseq |
GSM8181645 |
retinal organoid, NR2E3-null, D80, scRNAseq |
GSM8181646 |
retinal organoid, Isogenic Control, D80, scRNAseq |
GSM8181647 |
retinal organoid, ND Control, D120, scRNAseq |
GSM8181648 |
retinal organoid, NR2E3-null, D120, scRNAseq |
GSM8181649 |
retinal organoid, Isogenic Control, D120, scRNAseq |
GSM8181650 |
retinal organoid, ND Control, D160, scRNAseq |
GSM8181651 |
retinal organoid, NR2E3-null, D160, scRNAseq |
GSM8181652 |
retinal organoid, Isogenic Control, D160, scRNAseq |
GSM8181653 |
retinal organoid, ND Control, D260, scRNAseq |
GSM8181654 |
retinal organoid, NR2E3-null, D260, scRNAseq |
GSM8181655 |
retinal organoid, Isogenic Control, D260, scRNAseq |
GSM8181656 |
retinal organoid, NR2E3-null, D160, Multiome (GEX) |
GSM8181657 |
retinal organoid, Isogenic Control, D160, Multiome (GEX) |
GSM8181658 |
retinal organoid, NR2E3-null, D160, Multiome (ATAC) |
GSM8181659 |
retinal organoid, Isogenic Control, D160, Multiome (ATAC) |
GSM8181660 |
retinal organoid, NR2E3-null, D260, Multiome (GEX) |
GSM8181661 |
retinal organoid, Isogenic Control, D260, Multiome (GEX) |
GSM8181662 |
retinal organoid, NR2E3-null, D260, Multiome (ATAC) |
GSM8181663 |
retinal organoid, Isogenic Control, D260, Multiome (ATAC) |
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Relations |
BioProject |
PRJNA989235 |
Supplementary file |
Size |
Download |
File type/resource |
GSE236197_RAW.tar |
1.8 Gb |
(http)(custom) |
TAR (of CSV) |
SRA Run Selector |
Raw data not provided for this record |
Processed data provided as supplementary file |
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