Figure 3. Stabilization of beta-catenin does not promote a BBB-like state in cultured CNS ECs as determined by RNA-seq and ATAC-seq.

(A) Schematic outline of the genetic strategy for stabilizing beta-catenin (encoded by Ctnnb1). The Ctnnb1^flex3 allele contains loxP sites flanking exon 3. Cre-recombination produces an in-frame deletion of exon 3, and the resulting beta-catenin protein is stabilized and activates transcription in conjunction with LEF/TCF proteins. (B) Analysis of Ctnnb1 transcripts that include or omit exon 3. The six replicates of wild-type (WT) cultured adult brain ECs produced no RNA-seq reads that join exons 2+4 whereas each of the four replicates of Ctnnb1^flex3/+;Pdgfb-CreER;Tie2-GFP cultured adult brain ECs (in which exon 3 is deleted by Cre-mediated recombination) produced several hundred RNA-seq reads that join exons 2+4. (C) Scatter plots comparing cross-sample normalized RNA-seq read counts for protein-coding genes between beta-catenin stabilized and WT primary brain ECs cultured for 8 days. The left plot highlights (in red) EC-enriched genes that are expressed at similar levels in brain, liver, lung, and kidney EC subtypes. The right plot highlights (in blue) genes associated with the blood-brain barrier (BBB). Gene sets are as described for Figure 1B. Stabilizing beta-catenin does not increase the expression of BBB genes in cultured ECs. (D) Transcript abundances for seven genes from the ‘Regulated by beta-catenin signaling’ category. (E) PCA of protein-coding transcript abundances (RNA-seq; top) and PCA of ATAC-seq read density at all called ATAC-seq peaks (bottom) from independent biological replicates of WT and beta-catenin stabilized cultured adult brain ECs, acutely isolated adult and P7 brain ECs, and P7 liver, lung, and kidney ECs. The cyan-outlined red circles correspond to the beta-catenin stabilized cultured adult brain EC samples, and are indicated by cyan arrows. Black arrows point to cultured brain ECs without stabilized beta-catenin.

Figure 3—figure supplement 1. In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3.

Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019). The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1^flex3/+;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1^flex3/+;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.

Figure 3—figure supplement 2. WT and beta-catenin stabilized brain ECs in culture have nearly identical patterns of transcription and accessible chromatin.

Genome browser images showing ATAC-seq reads (top) and RNA-seq reads (bottom) for the same set of genes shown in Figure 2A for freshly isolated adult brain ECs, WT cultured adult brain ECs, and beta-catenin stabilized and cultured adult brain ECs. Read counts are averaged over the independent replicates. Arrows indicate ATAC-seq peaks that correlate with differential gene expression.