The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells

Abstract

The blood-brain barrier (BBB) maintains brain homeostasis and limits the entry of toxins and pathogens into the brain. Despite its importance, little is known about the molecular mechanisms regulating the development and function of this crucial barrier. In this study we have developed methods to highly purify and gene profile endothelial cells from different tissues, and by comparing the transcriptional profile of brain endothelial cells with those purified from the liver and lung, we have generated a comprehensive resource of transcripts that are enriched in the BBB forming endothelial cells of the brain. Through this comparison we have identified novel tight junction proteins, transporters, metabolic enzymes, signaling components, and unknown transcripts whose expression is enriched in central nervous system (CNS) endothelial cells. This analysis has identified that RXRalpha signaling cascade is specifically enriched at the BBB, implicating this pathway in regulating this vital barrier. This dataset provides a resource for understanding CNS endothelial cells and their interaction with neural and hematogenous cells.

Figure 1. Purification of endothelial cells from the cerebral cortex of Tie2GFP mice.

A) GFP is expressed in the vasculature of Tie2GFP transgenic mice. GFP (green) can be visualized in a vascular pattern in tissue cryosections of cerebral cortex of an adult Tie2GFP transgenic mouse. Nuclei were stained with DAPI (blue). Bar, 50 microns. B) FACS purification of GFP⁺ cells from the cerebral cortex of Tie2GFP mice. Single cell cortical suspensions were sorted for GFP fluorescence twice, isolating a GFP⁺ cell population that is greater than 99.3% pure. Y-axis represents side scatter co-efficient, X-axis represents GFP fluorescence, numerical values represent percent of total cells sorted that are within gated region. C) Purity of FACS sorted cells. GeneChip transcriptional profiling of GFP⁻ (parenchyma), GFP⁺ (vascular) and GFP⁺PDGFRbeta⁻ (endothelial) fractions from the cerebral cortex of Tie2GFP mice. Neuronal, astrocyte, oligodendrocyte, microglial, endothelial and pericyte markers were analyzed to detect the purity of sample for each FACS sorted sample. The GFP⁺ sample contains a mixture of endothelial cells and pericytes, such that an additional PDGFRbeta negative selection is needed to generate a pure endothelial cell population. Error Bars represent standard error of the mean (SEM).

Figure 2. Comparison of transcriptional profiles of brain endothelial cells with liver and lung endothelial cells.

A) Affymetrix GeneChips were utilized to compare gene expression in GFP⁺ cells isolated from cerebral cortex, liver and lung of Tie2GFP transgenic mice. The data is represented as a dot plot on a logarithmic scale, where each point reflects a probe set on the GeneChip. Black dots indicate probe sets identified as present in both samples, red dots indicate probe sets identified as present in the brain but not peripheral sample, blue dots indicate probe sets present in the peripheral but not brain sample, and grey dots represent probe sets identified as absent in both samples. Two biological replicates of the brain GFP⁺ samples were compared to identify variation between replicates. In addition, brain GFP⁺ cells were compared with profiles generated for neurons and glia by Cahoy et al (2008). Black diagonal lines represent cutoffs for two-fold, four-fold and eight-fold differences. B) Validation of transcriptional profiling by comparison of GeneChip expression with known CNS endothelial markers. Expression values are given relative to the brain GFP⁺ sample. Pan endothelial transcripts are expressed in all GFP⁺ samples, whereas BBB transcripts were enriched in the Brain GFP⁺ sample. Error bars represent SEM.

Figure 3. Selective expression of blood-brain barrier-enriched genes in CNS endothelial cells.

Expression of Pecam (A, B), Cldn5 (C, D), Apcdd1 (E, F), Abcb1a1 (G, H), Slco1c1 (I, J), Slco1a4 (K, L), Slc22a8 (M, N), Slc7a5 (O, P), Itih5 (Q, R) and Foxq1 (S, T) mRNA in the brain and liver of P20 mouse pups. The top four brain endothelial transcripts enriched compared with liver (Itih5, Apcdd1, Abcb1a and Slco1c1) and lung endothelial cells (Slc22a8, Slc7a5, Slco1a4 and Foxq1) were analyzed, excluding transcripts also expressed in the brain parenchyma (brain GFP⁺:brain GFP⁻<2), and those expressed at <10000 to ensure that the signal would be above the detection limit of the in situ hybridization. Note that Pecam1 and Cldn5 mRNAs are expressed in the endothelial cells in both brain and liver. However, the transcripts for the blood-brain barrier enriched genes are only present in brain but not liver endothelial cells. Bar, 200 microns.