Fig 1.
Transcriptomic characterization of cell types in the daytime rat pineal gland.
(A) t-Distributed stochastic neighbor embedding (t-SNE) visualization of 5,667 daytime rat pineal gland cells profiled by scRNA-seq. Cell types are color-coded by cluster assigned from the shared nearest neighbor (SNN) clustering algorithm. (B) Hierarchical clustering dendrogram showing transcriptomic similarity of cell types, including relationships of the two pinealocyte subtypes, the three astrocyte subtypes, the two microglia subtypes, and two vascular-associated cell types: VLMCs and endothelial cells. (C) Violin plots of select marker gene expression distribution for cells from each cell type. Y-Axis is natural log of normalized counts.
Fig 2.
IHC reveals cell type-specific patterns of expression.
Maximum intensity projections taken from IHC sections through the rat pineal gland midline with rostral stalk origin at the bottom. Images include the whole length and middle third of the width of the gland. Scale bar = 100 μm. (A) Asmt-positive pinealocytes are uniformly distributed. (B) Slc1a3-positive γ-astrocytes are most abundant in rostral region near the stalk. (C) S100b-positive cells are most abundant in the rostral region and appear elsewhere with distinctly lower density and expression strength. (D) Aif1-positive cells are unevenly distributed throughout pineal gland at low density. See S6 Fig for full images.
Fig 3.
scRNA-seq reveals two transcriptionally distinct pinealocyte populations.
(A) Heatmap of expression values for top 10 most differential expressed genes (by effect size) for α- and β-pinealocytes. Expression values are Z-scores of counts calculated between all cells of the two cell types. Each column represents one cell; random samples of 250 cells per cell type are shown. (B) Violin plots showing expression distribution differences between two pinealocyte subtypes for three functional groups and one gene, Asmt. Y-Axis is either normalized counts or natural log (ln) of normalized counts. Horizontal lines represent the mean. (*) indicates p<0.001, Wilcoxon rank sum test. All cells from each subtype are included (α-pinealocyte, n = 275; β-pinealocyte, n = 4,822). Mitochondrial group includes differentially expressed mitochondrial OxPhos genes (p<0.05, fold change ≥2.0), ribosomal group includes top 20 most differential ribosomal genes by effect size (p<0.05, fold change ≥2.0), G-protein γ-subunits include Gngt1, Gngt2, Gng10, and Gng13 (see S5 Fig for individual genes).
Fig 4.
scRNA-seq reveals three transcriptionally distinct astrocyte populations.
Heatmap of expression values for top 6 most differential expressed genes (by effect size) for α-, β-, and γ-astrocytes. Expression values are Z-scores of counts calculated between all cells of the three cell types. Each column represents one cell; random sample of 100 cells from α-astrocytes are shown; all β- and γ-astrocytes are shown. See also S1 Fig.
Fig 5.
Changes in gene expression between day and night occur in a cell type-specific manner.
(A) Number of differentially expressed (DE) genes upregulated by night or day by cell type. A gene is considered DE at p<0.01 (Wilcoxon rank sum), when expressed in at least 15% of cells in either of the two samples being tested, fold change ≥2.0, and effect size ≥0.35. (B) Heatmap summary of all 644 DE gene changes by cell type. Each column represents one gene. (C) Venn diagram of number of overlapping DE genes in α- and β-pinealocytes by day and night. (D) Heatmap summary of DE genes found in at least one non-pinealocyte and one other subtype. See also dot plots in SI.
Fig 6.
Comparison of differentially expressed genes between the nighttime pineal gland and isoproterenol-treated pineal gland.
Venn diagrams indicate the number of genes that were found to be significantly differentially expressed (DE) in the pineal gland (see methods). There was overlap between DE genes upregulated at night and by isoproterenol (Iso) treatment, as well as overlap between DE genes upregulated during the day and upregulated in the vehicle control treated (i.e. downregulated by isoproterenol treatment), in 3 cell types. Other cell types are not shown.
Fig 7.
Differences in melatonin synthesis between α- and β-pinealocytes inferred from scRNA-Seq.
(A-B) Opacity indicates relative strength of the pathway module; greater opacity indicates a more active pathway. (A) Conversion of N-acetylserotonin (NAS) to melatonin in α-pinealocytes is enhanced by increased ASMT activity and increased S-adenosyl methionine (SAM) availability, which is increased by greater ATP availability. ATP availability is increased by elevated ATP production from oxidative phosphorylation (OxPhos), as inferred by greater expression of mitochondrial genes in α-pinealocytes. ATP availability is also increased by reduced consumption by protein synthesis, as inferred by decreased expression of ribosomal genes in α-pinealocytes. (B) β-Pinealocytes also undergo melatonin synthesis, but do not have the same production increasing enhancements as α-pinealocytes. (C) Melatonin (M) is synthesized in both pinealocyte subtypes from N-acetylserotonin (NAS). NAS that is not converted to melatonin in β-pinealocytes enters the α-pinealocyte by passive diffusion through membranes and gap junctions (shown in blue). NAS is subsequently converted to melatonin by the high efficiency Asmt system in the α-pinealocyte, thereby maximizing melatonin production.