Sphingosine-1-phosphate signaling regulates the ability of Müller glia to become neurogenic, proliferating progenitor-like cells
Data files
May 01, 2025 version files 2.02 GB
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chick_retina_nmda_12_hrs.zip
135.57 MB
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chick_retina_nmda_3_hrs.zip
59.51 MB
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chick_retina_nmda_48_hrs_rep1.zip
89.48 MB
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chick_retina_nmda_48_hrs_SAHH_inhibitor.zip
104.67 MB
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chick_retina_nmda_48hrs_FABP_inhibitor.zip
98.55 MB
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chick_retina_nmda_48hrs_rep2.zip
123.26 MB
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chick_retina_saline_control.zip
73.15 MB
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chick_retina_saline_FABP_inhibitor.zip
73.71 MB
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chick_retina_saline_SAHH_inhibitor.zip
112.26 MB
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chick_retina_v2_2_doses_insulin_fgf2.zip
103.47 MB
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chick_retina_v2_3_doses_insulin_fgf2.zip
89.52 MB
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chick_retina_v2_nmda_24_hrs_rep1.zip
107.66 MB
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chick_retina_v2_nmda_24hrs_rep2.zip
148.35 MB
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chick_retina_v2_nmda_48_hrs_insulin_fgf2_rep1.zip
87.24 MB
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chick_retina_v2_nmda_48_hrs_insulin_fgf2_rep2.zip
68.61 MB
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chick_retina_v2_nmda_48_hrs_rep1.zip
22.58 MB
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chick_retina_v2_nmda_48_hrs_rep2.zip
123.26 MB
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chick_retina_v2_nmda_72_hrs_rep1.zip
144.27 MB
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chick_retina_v2_nmda_72_hrs_rep2.zip
105.18 MB
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chick_retina_v2_saline_control_rep1.zip
25.90 MB
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chick_retina_v2_saline_control_rep2.zip
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README.md
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Abstract
The purpose of these studies is to investigate how Sphingosine-1-phosphate (S1P) signaling regulates glial phenotype, dedifferentiation of Müller glia (MG), reprogramming into proliferating MG-derived progenitor cells (MGPCs), and neuronal differentiation of the progeny of MGPCs in the chick retina. We found that S1P-related genes are highly expressed by retinal neurons and glia, and levels of expression were dynamically regulated following retinal damage. Drug treatments that activate S1P receptor 1 (S1PR1) or increase levels of S1P suppressed the formation of MGPCs. Conversely, treatments that inhibit S1PR1 or decrease levels of S1P stimulated the formation of MGPCs. Inhibition of S1P receptors or S1P synthesis significantly enhanced the neuronal differentiation of the progeny of MGPCs. We report that S1P-related gene expression in MG is modulated by microglia and inhibition of S1P receptors or S1P synthesis partially rescues the loss of MGPC formation in damaged retinas missing microglia. Finally, we show that TGFβ/Smad3 signaling in the resting retina maintains S1PR1 expression in MG. We conclude that the S1P signaling is dynamically regulated in MG and MGPCs in the chick retina, and activation of S1P signaling depends, in part, on signals produced by reactive microglia.
https://doi.org/10.5061/dryad.tdz08kq8t
General Information
Dataset Overview
A detailed description of the general framework and specific methodology can be found in the relevant publication (https://doi.org/10.7554/eLife.102151.4).
For each dataset, barcode, feature, and matrix file from CellRanger output are provided. These files serve as inputs for preparing the Seurat objects used in this study. Barcode files contain a list of cell barcodes. Feature files contain gene names from the reference used for CellRanger and include 3 columns: ENSEMBL number, gene name, and the type of assay run ("GENE EXPRESSION"). Matrix files contain the sparse matrix containing UMI counts for each library.
Dissociated cells were loaded onto the 10X Chromium Cell Controller with Chromium 3’ V2, V3 or Next GEM reagents. Using Seurat toolkits (Powers and Satija, 2015; Satija et al., 2015), Uniform Manifold Approximation and Projection (UMAP) for dimensional reduction plots were generated from 9 separate cDNA libraries, including 2 replicates of control undamaged retinas, and retinas at different times after NMDA-treatment. Seurat was used to construct gene lists for differentially expressed genes (DEGs), violin/scatter plots, and dot plots.
Corresponding Author
Name: Andy J. Fischer
ORCID: 0000-0001-6123-7405
Email: andrew.fischer@osumc.edu
Affiliation: Department of Neuroscience, Ohio State University, Columbus, OH
Related Publications
Campbell W. A., Blum S.Reske A., Hoang T., Blackshaw S., Fischer A. J (2021b) Cannabinoid signaling promotes the de-differentiation and proliferation of Müller glia-derived progenitor cells Glia 69:2503–2521
Campbell W. A., Tangeman A., El-Hodiri H. M., Hawthorn E. C., Hathoot M., Blum S., Hoang T., Blackshaw S., Fischer A. J (2022) Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the chick retina Development 149
El-Hodiri H. M., Campbell W. A., Kelly L. E., Hawthorn E. C., Schwartz M., Jalligampala A., McCall M. A., Meyer K., Fischer A. J. (2022) Nuclear Factor I in neurons, glia and during the formation of Müller glia-derived progenitor cells in avian, porcine and primate retinas J Comp Neurol 530:1213–1230
El-Hodiri H. M., Bentley J. R., Reske A. G., Taylor O. B., Palazzo I., Campbell W. A., Halloy N. R., Fischer A. J (2023) Heparin-binding epidermal growth factor and fibroblast growth factor 2 rescue Müller glia-derived progenitor cell formation in microglia- and macrophage-ablated chick retinas Development 150
Hoang T., Wang J., Boyd P., Wang F., Santiago C., Jiang L., Yoo S., Lahne M., Todd L.J., Jia M.et al. (2020) Gene regulatory networks controlling vertebrate retinal regeneration Science 370
Notes for files
Description of files and naming convention
The compressed zip folders are titled as 'animal_tissue_reagent_treatment_tissue collection timepoint_replicate'. For example, chick_retina_v2_nmda_72_hrs_rep2.zip contains Gene-Cell matrices for Gallus gallus retinal tissue, collected 72 hours after NMDA-treatment, with 10X Chromium Controller V2 reagents, and as the 2nd biological replicate.
The compressed zip folders contain the following files:
- barcodes.tsv
- genes.tsv
- matrix.mtx
Usage
To perform single-cell RNA sequencing data analysis using the Seurat package, the following R packages must be installed:
- Seurat
- dplyr
Single-cell RNA sequencing data analysis pipeline is available at https://satijalab.org/seurat/articles/essential_commands
Access information
Other publicly accessible locations of the data:
We analyzed scRNA-seq libraries that were generated and characterized previously (Campbell et al., 2021b; Campbell et al., 2022; El-Hodiri et al., 2022; El-Hodiri et al., 2023, 2021; Hoang et al., 2020; Li et al., 2023; Lyu et al., 2023). Dissociated cells were loaded onto the 10X Chromium Cell Controller with Chromium 3’ V2, V3 or Next GEM reagents. Using Seurat toolkits (Powers and Satija, 2015; Satija et al., 2015), Uniform Manifold Approximation and Projection (UMAP) for dimensional reduction plots were generated from 9 separate cDNA libraries, including 2 replicates of control undamaged retinas, and retinas at different times after NMDA-treatment. Seurat was used to construct gene lists for differentially expressed genes (DEGs), violin/scatter plots, and dot plots. Significance of difference in violin/scatter plots was determined using a Wilcoxon Rank Sum test with Bonferroni correction. Genes that were used to identify different types of retinal cells included the following: (1) Müller glia: GLUL, VIM, SCL1A3, RLBP1, (2) MGPCs: PCNA, CDK1, TOP2A, ASCL1, (3) microglia: C1QA, C1QB, CCL4, CSF1R, TMEM22, (4) ganglion cells: THY1, POU4F2, RBPMS2, NEFL, NEFM, (5) amacrine cells: GAD67, CALB2, TFAP2A, (6) horizontal cells: PROX1, CALB2, NTRK1, (7) bipolar cells: VSX1, OTX2, GRIK1, GABRA1, and (7) cone photoreceptors: CALB1, GNAT2, GNB3, OPN1LW, and (8) rod photoreceptors: RHO, NR2E3, ARR3. The MG have an over-abundant representation in the scRNA-seq databases. This likely resulted from fortuitous capture-bias and/or tolerance of the MG to the dissociation process.