(A) Genome browser images showing CG methylation (top) and RNA expression (bottom) for two genes: Slc2a1, a glucose transporter expressed in brain ECs, and Fabp4, a fatty acid binding protein …
(A) A representative flow cytometry profile of ECs sorted from Tie2-GFP kidneys. The thresholds used to define GFP-positive CD11b-negative ECs (top left), singlets (top right), and viable propidium …
(A) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing …
(A) Examples of liver gene expression to assess purity of FACS-sorted fractions. Expression levels (TPMs) based on RNA-seq for genes known to be expressed in Kupffer cells (top row) or …
(A) Scatter plots comparing cross-sample normalized RNA-seq read counts of protein-coding genes for pairs of biological replicates from liver ECs, lung ECs, and kidney ECs. (B) Scatter plots …
(A) Scatter plots comparing the mean fraction of CG methylation in a 5 kb window immediately 3’ of the TSS for protein-coding genes from brain ECs versus liver, lung, and kidney ECs. Colored symbols …
(A) Scatter plots comparing the mean fraction of CG methylation in a 5 kb window immediately 3’ of the TSS for protein-coding genes for each of the peripheral EC biological replicates. (B) Scatter …
(A) Genome browser images showing CG methylation at differentially expressed TF genes. Colored bars indicate DMVs. Red arrows indicate illustrative examples of differential hypermethylation. (B) …
(A) Genome browser image showing CG methylation (top) and accessible chromatin (bottom) at the HOX-A gene cluster. HOX genes in this cluster are expressed in an anterior-posterior gradient …
Genome browser images showing CG methylation (top) and accessible chromatin (bottom) at the (A) HOX-B gene cluster, the (B) HOX-C gene cluster, and the (C) HOX-D gene cluster. HOX genes in each …
(A) Genome browser images showing CG methylation (top) and accessible chromatin (bottom) around ECTSGs. Colored bars under mCG tracks mark UMRs (upper row) and LMRs (lower row). For accessible …
(A) The fraction of mapped and properly paired reads within called peaks for each ATAC-seq replicate. (B) Peak saturation analysis for each ATAC-seq replicate. From the total number of mapped and …
(A) Expression levels (TPMs) based on RNA-seq for the genes shown in Figure 4A. (B) Heatmap depicting the percent of <100 bp ATAC-seq peaks (APs) that overlap between EC subtypes. Many of the …
(A) Scatter plots of normalized ATAC-seq read density (N) within ATAC-seq peaks called from <100 bp fragments between biological replicates. (B) Scatter plots of normalized ATAC-seq read density (N) …
(A) Heatmap depicting the percentage of various epigenetic features that overlap between EC subtypes. More UMRs are shared between EC subtypes than LMRs. A larger fraction of UMRs are accessible …
(A) HOMER-identified enriched motifs in ECTS-hypo-DMRs. Frequency of the indicated motif as a function of distance from the center of ECTS-hypo-DMRs. Shown above each individual plot is the position …
(A) HOMER-identified enriched motifs in EC-only LMRs and ATAC-seq peaks shared between all four EC subtypes. Frequency of the indicated motif as a function of distance from the center of these LMRs …
(A) Genome browser images showing CG methylation (top) and accessible chromatin (bottom) around Zic3, a brain EC-specific TF. (B–F) Genome browser images showing (from top to bottom): ECTS-hypo-DMRs …
(A–C) Coronal sections of E13.5 Tie2-Cre;R26-Tcf/Lef-LSL-H2B-GFP-6xMYC embryos near the cephalic flexure. The markers are: ICAM2 (pan-EC membrane protein), MYC (the canonical Wnt reporter), ERG …
(A) Quantification of Figure 6A. The values shown are the ratio of MYC +nuclei (i.e. Wnt reporter+) to ERG + nuclei. (B) Quantification of Figure 6C. The values shown are the number of either MYC +or…
(A) Coronal sections of E13.5 Tie2-Cre;R26-Tcf/Lef-LSL-H2B-GFP-6xMYC embryos near the cephalic flexure, immunostained for the indicated markers: ICAM2, MYC, LEF1, and DAPI. (A’) Higher magnification …
(A) t-SNE plot of 3946 P7 brain ECs showing six clusters corresponding to tip cells, and mitotic, venous, capillary-venous (Capillary-V), capillary-arterial (Capillary-A), and arterial ECs. (B) The …
(A–F) Coronal section of a P7 C57BL/6 brain stained for either GS lectin (green) to mark blood vessels or DOPA decarboxylase (DDC; magenta). Rightmost column shows merged images. Images are from the …
(A–E) Whole mount retina in situ hybridization (ISH) for known tip cell marker Apln (A); novel tip cell markers Mcam (B), Lamb1 (C), and Trp53i11 (D); and a novel arterial marker Tm4sf1 (E). The …
(A) Plot showing the position of cells in each EC cluster on the constructed cell trajectory. (B) Summary of the two branch points (labeled ‘1’ and ‘2’) in the cell trajectory analysis. M, mitotic; …
(A) t-SNE plot from Figure 7A showing expression of several interesting genes. Cyclin-dependent kinase inhibitor Cdkn1a was enriched in mitotic cells, tip cells and capillary-A ECs, Cdkn1c was …
Abbreviation | Definition |
---|---|
ATAC | Assay for Transposase Accessible Chromatin |
BBB | Blood-brain barrier |
CRE | Cis-regulatory element |
DEG | Differentially expressed gene |
DMR | Differentially methylated region |
DMV | DNA methylation valley (UMR > 3 kb) |
EC | Endothelial cell |
ECTS-hypo-DMR | Endothelial cell tissue-specific hypo-DMR |
ECTSAP | Endothelial cell tissue-specific ATAC-seq peak |
ECTSG | Endothelial cell tissue-specific gene |
GO | Gene ontology |
LMR | Low-methylated region (hypomethylated, CG-poor) |
PCA | Principal component analysis |
scRNA-seq | Single-cell RNA-seq |
TF | Transcription factor |
TSS | Transcription start site |
UMR | Unmethylated region (hypomethylated, CG-rich) |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (Mus musculus; Male) | Tie2-GFP | The Jackson Laboratory | Stock No: 003658; RRID:IMSR_JAX:003658 | |
Genetic reagent (M. musculus; Male) | Tie2-Cre | The Jackson Laboratory | Stock No: 008863 | |
Genetic reagent (M. musculus; Male) | R26-Tcf/Lef-LSL- H2B-GFP-6xMYC | PMID: 28803732 | ||
Antibody | anti-CD11b BV421 (rat monoclonal) | Biolegends | Cat No: 101235; RRID:AB_10897942 | 1:1000 |
Antibody | anti-ICAM2 (rat monoclonal) | BD Biosciences | Cat No: 553326 | 1:300 |
Antibody | anti-6xMYC (chicken polyclonal) | PMID: 24411735 | 1:10000 | |
Antibody | anti-GLUT1 (rabbit polyclonal) | Thermo Fisher Scientific | RB-9052 | 1:500 |
Antibody | anti-ERG (rabbit polyclonal) | Cell Signaling | A7L1G | 1:500 |
Antibody | anti-dopa decarboxylase (goat polyclonal) | R and D Systems | AF3564 | 1:500 |
Antibody | anti-ZIC3 (rabbit polyclonal) | PMID: 23217714 | 1:50 | |
Other | Alexa Fluor 594-conjugated GS Lectin | Thermo Fisher Scientific | L21416 | 1:200 |
Peptide, recombinant protein | Tn5 transposase | Illumina | FC-121–1030 | |
Commercial assay or kit | Worthington Papain Dissociation Kit | Worthington Biochemical Corporation | LK003160 | |
Commercial assay or kit | Rneasy Micro Plus Kit | Qiagen | 74034 | |
Commercial assay or kit | Dneasy Blood and Tissue Kit | Qiagen | 69504 | |
Commercial assay or kit | MinElute GelExtraction Kit | Qiagen | 28604 | |
Commercial assay or kit | Agencourt AMPure XP beads | Beckman Coulter | A63880 | |
Commercial assay or kit | EZ DNA Methylation-Direct Kit | Zymo | D5021 | |
Software, algorithm | RSEM | PMID: 21816040 | RRID:SCR_013027 | |
Software, algorithm | DESeq2 | PMID: 25516281 | RRID:SCR_015687 | |
Software, algorithm | Bowtie2 | PMID: 22388286 | ||
Software, algorithm | MACS2 | PMID: 18798982 | ||
Software, algorithm | DiffBind | PMID: 22217937 | RRID:SCR_012918 | |
Software, algorithm | deepTools | PMID: 27079975 | ||
Software, algorithm | HOMER | PMID: 20513432 | ||
Software, algorithm | Monocle | PMID: 24658644 | ||
Software, algorithm | Methylpy | PMID: 26030523 | ||
Software, algorithm | BEDTools | PMID: 20110278 | RRID:SCR_006646 |
Characteristics of each sequencing sample.
(A) RNA-seq. (B) ATAC-seq. (C) MethylC-seq
Gene expression data.
(A-C) Transcript abundances in raw expected read counts (A), cross-sample normalized read counts (B), and TPMs (C). (D) List of EC-enriched transcripts. (E) List of all differentially expressed genes between one or more pairs of EC subtypes. (F) List of ECTSGs
Gene ontology (GO) enrichment analysis
(A) GO enrichment analysis of brain ECTSGs. (B) GO enrichment analysis of liver ECTSGs. (C) GO enrichment analysis of lung ECTSGs. (D) GO enrichment analysis of kidney ECTSGs.
Hypomethylated features in each EC subtype
(A-D) UMRs for each EC subtype. (E-H) LMRs for each EC subtype. (I-L) DMRs for each EC subtype. (I’-L’) ECTS-hypo-DMRs for each EC subtype. (M-P) DMVs for each EC subtype. (Q-T) ECTS-large hypo-DMRs for each EC subtype. (U) LMRs found only in ECs relative to photoreceptors and cortical neurons. (V) LMRs from (U) that are shared between EC subtypes
Numbers underlying heatmaps shown throughout figures.
Accessible chromatin peaks in each EC subtype.
(A-D) ATAC-seq peaks for each EC subtype called using the full range of ATAC-seq fragment lengths. (E-H) ATAC-seq peaks for each EC subtype called using <100 nt ATAC-seq fragments. (I-L) Differential ATAC-seq peaks (<100 nt) for each EC subtype. (I’-L’) ECTSAPs (<100 nt) for each EC subtype. (M) ATAC-seq peaks (<100 nt) found only in ECs relative to photoreceptors and cortical neurons. (N) ATAC-seq peaks from (M) that are shared between EC subtypes.
HOMER motif files used in this study.
(A) HOMER motif files for enriched k-mers identified in ECTS-hypo-DMRs and ECTSAPs. (B) HOMER motif file used for representative member of TF families. (C) HOMER motif file used for paired ETS:ZIC motif.
Top 25 genes for each single-cell RNA-seq cluster.
(A) Arterial cluster markers. (B) Capillary-A cluster markers. (C) Capillary-V cluster markers. (D) Mitotic cluster markers. (E) Tip cell cluster markers. (F) Venous cluster markers.