Research ResourceVASCULAR BIOLOGY

Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels

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Sci. Signal.  11 Jul 2017:
Vol. 10, Issue 487, eaag2476
DOI: 10.1126/scisignal.aag2476
  • Fig. 1 TRAP-seq to study brain-specific EC differentiation.

    (A) Schematic of the experimental procedure to compare the molecular profiles of seven different vascular beds in E14.5 mouse embryos expressing the mCherry-Rpl10a fusion protein in ECs (red). mCherry+ cells were isolated from the indicated tissues for analysis. (B) PCA of the seven EC populations profiled showing individual replicates. PC1, first principal component explaining 22% of variance; PC2, second principal component explaining 17% of variance. n = 3 biological replicates. (C) Number of transcripts either specifically expressed, enriched, or depleted in ECs derived from different organs. (D) Venn diagram showing transcripts considered as translated in the brain endothelium at E14.5. (E and F) Expression of transporters (E) and transcription factors (F) that were highly enriched in the brain vasculature compared to vascular beds from other organs and to whole-tissue samples.

  • Fig. 2 CNS vascular maturation during embryonic development.

    (A) Schematic of the experimental procedure to compare molecular profiles of the brain vasculature at different developmental stages in embryos expressing the mCherry-Rpl10a fusion protein in the vasculature (red). The tissues shaded in blue were isolated for analysis. (B) PCA of temporally distinct EC populations profiled showing three biological replicates. PC1, first principal component explaining 31% of variance; PC2, second principal component explaining 11% of variance. (C) Diagram depicting significant (DESeq padj < 0.001) changes of different groups of transcripts translated in the CNS vasculature over time during embryonic development. (D and E) Box plots of calculated z scores for expression during developmental stages E11.5 to E17.5 for (D) 63 genes identified as enriched in E14.5 brain ECs or (E) 20 genes encoding markers of angiogenesis. n = 3 biological replicates. Statistical significance was calculated using nonparametric Friedman with post hoc Nemenyi test (***P < 0.001, **P < 0.01, *P < 0.05).

  • Fig. 3 Loss of Ctnnb1 in brain ECs reverses their BBB maturation progress.

    (A) Schematic of the experimental procedure to compare molecular profiles of brain ECs from wild-type (WT) and Ctnnb1-deleted (KO) E14.5 and E17.5 embryos expressing the mCherry-Rpl10a fusion protein in the vasculature (red). Ctnnb1 deletion was induced by tamoxifen (Tm) treatment 2 days before isolating the tissue for analysis. (B) PCA for WT and KO EC populations at E14.5 and E17.5 showing individual replicates. n = 3 to 4. PC1, first principal component explaining 25% of variance; PC2, second principal component explaining 11% of variance. (C) Venn diagram showing depleted transcripts in KO compared to control ECs at E14.5 and E17.5. (D and E) GO analysis of depleted transcripts in KO compared to control ECs at E14.5 (D) and E17.5 (E). (F) Venn diagram showing enriched transcripts in KO compared to control ECs at E14.5 and E17.5. (G and H) GO analysis of enriched transcripts in KO compared to control ECs at E14.5 (G) and E17.5 (H). (I and J) Box plots of calculated z scores for expression during developmental stages E11.5 to E17.5 in WT or KO ECs at E14.5 and E17.5 for (I) 63 genes identified as enriched in brain ECs or (J) 20 genes encoding angiogenesis markers. n = 3 to 4 biological replicates. Statistical significance was calculated using nonparametric Friedman with post hoc Nemenyi test (***P < 0.001, *P < 0.05).

  • Fig. 4 Single-cell RNA-seq analysis distinguishes ECs from other cell lineages.

    (A) Schematic of the experimental procedure to compare molecular profiles of individual single brain ECs from two E14.5 embryos expressing the mCherry-Rpl10a fusion protein in the vasculature (red). (B) Unsupervised hierarchical clustering of expression in 80 individual cells (columns) across all genes (rows) with ≥5 RPKM in at least one sample. The following groups of cells were identified: 35 ECs (EC 1, red), 26 ECs with a proliferation signature (EC 2, yellow), 4 microglia (blue) and 12 neurons (green). The identity of three cells remained unclear. (C) PCA for 80 profiled individual cells from E14.5 embryos.

  • Fig. 5 EC differentiation correlates with expression of Foxf2, Foxq1, Ppard, or Zic3.

    (A) Unsupervised hierarchical clustering of 61 E14.5 individual ECs across brain vasculature–enriched transcripts. Colors indicate the two groups of single cells that exhibit low (blue) or high (yellow) expression of genes encoding differentiation markers. (B) PCA for profiled ECs using brain-only transcripts enriched in the endothelium. Colors indicate low (blue) or high (yellow) expression of genes encoding differentiation markers. (C to G) Box plots of calculated z scores of genes encoding differentiation (C), general EC (D), angiogenesis (E), proliferation and cell cycle (F) markers, or individual brain EC–enriched transcription factors (G) showing low (blue) and high (yellow) abundance of brain EC–enriched transcripts. Statistical significance was calculated using Welch’s two-sample t test for normally or Wilcoxon signed-rank test for not normally distributed data (***P < 0.001, **P < 0.01, *P < 0.05). Normal distribution was tested using the Shapiro-Wilk test.

  • Fig. 6 Foxf2 and ZIC3 induce expression of BBB-related transcripts in vitro.

    (A) qRT-PCR analysis of purified RNA from HBMEC, HUVEC, and HEK293FT cells expressing Foxf2, Foxq1, ZIC3, or all three of these transcription factors (TFs) revealed transcription factor–mediated induction of BBB markers. Values are means ± SD of n = 3 independent experiments. (B) PCA for HUVECs transfected with control (empty) vector or vector containing Foxf2, Foxq1, ZIC3, or all three transcription factors combined, showing three biological replicates. PC1, first principal component explaining 30% of variance; PC2, second principal component explaining 11% of variance. (C) Venn diagram showing the number of transcripts induced (fold change > 4, RPKM > 5) by Foxf2, ZIC3, or the three transcription factors combined in HUVECs. (D) Expression of increased BBB transcripts in HUVECs transduced with control (empty vector), Foxf2, Foxq1, ZIC3, or all three transcription factors combined. n = 3 independent experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/487/eaag2476/DC1

    Fig. S1. Gene expression in the embryonic CNS vasculature at E14.5.

    Fig. S2. Translated transcripts encoding transporters in the embryonic CNS vasculature.

    Fig. S3. Translated transcripts encoding molecular markers related to physical barrier properties in the embryonic CNS vasculature.

    Fig. S4. Translated transcripts encoding transcription factors in embryonic vasculature.

    Fig. S5. Cdh5CreERT2 driver line activity is not strictly limited to ECs only.

    Fig. S6. Validation of TRAP-seq results by in situ hybridization.

    Fig. S7. Brain EC maturation during embryonic development.

    Fig. S8. Ctnnb1 deletion–dependent molecular changes in brain ECs.

    Fig. S9. Single-cell sequence reads and mapping.

    Fig. S10. High EC heterogeneity at the single-cell level for genes encoding general EC markers or mCherry.

    Fig. S11. High EC heterogeneity at the single-cell level for genes encoding specific BBB markers.

    Fig. S12. High level of EC heterogeneity.

    Fig. S13. High EC heterogeneity at the single-cell level for genes encoding transcription factors identified as enriched in CNS vasculature by TRAP-seq.

    Table S1. Specifically translated genes in brain ECs.

    Table S2. Brain EC–enriched transcripts.

    Table S3. Transcripts shared between ECs from all other organs.

    Table S4. Brain EC–depleted transcripts.

    Table S5. Specifically translated genes in brain ECs during embryonic development.

    Table S6. Regulated transcripts in brain ECs during embryonic development.

    Table S7. Transcripts with increasing translation in brain ECs during embryonic development.

    Table S8. Transcripts with decreasing translation in brain ECs during embryonic development.

    Table S9. Genes with increasing and subsequently decreasing translation in brain ECs during embryonic development.

    Table S10. Genes with decreasing and subsequently increasing translation in brain ECs during embryonic development.

    Table S11. E14.5 brain EC–enriched transcripts during embryonic development.

    Table S12. E14.5 shared EC transcripts during embryonic development.

    Table S13. E14.5 brain EC–depleted genes during embryonic development.

    Table S14. Genes encoding angiogenesis marker in brain ECs during embryonic development.

    Table S15. Genes encoding proliferation and cell cycle marker in brain ECs during embryonic development.

    Table S16. Ctnnb1 KO–deleted transcripts in brain ECs at E14.5.

    Table S17. Ctnnb1 KO–deleted transcripts in brain ECs at E17.5.

    Table S18. E14.5 brain EC–enriched transcripts in Ctnnb1 KO brain ECs.

    Table S19. Ctnnb1 KO–enriched transcripts in brain ECs at E14.5.

    Table S20. Ctnnb1 KO–enriched transcripts in brain ECs at E17.5.

    Table S21. E14.5 shared EC transcripts in Ctnnb1 KO brain ECs.

    Table S22. Genes encoding angiogenesis marker in Ctnnb1 KO brain ECs.

    Table S23. E14.5 brain EC–depleted genes in Ctnnb1 KO brain ECs.

    Table S24. Genes encoding proliferation and cell cycle marker in Ctnnb1 KO brain ECs.

    Table S25. Genes encoding housekeeping markers in single cells.

    Table S26. Genes encoding EC markers in single cells.

    Table S27. Genes encoding neuronal markers in single cells.

    Table S28. Genes encoding housekeeping markers in single HEK293T cells.

  • Supplementary Materials for:

    Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels

    Mike Hupe,* Minerva Xueting Li, Susanne Kneitz, Daria Davydova, Chika Yokota, Julianna Kele, Belma Hot, Jan M. Stenman, Manfred Gessler

    *Corresponding author. Email: mike.hupe{at}uni-wuerzburg.de

    This PDF file includes:

    • Fig. S1. Gene expression in the embryonic CNS vasculature at E14.5.
    • Fig. S2. Translated transcripts encoding transporters in the embryonic CNS vasculature.
    • Fig. S3. Translated transcripts encoding molecular markers related to physical barrier properties in the embryonic CNS vasculature.
    • Fig. S4. Translated transcripts encoding transcription factors in embryonic vasculature.
    • Fig. S5. Cdh5CreERT2 driver line activity is not strictly limited to ECs only.
    • Fig. S6. Validation of TRAP-seq results by in situ hybridization.
    • Fig. S7. Brain EC maturation during embryonic development.
    • Fig. S8. Ctnnb1 deletion–dependent molecular changes in brain ECs.
    • Fig. S9. Single-cell sequence reads and mapping.
    • Fig. S10. High EC heterogeneity at the single-cell level for genes encoding general EC markers or mCherry.
    • Fig. S11. High EC heterogeneity at the single-cell level for genes encoding specific BBB markers.
    • Fig. S12. High level of EC heterogeneity.
    • Fig. S13. High EC heterogeneity at the single-cell level for genes encoding transcription factors identified as enriched in CNS vasculature by TRAP-seq.
    • Legends for tables S1 to S28

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 2.99 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Specifically translated genes in brain ECs.
    • Table S2 (Microsoft Excel format). Brain EC–enriched transcripts.
    • Table S3 (Microsoft Excel format). Transcripts shared between ECs from all other organs.
    • Table S4 (Microsoft Excel format). Brain EC–depleted transcripts.
    • Table S5 (Microsoft Excel format). Specifically translated genes in brain ECs during embryonic development.
    • Table S6 (Microsoft Excel format). Regulated transcripts in brain ECs during embryonic development.
    • Table S7 (Microsoft Excel format). Transcripts with increasing translation in brain ECs during embryonic development.
    • Table S8 (Microsoft Excel format). Transcripts with decreasing translation in brain ECs during embryonic development.
    • Table S9 (Microsoft Excel format). Genes with increasing and subsequently decreasing translation in brain ECs during embryonic development.
    • Table S10 (Microsoft Excel format). Genes with decreasing and subsequently increasing translation in brain ECs during embryonic development.
    • Table S11 (Microsoft Excel format). E14.5 brain EC–enriched transcripts during embryonic development.
    • Table S12 (Microsoft Excel format). E14.5 shared EC transcripts during embryonic development.
    • Table S13 (Microsoft Excel format). E14.5 brain EC–depleted genes during embryonic development.
    • Table S14 (Microsoft Excel format). Genes encoding angiogenesis marker in brain ECs during embryonic development.
    • Table S15 (Microsoft Excel format). Genes encoding proliferation and cell cycle marker in brain ECs during embryonic development.
    • Table S16 (Microsoft Excel format). Ctnnb1 KO–deleted transcripts in brain ECs at E14.5.
    • Table S17 (Microsoft Excel format). Ctnnb1 KO–deleted transcripts in brain ECs at E17.5.
    • Table S18 (Microsoft Excel format). E14.5 brain EC–enriched transcripts in Ctnnb1 KO brain ECs.
    • Table S19 (Microsoft Excel format). Ctnnb1 KO–enriched transcripts in brain ECs at E14.5.
    • Table S20 (Microsoft Excel format). Ctnnb1 KO–enriched transcripts in brain ECs at E17.5.
    • Table S21 (Microsoft Excel format). E14.5 shared EC transcripts in Ctnnb1 KO brain ECs.
    • Table S22 (Microsoft Excel format). Genes encoding angiogenesis marker in Ctnnb1 KO brain ECs.
    • Table S23 (Microsoft Excel format). E14.5 brain EC–depleted genes in Ctnnb1 KO brain ECs.
    • Table S24 (Microsoft Excel format). Genes encoding proliferation and cell cycle marker in Ctnnb1 KO brain ECs.
    • Table S25 (Microsoft Excel format). Genes encoding housekeeping markers in single cells.
    • Table S26 (Microsoft Excel format). Genes encoding EC markers in single cells.
    • Table S27 (Microsoft Excel format). Genes encoding neuronal markers in single cells.
    • Table S28 (Microsoft Excel format). Genes encoding housekeeping markers in single HEK293T cells.

    [Download Tables S1 to S28]


    Citation: M. Hupe, M. X. Li, S. Kneitz, D. Davydova, C. Yokota, J. Kele, B. Hot, J. M. Stenman, M. Gessler, Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels. Sci. Signal. 10, eaag2476 (2017).

    © 2017 American Association for the Advancement of Science