Sphingosine-1-phosphate signaling regulates the ability of Müller glia to become neurogenic, proliferating progenitor-like cells

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.

Methods
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.