A cellular and spatial map of the choroid plexus across brain ventricles and ages

Abstract

The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1β (IL-1β) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.

Keywords: aging; brain barrier; cerebrospinal fluid; choroid plexus; development; single-cell RNA sequencing; single-nucleus RNA sequencing.

Figure 1.. Single cell and single nucleus RNA-Seq of embryonic, adult, and aged ChPs.

(A) E16.5 brain and ChP from each ventricle in E16.5, adult, and aged brain. Arrows: anterior-posterior (A/P), dorso-ventral (D/V) and medial-lateral (M/L) axes. (B) Workflow. (C) Major cell types of E16.5 ChP. T-SNE of 15,620 single cell profiles (n = 9 mice, in pools of 3 animals per ventricle), colored by post hoc annotated cell type. (D) TUBB3-positive neurons (arrowheads) in E16.5 LV ChP. Inset a: High-magnification image. Schematic: dotted outline denotes region shown by immunostaining. Arrows: A/P and D/V axes. (E) Major cell type markers expressed in ChP. Top: H&E; bottom: Epithelial (AQP1), endothelial (PECAM1), mesenchymal (COL1A1) and immune cell (SPI1, arrowheads) immunostaining; Hoechst counterstain. (F) Major cell types of E16.5, adult, and aged ChP. UMAP of 83,040 single nucleus profiles (snRNA-seq, n=17 mice. 2-4 animals per replicate of LV, 3V and 4V ChP from E16.5, adult and aged mice), colored by post hoc annotated cell type. (G-H) UMAP as in F, colored by ventricle (in G) or age (in H). (I) Cell type marker genes by snRNA-seq. Median expression level in expressing cells (color) and proportion of expressing cells (circle size) of selected genes (rows) in each major cell population (columns). See also Figure S1.

Figure 2.. Epithelial differentiation trajectory reveals progenitor and ciliogenesis programs of ChP epithelial cells during development.

(A) Embryonic neuronal and glia-like cell subsets. T-SNE of embryonic neuronal and glia-like single cell profiles (scRNA-seq, 955 cells). (B) Diffusion map embedding (components 1 and 3) of neuronal, glia-like and epithelial cell profiles (dots) from 3V ChP, colored by log₂(TP10K+1) expression of marker genes of neurons (Tubb3), progenitors (Pax6, Rspo2), cycling (Mki67), ciliogenesis (Ccdc67), and mature epithelial (Krt18) cells. Right: Suggested differentiation trajectories. (C) Schematic of embryonic hindbrain; upper rhombic lip (URL), lower rhombic lip (LRL). Dotted box: area shown in panels (D-K). (D) Proliferating (KI67) progenitors (PAX6) in URL. (E) SmFISH markers of progenitors (Rspo2, Rspo3) and Wnt1. (F) SmFISH of Wnt1 and FoxJ1. (G) High magnification image of (F). (H) Confetti-labeling schematic. (I) E16.5 hindbrain region from Wnt1-CRE2 crossed with Confetti mouse shows distribution of Wnt1-derived cells. (J) Wnt1-derived cRFP or mCFP epithelial cells adjacent to URL. (K) Wnt1-derived cRFP and TUBB3 neurons in URL with DAPI. Inset: split cRFP (top) and TUBB3 (bottom). (L) Confetti labeled cells in 3V ChP. (M) Proliferating cells enriched at LV ChP base. Left: BrdU labeling; Middle: ISH of Ccdc67; Right: AQP1. (N) Left: Scanning EM of multi-ciliated epithelial cell. Right: Median expression level in expressing cells (color) and proportion of expressing cells (circle size) of markers of ciliogenesis (columns) in ciliogenic (developing) and mature epithelial cells (rows). (O) Left: LV ChP immunostained for Ac-Tubulin and CCDC67/DEUP1. Middle and right: Ac-Tubulin with CCDC67 (middle) or SHISA8 (right). (P) Schematic: LV ChP maturation regions. (Q-R) Spatial mapping of maturation domains in 3V and 4V ChP marked by progenitors (Rspo2), ciliogenesis (Ccdc67), differentiated epithelial cells (Krt18) (Genepaint). Schematic: ChP regions with maturing epithelial cells. See also Figure S2.

Figure 3.. Regionalized epithelial transcriptional programs across ventricles and ages.

(A) Distinct embryonic epithelial cells clusters by ventricle. T-SNE of embryonic epithelial cell profiles (dots, scRNA-seq), colored by ventricle. (B) For each ventricle associated topic, shown is a bar plot of topic scores for top ranked genes (left), and t-SNE of the single cell profiles (as in A) colored by topic’s weight per cell (right). (C-D) Regionalized genes in embryonic epithelial cells across ventricles. (C) Distribution of log₂(TP10K+1) expression of each gene across ventricles. (D) Top rows: Regionalized mRNA expression from Genepaint. Bottom row: Ins2 smFISH. (E) Regionalized mRNA expression from Genepaint in 4V ChP with schematic. (F-G) T-SNE of single epithelial nucleus profiles colored by age (in F) or ventricle (in G). (H) Expression level scaled per gene (color scale) of transport and secretion genes, differential across ages (FDR<0.01) (see also (Xu et al., 2021) . (I-J) Heatmap of snRNA-seq data of differentially expressed genes across ventricles (FDR<0.01) that are age-invariant (I) or age-dependent (J). Scaled expression level per gene (columns). Left color bar: ventricle. Right color bar: Age. (K-L) Age-dependent genes (snRNA-seq, FDR<0.01) between embryonic and mature (adult and aged, in K) or between adult and aged (in L). Average expression per sample across ventricles, scaled per gene (rows). Right: Enriched pathways per age-signature (hypergeometric p-value). See Figure S3.

Figure 4.. Regionalized mesenchymal transcriptional programs across ventricles and ages.

(A) Embryonic mesenchymal cells largely cluster by ventricle. T-SNE of embryonic mesenchymal cell profiles (dots) colored by ventricle. (B) Ventricle associated topics with a mesenchymal subtype: a bar plot of topic scores for top ranked genes (left), and t-SNE (as in A) colored by topic’s weight per cell (right) (topics associated with cycling cells in Figure S4B). (C-E) T-SNE of mesenchymal single nucleus profiles (dots), colored by cell type (C), age (D), and ventricle (E), and log₂(TP10K+1) expression of pericyte markers Rgs5 and Des (F). (G-H) Heatmap of snRNA-seq data of differentially expressed mesenchymal genes across ventricles (FDR<0.01) that are age-invariant (G) or age-dependent (H). Scaled expression level per gene (columns). Left color bar: ventricle. Right color bar: Age. (I) Age-dependent genes (snRNA-seq, FDR<0.01) between embryonic and mature (adult and aged). Average expression per sample across ventricles, scaled per gene (rows). Right: Enriched pathways per age-signature (hypergeometric p-value). (J) SARS-CoV-2 cell entry and associated genes in embryonic scRNA-seq (left) and snRNA-seq (right). Median expression level in expressing cells (color) and proportion of expressing cells (circle size) of markers (rows) across cell types (columns). (K) ACE2 expression in mesenchymal cells (COL1A1+) in 4V ChP. Coarse/fine dotted lines indicate apical/basal boundaries of epithelia. Arrowhead: ACE2+ cells. (L) ACE2 in adult epithelial cells (AQP1). Dotted line: base of epithelial cell. Arrowhead: stromal cell. Arrow: epithelial cell. (M) NRP1 expression in mesenchymal cells (COL1A1). See also Figure S4.

Figure 5.. Immune cell diversity and macrophage niches within and across ChP

(A) Embryonic immune cell subsets in the ChP. T-SNE of embryonic immune single cell profiles, colored by cluster. (B) Median expression level in expressing cells (color) and proportion of expressing cells (circle size) of selected marker genes (columns) across immune cell subsets (rows). (C) Embryonic macrophage diversity. Diffusion map embedding of single cell macrophages, colored by log₂(TP10K+1) expression of general macrophage marker (Cx3cr1) or of state specific genes (Lyve1, Spp1, Slc40a1, Spic). (D) Cx3cr1^+/GFP mouse LV ChP immunostained with PECAM1 (red) and Hoechst (blue). Arrowhead: epiplexus cells. Dotted line: ChP apical surface. (E-F) Cx3cr1^+/GFP LV ChP explant immunostained with PECAM1. (F) SLC40A1/FPN+ macrophages (arrowheads) along blood vessels. (G) Left: LYVE1+ epiplexus macrophages. Right: cross section at positions indicated by letters a, b and c in main panel. Arrows: stromal ChP macrophages. (H) UMAP of single immune nucleus profiles (dots), colored by immune cell type identity. (I) Volcano plot comparing embryonic to mature macrophages (snRNA-seq). (J) T-SNE of single immune nuclei (dots) colored by log₂(TP10K+1) expression of genes marking macrophages (Cx3cr1), embryonically enriched gene (Mrc1/CD206) and adult enriched gene (Cd74). (K) Cx3cr1^+/GFP 4V ChP immunostained with MRC1 in embryo (E16.5, left) and adult (right). (L) 4V ChP immunostained with CD74 and IBA1 (E16.5, left) and adult (right). (K-L) Arrowheads: stromal macrophages. Dotted line: epithelial apical boundary. See also Figure S5.

Figure 6.. Vascular identity and BBB protein zonation within the ChP across ages.

(A) Embryonic endothelial cell transcriptional programs. For each topic, topic scores for top ranked genes (left) and t-SNE of single cell profiles colored by topic’s weight per cell (right). (B) LV ChP immunostained with PECAM1, ACTA2 and VWF, marking the arterial (ACTA2+) and venous (ACTA2-, VWF+) vessels. (C) Angiogenic zonation. Top: LV ChP immunostained with PECAM1 and ESM1 (green). Middle: Dotted line: LV ChP free margin. Double headed arrows: A/P and D/V axes. Bottom: ESM1, PECAM1 and Hoechst. (D) UMAP of single endothelial nucleus profiles (dots) colored by age (top), endothelial subtype score (bottom). (E) LV ChP immunostained as in (B) reveal arterio-venous organization in adult. Dotted line: region of interest as inset. (F) Aged LV ChP immunostained as in (B,E). Arrowheads: VWF accumulation in vessels. Dotted line: region of interest as inset. Inset: elevated VWF expression. (G) UMAP (as in D), colored by log₂(TP10K+1) expression of genes Vwf and Selp. (H) Subset of aged vessels express VWF and SELP. (I) Distribution of Cldn5 expression across ages. (J) Embryonic LV ChP immunostained with CLDN5 and PECAM1. Dotted line: brain-ChP border. Bottom: Vessel at position ‘a’. (K) Adult LV ChP immunostained with CLDN5 and PECAM1. Arrowhead: CLDN5+ vessel at base of ChP. Dotted line: region enlarged on right. See also Figure S6.

Figure 7.. Mesenchymal, endothelial and immune cells contribute to cellular crosstalk in ChP.

(A) Number of statistically significant ligand-receptor pairs (LRPs, from CellPhoneDB (Efremova et al., 2020), STAR Methods) between pairs of ChP cell types, across ventricles in adult (top) and aged (bottom, embryo in Figure S7A). Significant pairs of cell types based on a randomization test marked as: *p≤0.05, **p≤0.01, ***p≤0.001. (B) Differential LRPs across ventricles or ages. Middle bar: cell types expressing the ligand (color coded). Right/left bars: cell types expressing the corresponding receptor. Edge width: Number of LRPs in each cell type pair that are specific to the specific ventricle (top) or age (bottom). Edge color: cell-type (middle bar). (C-E) Selected ventricle-dependent and age-dependent LRPs. Median expression level in expressing cells (color) and proportion of expressing cells (circle size) in major cell population, across all ventricles (color coded) and ages (top: E16.5; middle: adult; bottom: aged). Dashed square: ventricle-dependent expression, solid line: age-dependent. Aging-dependent IL-1β-IL1R1 signaling (in E). (F) IL-1β immunostained macrophage in vessel like structures connecting ChP and brain parenchyma in aged brain. Inset: IL-1B macrophage. (G) Left: Whole mount aged hindbrain. Dotted lines outline 4V ChP. Right: IL1R1 immunostained endothelial cells (PECAM1) . Arrowheads: IL1R1+ blood vessels originating from the brain parenchyma and entering the ChP. See also Figure S7.