DOI: 10.5061/dryad.tdz08kq8t
title: Sphingosine-1-phosphate signaling regulates the ability of Müller glia to become neurogenic, proliferating progenitor-like cells

description: 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.
description: 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: <em>GLUL, VIM, SCL1A3, RLBP1</em>, (2) MGPCs:
 <em>PCNA, CDK1, TOP2A, ASCL1</em>, (3) microglia:
 <em>C1QA, C1QB, CCL4, CSF1R, TMEM22</em>, (4) ganglion cells:
 <em>THY1, POU4F2, RBPMS2, NEFL, NEFM</em>, (5) amacrine cells:
 <em>GAD67, CALB2, TFAP2A</em>, (6) horizontal cells:
 <em>PROX1, CALB2, NTRK1</em>, (7) bipolar cells:
 <em>VSX1, OTX2, GRIK1, GABRA1</em>, and (7) cone
 photoreceptors: <em>CALB1, GNAT2, GNB3, OPN1LW</em>, and (8)
 rod photoreceptors: <em>RHO, NR2E3, ARR3. </em>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.
description: # Sphingosine-1-phosphate signaling regulates the ability of Müller glia
 to become neurogenic, proliferating progenitor-like cells
 [https://doi.org/10.5061/dryad.tdz08kq8t](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](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](https://elifesciences.org/reviewed-preprints/102151#c45); [Satija et al., 2015](https://elifesciences.org/reviewed-preprints/102151#c50)), 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](mailto: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](https://satijalab.org/seurat/articles/essential_commands) ## Access information Other publicly accessible locations of the data: [https://osumc.sharepoint.com/sites/Links/FischerLab/Forms/AllItems.aspx?ct=1741028626517&or=OWA%2DNT%2DMail&ga=1&LOF=1&id=%2Fsites%2FLinks%2FFischerLab%2Fchick%20retina%20scRNA%2Dseq%20Cell%20Ranger%20output%20files&viewid=3807c9c4%2D0481%2D4290%2Dad09%2Deeb5748b62b2](https://osumc.sharepoint.com/sites/Links/FischerLab/Forms/AllItems.aspx?ct=1741028626517&or=OWA%2DNT%2DMail&ga=1&LOF=1&id=%2Fsites%2FLinks%2FFischerLab%2Fchick%20retina%20scRNA%2Dseq%20Cell%20Ranger%20output%20files&viewid=3807c9c4%2D0481%2D4290%2Dad09%2Deeb5748b62b2)