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Cited by 392Single-cell and spatial transcriptomicsCerebrospinal fluid and hydrocephalusBarrier Structure and Function Studies

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

Open access
Dani, Neil, Herbst, Rebecca H., McCabe, Cristin, Green, Gilad S., Kaiser, Karol, Head, Joshua P., Cui, Jin, Shipley, Frederick B. +14 more·2021·Cell
DOI 10.1016/j.cell.2021.04.003PMC PMC8214809

Published version of a preprint

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Abstract

SUMMARY 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 showed 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 upregulated host defense programs and resident macrophages upregulated IL-1β signaling genes. Our atlas revealed cellular diversity, architecture and signaling across ventricles during development, maturation and aging of the ChP brain barrier.

AI snapshot

Written from the paper’s full text.

TL;DR

This paper presents a single-cell and spatial atlas of the mouse choroid plexus across lateral, third, and fourth ventricles in developing, adult, and aged brains. It identifies ventricle- and age-dependent epithelial/mesenchymal programs, a Wnt1+ common progenitor for epithelial and neuronal cells, and aging-associated immune signaling including IL-1β.

Key findings

  • Profiled 15,620 single cells and 83,040 single nuclei from lateral, third, and fourth ventricle choroid plexuses of developing (E16.5, n = 13), adult (4 months, n = 4), and aging (20 months, n = 9) mice, defining major cell classes and subtypes across ages and ventricles.
  • Trajectory inference and lineage tracing supported a common progenitor pool for epithelial and neuronal cells; Wnt1+ progenitors near FoxJ1+ epithelial cells gave rise to both epithelial and neuronal progeny in the 4V and 3V ChP.
  • Epithelial and fibroblast cells showed ventricle-regionalized expression starting embryonically and persisting with age; maturation produced a large transcriptional shift, and aged epithelial cells upregulated host-defense programs while aged macrophages upregulated IL-1β signaling genes.
  • Mesenchymal cells expressed ACE2 and accessory SARS-CoV-2 entry components, and ligand-receptor analysis predicted mesenchymal cells as an interaction hub, with IL-1β signaling from aged macrophages to endothelial and mesenchymal cells.

Methods

  • Mouse ChP from LV, 3V, and 4V was dissected at E16.5, 4 months, and 20 months; embryonic samples were profiled by scRNA-seq, and all ages by snRNA-seq, followed by smFISH, immunostaining, transgenic lineage tracing, and whole-tissue imaging.
  • Analyses included clustering, diffusion maps, RNA velocity, topic modeling, pseudobulk differential expression, and CellPhoneDB-based ligand-receptor interaction inference across ventricles and ages.
  • Validation used marker immunostaining, RNAscope, Cx3cr1-GFP and Wnt1-CRE2::R26R-Confetti mice, electron microscopy, and published in situ hybridization data.

Limitations

  • The paper states: small tissue size required pooling samples across donors, especially for immune cells; dissection purity was limited by anatomically defined borders, particularly for 3V ChP; adult/aged cellular health led to differential processing (scRNA-seq for embryonic vs. frozen samples and snRNA-seq for adult/aged, also applied to embryonic), introducing computational comparison challenges; continuous cell-state and age transitions required topic modeling; and lowly expressed genes may be missed due to coverage depth.
  • Evident from the text: the atlas is mouse-only, and several conclusions about signaling and progenitor relationships rely on inferred trajectories and predicted ligand-receptor interactions, with validation focused on selected markers and lineages.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.cell.2021.04.003

Cites

134 of 134 references verified0 citation errors · 0 not checkable

Statistics

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Claims the paper makes

0 of 91 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    In embryos, subsets of glial cells expressed markers of neural progenitor/stem-like cells or of oligodendrocyte precursor cells.

    “In embryos, subsets of glial cells expressed markers of neural progenitor/stem-like cells (e.g. Pax6, Nes, Sox2, Rspo2/3) or of oligodendrocyte precursor cells (OPCs, e.g. Olig1, Olig2) (Kriegstein and Alvarez-Buylla, 2009) (Figure 2A, S2A,B).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kriegstein and Alvarez-Buylla, 2009 source

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  • Findingown data

    The study profiled 15,620 single cells and 83,040 single nuclei from the lateral, third, and fourth brain ventricles of developing, adult, and aging mice.

    “we profiled 15,620 single cells (scRNA-seq) and 83,040 single nuclei (snRNA-seq) from the lateral (LV), third (3V), and fourth (4V) brain ventricles of developing (embryonic day [E]16.5, n = 13), adult (4 months, n = 4) and aging (20 months, n = 9) mice”
    Quote in the paperSubstantial claim
  • Findingcitation

    The boundary between the 3V ChP and developing pineal gland was mapped using published ISH of marker genes from the data.

    “We leveraged the inclusion of adjacent brain tissue in our atlas to map the boundary between the 3V ChP and developing pineal gland using published ISH (Lein et al., 2007; Visel et al., 2004) of marker genes from our data (Figure S2C).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Lein et al., 2007 source

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    • cites Visel et al., 2004 source

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  • Findingown data

    Embryonic cells partitioned into clusters identifying epithelial, mesenchymal (mural and fibroblast), endothelial, immune, neuronal and glial cells in each ChP from all ventricles.

    “Embryonic cells partitioned into clusters identifying epithelial, mesenchymal (mural and fibroblast), endothelial, immune, neuronal and glial cells in each ChP from all ventricles (by marker genes expression, Figure 1C, S1A,B,C, Table S1, STAR Methods).”
    Quote in the paperSubstantial claim
  • Findingown data

    Embryonic ChP scRNA-seq profiles of epithelial, glial, and neuronal cells were not discretely separable by low-dimensional embedding.

    “The embryonic ChP scRNA-seq profiles of epithelial, glial, and neuronal cells were not discretely separable by low-dimensional embedding (Figure 1C, STAR Methods), suggesting a common progenitor pool for both epithelial and neuronal cells may exist in the ChP.”
    Quote in the paperSubstantial claim
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    Findingown data

    Tissue maturation was accompanied by a distinct shift in gene expression between embryo and adult/aged ChP, while canonical cell-type markers were conserved across ages and ventricles.

    “While tissue maturation was accompanied by a distinct shift in gene expression between embryo and adult/aged ChP (Figure 1F-H, S1F,G), canonical cell-type markers were conserved across ages and ventricles (Figure 1I, Figure S1H,I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The diversity of embryonic glia and neuronal populations observed in scRNA-seq was confirmed by snRNA-seq and expanded to adult and aged ChP.

    “The diversity of embryonic glia and neuronal populations observed in scRNA-seq was confirmed by snRNA-seq and expanded to adult and aged ChP (Figure S2D,E) (Dulken et al., 2017; Mizrak et al., 2019; Shah et al., 2018).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Dulken et al., 2017 source

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    • cites Mizrak et al., 2019 source

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    • cites Shah et al., 2018 source

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  • May combine two claims.

    Findingown data

    Actively cycling cell subsets were present within each of the six major cell classes in the developing ChP and were enriched along the base of the ChP proximal to the brain.

    “Notably, actively cycling cell subsets were present within each of the six major cell classes in the developing ChP (Figure S1D) and were enriched along the base of the ChP proximal to the brain (Figure S1E).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The predicted progenitors expressed proliferation (Mki67) and known markers (Pax6, Rspo2/3, Wnt5a/3a/1, Fabp7).

    “The predicted progenitors expressed proliferation (Mki67) and known markers (Pax6, Rspo2/3, Wnt5a/3a/1, Fabp7, clusters 7 and 8 in Figure 2A,B S2B; STAR Methods), previously reported for precursors of ependymal cells and cortical neurons (Kriegstein and Alvarez-Buylla, 2009), with Wnt1 having the most specific expression (Figure S2G).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kriegstein and Alvarez-Buylla, 2009 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Neuronal and glia-like cells were captured in the ChP across all ages, validated by immunostaining.

    “We captured neuronal (Tubb3 expressing) and glia-like (Slc1a3/EAAT1 or GFAP expressing) cells in the ChP across all ages, validated by immunostaining (Figures 1C,D,I, S1J-L), in agreement with previous studies (Lindvall et al., 1978).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Lindvall et al., 1978 source

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    Findingown data

    Subsets of neurons expressed markers of intermediate progenitors, immature neurons, and various neuropeptides.

    “Subsets of neurons expressed markers of intermediate progenitors (e.g. Eomes/Tbr2) or of immature neurons (e.g. Neurod1/2, Dlx1/2), and various neuropeptides (e.g. Vgf, Chgb) (Figure S2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    SnRNA-seq of embryonic, adult, and aged ChP identified the same major cell classes in each ventricle, with conservation of overall cellular composition with age.

    “SnRNA-seq of embryonic, adult, and aged ChP identified the same major cell classes in each ventricle (as in scRNA-seq of embryonic samples, Figure 1F), with conservation of overall cellular composition with age.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Comparing patterns across three age groups profiled by snRNA-seq, both age-invariant and age-dependent expression patterns were found, including regionalized patterns, with the main shift occurring from embryo to adult and then persisting into aging.

    “Comparing patterns across three age groups profiled by snRNA-seq, we found both age-invariant and age-dependent expression patterns, including regionalized patterns, with the main shift occurring from embryo to adult and then persisting into aging (Figure 3F,G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Several topics reflected cross-ventricle regionalized gene programs, having differential weights between cells from different ventricles.

    “Several topics reflected cross-ventricle regionalized gene programs (Topics 3, 6, 11, 14, 16, and 23), having differential weights between cells from different ventricles (Figure 3B, S3C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among age-dependent ventricle-invariant genes, embryonic fibroblasts expressed higher levels of developmental and morphogenesis genes, whereas adult and aged fibroblasts upregulated transporters, receptors, peptidase and proteolytic activity genes.

    “Among age-dependent ventricle-invariant genes, embryonic fibroblasts expressed higher levels of developmental and morphogenesis genes, whereas the adult and aged fibroblasts upregulated transporters, receptors, peptidase and proteolytic activity genes (Figure 4I, S4H,I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The proportion of replicating epithelial cells was reduced from ~10% in embryonic ChP to ~1% in adult ChP, with an overall shift towards more mature cells compared to ciliogenic epithelial cells (p < 0.05).

    “Notably, the proportion of replicating epithelial cells was reduced from ~10% in embryonic ChP to ~1% in adult ChP, with an overall shift towards more mature cells compared to ciliogenic epithelial cells (p-value < 0.05, t-test on fraction of cells, Figure S2L).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Wnt1-derived epithelial cells in the maturing 4V ChP extended away from the URL, and Wnt1-derived neurons were in close proximity to progenitors in the URL.

    “Wnt1-derived epithelial cells in the maturing 4V ChP extended away from the URL, and Wnt1-derived neurons were in close proximity to progenitors in the URL (Figure 2J,K).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In fibroblasts, both age-invariant and age-dependent patterns were found, including regionalized patterns.

    “In fibroblasts, we found both age-invariant and age-dependent patterns, including regionalized patterns (Figure 4G,H, Table S5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Embryonic epithelia upregulated genes for developmental processes, while aged epithelia upregulated host-defense programs.

    “These were enriched for specialized programs across ages: Embryonic epithelia upregulated genes for developmental processes (Figure 3K, S3K), while aged epithelia upregulated host-defense programs (Figure 3L, FDR q-value < 0.01, GOrilla (Eden et al., 2007, 2009;Baruch et al., 2014).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Eden et al., 2007 source

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    • cites Eden et al., 2009 source

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    • cites Baruch et al., 2014 source

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  • May combine two claims.

    Findingown data

    In adult ChP, Slc12a2 (NKCC1), Kcnj13 (Kir7.1), and Slc4a5 (NBC4) were upregulated, while Aqp1 was modestly downregulated.

    “For example, in adult ChP Slc12a2 (NKCC1), Kcnj13 (Kir7.1), and Slc4a5 (NBC4) were upregulated (see also (Xu et al., 2021)), while Aqp1 was modestly downregulated (Figure 3H, S3G).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Xu et al., 2021

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  • May combine two claims.

    Findingown data

    Differentiating epithelial populations mapped to distinct spatial boundaries within each ventricle, with progenitor cells located near the ChP-brain boundary.

    “We mapped the differentiating populations to distinct spatial boundaries within each ventricle (Figure 2P-R), with progenitor cells located near the ChP-brain boundary (by smFISH of Rspo1/2/3; Wnt2b/3a/5a; Axin2) (Figure S2H-K).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Age-dependent fibroblast genes typically changed postnatally and were maintained in aged ChP.

    “As in epithelial cells, age-dependent fibroblast genes typically changed postnatally and were maintained in aged ChP (Figure 4H).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Macrophages were the most prevalent and were associated with blood vessels across all ages.

    “Macrophages were the most prevalent and were associated with blood vessels across all ages (Figure S5G; Shipley et al., 2020).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Shipley et al., 2020 source

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  • May combine two claims.

    Findingown data

    In the embryo, epithelial cells formed the largest cell class in each ChP and partitioned into subsets representing cycling, newly differentiated ciliogenic, and mature cells.

    “In the embryo, epithelial cells formed the largest cell class in each ChP (by scRNA-seq, Figure S1B) and partitioned into subsets representing cycling, newly differentiated cells undergoing ciliogenesis (above, Figure 2N), and mature cells (Figure 3A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Cx3cr1-GFP-expressing cells tiled across the ChP, with the subset expressing SLC40A1 located proximal to major vessels.

    “Cx3cr1-GFP-expressing cells tiled across the ChP (Figure 5E; Cui et al., 2020), with the subset expressing SLC40A1 located proximal to major vessels, suggesting roles in regulating iron homeostasis (Figure 5F).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Cui et al., 2020 source

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  • May combine two claims.

    Findingown data

    In contrast to Van Hove et al., the separation of BAM subsets was age-dependent and potentially represented a maturation-dependent shift from MHC IIlow to MHC IIhigh.

    “However, in contrast to Van Hove et al., our data revealed that this separation was age-dependent and potentially represented a maturation-dependent shift from MHC IIlow to MHC IIhigh (Figure 5I, S5I,J, Table S6).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Van Hove et al., 2019 source

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  • May combine two claims.

    Findingown data

    The stereotyped vascular organization was retained in aged ChP, where Vwf and Selp (P-selectin) expression were also detected.

    “The stereotyped vascular organization was retained in aged ChP (Figure 6F), where Vwf and Selp (P-selectin) expression were also detected (Figure 6G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Topic modeling of embryonic ChP cells uncovered a previously unknown brain barrier transition zone embedded in the molecular and vascular map of the ChP, validated across ages.

    “Notably, topic modeling of embryonic ChP cells also uncovered a previously unknown brain barrier transition zone embedded in the molecular and vascular map of the ChP, which we validated across ages.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mesenchymal cells (fibroblasts and pericytes) expressed Pdgfra and Pdgfrb respectively, and epithelial and endothelial cells expressed their cognate ligands, Pdgfa and Pdgfb respectively.

    “For example, mesenchymal cells (fibroblasts and pericytes) expressed Pdgfra and Pdgfrb respectively, and epithelial and endothelial cells expressed their cognate ligands, Pdgfa and Pdgfb respectively (Figure S7C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In adult and aged LV ChP, IHC showed that arteries and veins were regularly segmented across the tissue and connected by an expanded capillary-like network.

    “In adult and aged LV ChP, IHC showed that arteries and veins were regularly segmented across the tissue (Figure 6E,F; Shipley et al., 2020) and connected by an expanded capillary-like network (Figure S6D) similar to human tissue (Hudson, 1960).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Shipley et al., 2020 source

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    • cites Hudson, 1960 source

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    Findingown data

    CD206 (Mrc1) expressed by embryonic macrophages was downregulated in adults, whereas CD74 was upregulated in adult and aged macrophages vs. embryos.

    “For example, CD206 (Mrc1, mannose receptor C-type I) expressed by embryonic macrophages was downregulated in adults, whereas CD74 (MHC class II associated protein) was upregulated in adult and aged macrophages vs. embryos (Figure 5J-L, Figure S5H).”
    Quote in the paperSubstantial claim

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    Findingown data

    Comparing significant interactions across ventricles or ages revealed age- or ventricle-dependent interactions and overall differences in the number of interactions.

    “Comparing significant interactions across ventricles or ages revealed age- or ventricle-dependent interactions and overall differences in the number of interactions (Figure 7B-D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cldn5 expression persisted in a subset of endothelial cells in snRNA-seq from all ages, and CLDN5 protein was prominent in the major arteries bifurcating from the choroidal artery and extending into the ChP.

    “Cldn5 expression persisted in a subset of endothelial cells in snRNA-seq from all ages (Figure 6I, S6C), and CLDN5 protein was prominent in the major arteries bifurcating from the choroidal artery and extending into the ChP (Figure 6J, K, S6I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IHC revealed VWF and SELP colocalized to enlarged luminal domains of vessels in aged mice, suggesting age-associated vascular damage.

    “IHC revealed VWF and SELP colocalized to enlarged luminal domains of vessels in aged mice (Figure 6H), suggesting age-associated vascular damage.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The majority of significantly interacting cell type pairs involved mesenchymal cells (60%, 70% and 85% in embryonic, adult and aged, respectively), followed by epithelial cells (45%, 55% and 42% in embryonic, adult and aged, respectively).

    “The majority of significantly interacting cell type pairs involved mesenchymal cells (FDR<0.05, 60%, 70% and 85% in embryonic, adult and aged, respectively), followed by epithelial cells (FDR<0.05, 45%, 55% and 42% in embryonic, adult and aged, respectively).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Adult regionalization occurred either by activation of expression of genes not expressed in the embryo or by restriction of expression of genes expressed across ventricles in the embryo.

    “either by activation of expression (for genes not expressed in the embryo) or by restriction of expression (for genes expressed across ventricles in the embryo).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Vegfa was expressed in epithelial and mesenchymal cells and its receptor Flt1 on endothelial cells.

    “Other notable interactions included Vegfa in epithelial and mesenchymal cells and its receptor Flt1 on endothelial cells (Figure S7C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Histological analyses revealed arteries developing obliquely across the ChP and veins running along the ventricular free margin of the tissue.

    “Histological analyses revealed arteries developing obliquely across the ChP and veins running along the ventricular free margin of the tissue (Figure 6B).”
    Quote in the paperSubstantial claim

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    Findingown data

    In a more conservative, degree-preserving permutation test, the number of predicted interactions between most cell type pairs was not statistically significant, indicating the dominance of a cell type in the crosstalk across all cells and not specific pairs.

    “Notably, in a more conservative, degree-preserving permutation test, the number of predicted interactions between most cell type pairs was not statistically significant (Figure S7D), indicating the dominance of a cell type in the crosstalk across all cells and not specific pairs.”
    Quote in the paperSubstantial claim

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    Findingown data

    Significant individual interactions were found between each pair of cell types across all ventricles and ages, with specific cell type pairs having a significant enrichment in the number of interactions.

    “We found significant individual interactions between each pair of cell types across all ventricles and ages, with specific cell type pairs having a significant enrichment in the number of interactions (Figure 7A, S7A,B, Table S8).”
    Quote in the paperSubstantial claim

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    Findingcitation

    While no overt regionalization of endothelial cell programs was observed, venous markers were enriched in the 3V ChP across all ages.

    “While we did not observe overt regionalization of endothelial cell programs, venous markers were enriched in the 3V ChP across all ages (Figure 6D, S6F,G), likely resulting from capture of the proximal vein of Galen (Mancini et al., 2015) (Figure S6H).”
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    • cites Mancini et al., 2015 source

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    Findingown data

    Plvap scored highly in Topic 3 from scRNA-seq of embryonic cells and was also identified in adult and aged snRNA-seq profiles.

    “Moreover, Plvap, which marks diaphragms of fenestrated endothelial cells (Herrnberger et al., 2012), scored highly in Topic 3 from scRNA-seq of embryonic cells (Figure 6A), and was also identified in adult and aged snRNA-seq profiles (Figure S6C).”
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    • cites Herrnberger et al., 2012 source

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    Findingown data

    Wnt5a was most robustly expressed by mesenchymal cells and its receptor Ror1 was expressed, amongst others, by epithelial cells, suggesting lifelong contributions to ChP function.

    “Wnt5a, a regulator of 4V ChP and hindbrain development (Kaiser et al., 2019), was most robustly expressed by mesenchymal cells and its receptor Ror1 was expressed, amongst others, by epithelial cells, suggesting lifelong contributions to ChP function (Figure S7C).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kaiser et al., 2019 source

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  • May combine two claims.

    Findingcitation

    Essentially all major choroid plexus cell types, and in particular choroid plexus fibroblasts, expressed many secreted factors found in cerebrospinal fluid, including Igf2.

    “Essentially all major ChP cell types, and in particular ChP fibroblasts, also expressed many secreted factors found in CSF, including Igf2 (Lehtinen et al., 2011).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lehtinen et al., 2011 source

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    Findingown data

    Adult macrophages upregulated CD74, which was even further upregulated in the aged choroid plexus.

    “adult macrophages upregulated CD74, a chaperone of antigen presentation protein that was even further upregulated in the aged ChP.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Epithelial cell clusters scored highly for immediate early genes.

    “the epithelial cell clusters scoring highly for IEG genes (Figure S2D)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Macrophage cells of replicate 3 had very high expression of mitochondrial genes.

    “because they had very high expression of mitochondrial genes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Choroid plexus immune cell populations show spatial organization and age-dependent expression across ventricles and within the choroid plexus.

    “Our profiles of ChP immune cell populations revealed spatial organization and age-dependent expression across ventricles and within the ChP.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The random forest classifier trained on the embryo snRNA-seq dataset had an out-of-bag error of 1.7%.

    “The out-of-bag error was 1.7%.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The choroid plexus in each brain ventricle produces cerebrospinal fluid and forms the blood-CSF barrier.

    “The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier.”
    • Also stated in: § INTRODUCTION · boosted
      “The choroid plexus (ChP) is an essential brain barrier known primarily as a source of cerebrospinal fluid (CSF) and comprises an analogous set of four tissue structures located in each brain ventricle.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Damkier et al., 2013 source

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    • cites Ghersi-Egea et al., 2018 source

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    • cites Lun et al., 2015a

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    Premisecitation

    Bulk profiling indicates that each ChP retains a developmental blueprint of its origins along the body axis, with ventricle-specific transcriptomes and secretomes for the lateral and 4th ventricle ChPs.

    “Bulk profiling of whole tissue (Liddelow et al., 2010; Lun et al., 2015b; Silva-Vargas et al., 2016) or sorted subpopulations (Lun et al., 2015b) indicates that each ChP retains a developmental blueprint of its origins along the body axis, with ventricle-specific transcriptomes and secretomes for the lateral and 4th ventricle ChPs (Lun et al., 2015a).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Liddelow et al., 2010 source

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    • cites Lun et al., 2015b

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    • cites Silva-Vargas et al., 2016 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The ChP epithelial sheet, together with vasculature and diverse cell types, regulates CSF secretion and composition by de novo protein synthesis and selective transcytosis of blood-borne factors.

    “The foundation of the ChP is an epithelial sheet that, together with a network of vasculature and diverse cell types, regulates CSF secretion and governs its composition by de novo protein synthesis and selective transcytosis of blood-borne factors (Damkier et al., 2013; Ghersi-Egea et al., 2018; Lun et al., 2015a; Saunders et al., 2018b).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Damkier et al., 2013 source

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    • cites Ghersi-Egea et al., 2018 source

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    • cites Lun et al., 2015a

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Each ChP develops independently from distinct locations along the roofplate, where capillaries, mesenchymal and neural crest cells invaginate the neuroepithelium.

    “Each ChP develops independently from distinct locations along the roofplate, where capillaries, mesenchymal and neural crest cells invaginate the neuroepithelium (Hunter and Dymecki, 2007; Lun et al., 2015a; Wilting and Christ, 1989).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Hunter and Dymecki, 2007

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    • cites Lun et al., 2015a

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    • cites Wilting and Christ, 1989

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  • May combine two claims.

    Interpretationcitation

    These data highlight the potential of mesenchymal and epithelial ChP cells to facilitate viral particle entry into the CSF in all ventricles and ages and to contribute to neurological sequelae associated with SARS-CoV-2 infection.

    “These data highlight the potential of mesenchymal and epithelial ChP cells to facilitate viral particle entry into the CSF (Pellegrini et al., 2020b, 2020a; Wu et al., 2020) in all ventricles and ages and to contribute to neurological sequelae associated with SARS-CoV-2 infection (Ellul et al., 2020).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Pellegrini et al., 2020b

      Not checked in the free run.

    • cites Pellegrini et al., 2020a

      Not checked in the free run.

    • cites Wu et al., 2020

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    Differences between predicted significant interactions from scRNA-seq vs. snRNA-seq for embryo ChP may reflect differences in cell type recovery between methods.

    “In addition, there were some differences between predicted significant interactions from scRNA-seq vs. snRNA-seq for embryo ChP, which may reflect differences in cell type recovery between methods (Figure S7A).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Multiple comparisons ANOVA was employed and P < 0.05 was considered significant.

    “Multiple comparisons ANOVA was employed using statistical software package Graphpad PRISM. Data are represented as mean ± SEM. P < 0.05 was considered significant.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The study cannot rule out missing some lower expressed genes of interest to choroid plexus biology due to limitations in depth of coverage.

    “We cannot rule out the possibility that we may miss some lower expressed genes of interest to ChP biology due to the limitations in depth of coverage.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Nuclei with more than 10% of total UMIs mapped to mitochondrial genes were excluded.

    “we excluded nuclei that had more than 10% of total UMI’s mapped to mitochondrial genes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CellPhoneDB is developed for human ligand-cognate receptor pairs.

    “As CellPhoneDB is developed for human ligand-cognate receptor pairs”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Efremova et al., 2020 source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductreasoning

    Cellular health was particularly challenging for adult and aged tissues, leading the authors to favor differential sample processing.

    “cellular health was particularly challenging for adult and aged tissues, ultimately leading us to favor differential sample processing”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Topic 19 may reflect a response to tissue dissociation.

    “and may reflect a response to tissue dissociation (van den Brink et al., 2017)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites van den Brink et al., 2017 source

      Not checked in the free run.

    • cites Lacar et al., 2016 source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The choroid plexus is a primary producer of cerebrospinal fluid and cerebrospinal fluid-distributed factors.

    “The ChP is considered to be a primary producer of CSF and CSF-distributed factors (Ghersi-Egea et al., 2018; Lun et al., 2015a; Saunders et al., 2018b)”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Ghersi-Egea et al., 2018 source

      Not checked in the free run.

    • cites Lun et al., 2015a

      Not checked in the free run.

    • cites Saunders et al., 2018b

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All mouse work was performed in accordance with the Institutional Animal Care and Use Committees and relevant guidelines of Boston Children's Hospital and Masaryk University.

    “All mouse work was performed in accordance with the Institutional Animal Care and Use Committees (IACUC) and relevant guidelines of Boston Children’s Hospital and Masaryk University.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Doublets were identified and removed from scRNA-seq data.

    “Doublets were clearly identifiable as forming their own clusters with dual expression of marker genes of distinct subsets (e.g., epithelial genes and endothelial genes) and were removed from further analysis and any visualizations.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Batch correction was performed for each cell type separately using ComBat.

    “Batch correction was performed for each cell type separately using ComBat as implemented in the R package sva (Leek et al., 2012), using the default parametric adjustment mode.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Leek et al., 2012 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    A total of four independent explants were quantified for regionalized proliferation in the LV choroid plexus.

    “A total of four independent explants were quantified.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims. The quote only partly states this claim.

    Study design and conductcitation

    Nucleus doublets were identified using DoubletFinder and removed from downstream analysis.

    “Nucleus doublets were identified using the R package DoubletFinder (version DoubletFinder_2.0.2, (McGinnis et al., 2019).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites McGinnis et al., 2019 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Differential sample processing introduced additional computational challenges in comparison of the two datasets.

    “This strategy subsequently introduced additional computational challenges in comparison of the two datasets.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    CellBender was applied to each sample separately to address ambient RNA and other confounders.

    “To address ambient (background) RNA and other confounders we applied CellBender (Fleming et al., 2019) to each sample separately”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Fleming et al., 2019

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The number of topics and tolerance values were chosen by fitting a range of values and selecting those that gave robust topics and mostly saturated the number of informative topics found.

    “we fit a range of K and tolerance values and picked values that gave us robust topics and where we mostly saturated the number of informative topics found.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    The analysis required adjustments to computational tools including the use of topic modeling.

    “required adjustments to our computational tools including the use of topic modeling.”
    Quote in the paperNot a substantial claim
66 of 66

  • The paper relies on this without support.

    The third ventricle is the least explored of the choroid plexuses.

    “The less-accessible third ventricle is the least explored, and boundaries of all ChPs with adjacent neural tissues remain poorly defined.”

  • The paper relies on this without support.

    The boundaries of all choroid plexuses with adjacent neural tissues remain poorly defined.

    “The less-accessible third ventricle is the least explored, and boundaries of all ChPs with adjacent neural tissues remain poorly defined.”

  • The paper relies on this without support.

    Earlier efforts to address deficiencies in choroid plexus knowledge were limited by the lack of techniques to access, isolate, and comprehensively profile the ChP across brain ventricles and ages.

    “Earlier efforts to address these deficiencies were limited by the lack of techniques to access, isolate, and comprehensively profile the ChP across brain ventricles and ages.”

  • The paper relies on this without support.

    The lack of a detailed cellular and molecular map for the choroid plexus has been a major obstacle to elucidating its roles in instructing brain development, brain function, understanding its barrier properties, and unlocking its lifelong therapeutic potential.

    “The lack of a detailed cellular and molecular map for the ChP has been a major obstacle to elucidating its roles in instructing brain development, brain function, understanding its barrier properties, and unlocking its lifelong therapeutic potential.”

  • The paper relies on this without support.

    The choroid plexus-cerebrospinal fluid system is implicated in a growing number of neurologic conditions.

    “Since the ChP-CSF system is implicated in a growing number of neurologic conditions”

  • The paper relies on this without support.

    Transitions between cell states and ages are continuous.

    “Transitions between cell states and ages are continuous”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Rspo3 probe is labeled as Rspo1

    Minor

    The probe listed for Rspo3 is labeled "mm-Rspo1," even though a separate Rspo1 probe is listed with a different reference number. The Rspo3 probe identity is therefore inconsistent within the probe list.

1 of 1

Cites

Flagged first, then by citations

2 of the 136 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Fleming, 2019, CellBender remove-background: A deep generative model for unsupervised removal of background noise from scRNA-seq datasets, BioRxiv, 791699

Cited by

Sorted by citations

Related

Authors
Dani, N., Herbst, R. H., McCabe, C., Green, G. S., Kaiser, K., Head, J. P., Cui, J., Shipley, F. B., Jang, A., Dionne, D., Nguyen, L., Rodman, C., Riesenfeld, S. J., Prochazka, J., Prochazkova, M., Sedlacek, R., Zhang, F., Bryja, V., Rozenblatt-Rosen, O., Habib, N., Regev, A., & Lehtinen, M. K.
Affiliations
  • Boston Children's Hospital (US)
  • Broad Institute (US)
  • Hebrew University of Jerusalem (IL)
  • Masaryk University (CZ)
Corresponding author
  • Habib, Naomi
  • Regev, Aviv
  • Lehtinen, Maria K.
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2021-05
Volume
184
Issue
11
Pages
3056-3074.e21
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2021.04.003
PMC
PMC8214809
OpenAlex
W3157400350
Semantic Scholar
2401a5c95b12f729e979e5eaaabc8c3df3e80d3b
Cited by
388
References
136
Citations by year
2026: 71 · 2025: 97 · 2024: 79 · 2023: 64 · 2022: 41 · 2021: 22 · 2020: 11 · 2019: 3
FWCI
16.54
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
Funders
  • Grantová Agentura České Republiky (LM2018129, GX19-28347X)
  • Israel Science Foundation (1709/19)
  • New York Stem Cell Foundation
  • HHMI
  • National Science Foundation
  • Helen Hay Whitney Foundation
  • NSF
  • Broad Institute
  • NIH (1U54HD090255, T32 HL110852)
  • AFAR
  • Ministerstvo Školství, Mládeže a Tělovýchovy (RVO 68378050)
  • European Regional Development Fund
  • Akademie Věd České Republiky (LM2018126l CZ.1.05/2.1.00/19.0395, CZ.02.1.01/0.0/0.0/16_013/0001789)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Cerebrospinal fluid and hydrocephalus
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
choroid plexus, cerebrospinal fluid, blood-CSF barrier, brain ventricles, single-cell atlas, spatial atlas, mouse brain development, brain aging, choroid plexus epithelial cells, progenitor cells, lineage tracing, fibroblasts, macrophages, interleukin-1β signaling, host defense genes
MeSH terms
Age Factors, Aging, Animals, Blood-Brain Barrier, Brain, Brain Diseases, Cell Differentiation, Choroid Plexus, Epithelial Cells, Female, Male, Mice, Inbred C57BL, Signal Transduction, Cell Lineage, Mice, Single-Cell Analysis

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

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