Pericytes are required for blood–brain barrier
integrity during embryogenesis
Richard Daneman1, Lu Zhou2, Amanuel A. Kebede1& Ben A. Barres2
barrier that restricts the movement of molecules and ions between
ensure proper neuronal function and protect the CNS from injury
and disease1. Transplantation studies have demonstrated that the
BBB is not intrinsic to the endothelial cells, but is induced by inter-
actions withtheneural cells2. Owingto theclose spatial relationship
between astrocytes and endothelial cells, it has been hypothesized
of BBB formation has been controversial4–9. Here we demonstrate
that the barrier is formed during embryogenesis as endothelial cells
invade the CNS and pericytes are recruited to the nascent vessels,
over a week before astrocyte generation. Analysing mice with null
and hypomorphic alleles of Pdgfrb, which have defects in pericyte
generation, we demonstrate that pericytes are necessary for the
formation of the BBB, and that absolute pericyte coverage deter-
minesrelativevascularpermeability.We demonstrate that pericytes
regulate functional aspects of the BBB, including the formation of
tight junctions and vesicle trafficking in CNS endothelial cells.
Pericytes do not induce BBB-specific gene expression in CNS
vascular permeability and CNS immune cell infiltration. These data
indicate that pericyte–endothelial cell interactions are critical to
actions may lead to BBB dysfunction and neuroinflammation
during CNS injury and disease.
To understand the mechanisms of BBB formation, we investigated
the sequence of cell generation and BBB formation in the developing
CNS (Fig. 1 and Supplementary Figs 1–7). In the rat cerebral cortex,
angiogenesis begins at embryonic day 12 (E12), as endothelial cells
invade the neural tissue fromthe surrounding vascular plexus (Fig. 1a
and Supplementary Fig. 1). Pericytes—platelet-derived growth factor
receptor-b (PDGFR-b)1NG21vascular support cells—are found
associated with endothelial tubes as nascent vessels are generated
(Fig. 1b and Supplementary Fig. 1). Neural cells are produced from
progenitorsin a defined sequence, with neuronsgeneratedbefore glia.
are first observed at E19 and migrate throughout the cortex by birth
(Fig. 1c). Astrocytes are first generated directly after birth and extend
and Supplementary Fig. 2). If astrocytes induce the BBB, barrier
properties should only be acquired after birth.
We next examined endothelial cell protein expression and barrier
function during rat cortical development. BBB-forming endothelial
cells are characterized by tight junctions, low rates of transcytosis,
and the expression of specialized influx and efflux transporters.
Tight junction molecules occludin, claudin 5 and ZO-1 were each
tary Fig. 4 and not shown). The same was observed with the BBB-
specific influx transporter Glut1 (Fig. 1f and Supplementary Fig. 3).
Notably, the BBB-specific efflux transporter Pgp is expressed at low
levels during embryogenesis, but increases during postnatal develop-
ment (Fig. 1g), indicating a distinct regulation mechanism for efflux
transport. Similar timing of cell generation and BBB gene expression
was observed in the developing mouse, with vascularization of the
cortexstarting atE11(Supplementary Fig. 6).The expression of genes
that increasevascularpermeability,includingtranscytosis(Plvap) and
leukocyte infiltration (Icam1), decreased after initial high expression
(Supplementary Fig. 7). The developmental timing of BBB function
was examined by trans-cardiac perfusion with tracers. In adults, the
the tracer diffuses throughout the extracellular space (Fig. 1h–j). We
used this method to examine BBB function in postnatal animals and
vessels excluded the tracer from the CNS parenchyma (Fig. 1h–j
and not shown). In embryonic time points, the tracer was excluded
frommost of the CNS; however,distinct regions of the CNS displayed
leakiness, including regions close to the pia (Supplementary Fig. 5),
ings or choroid plexus. Thus, a functional BBB is present during
embryogenesis before astrocyte generation.
Because pericyte recruitment to CNS vessels temporally correlates
cells in regulating BBB function, structure and gene expression.
Although pericytes are associated with the vasculature throughout
the body, and thus are unlikely candidates to regulate brain-specific
vascular properties, recent studies have demonstrated that CNS peri-
cytes haveadifferentdevelopmentaloriginfromother pericytes10,and
several studies have suggested that pericytes are capable of regulating
BBB properties in vitro11–13. Todetermine ifpericytes are necessary for
BBB formation we compared the vascular permeability of Pdgfrb2/2
or receptor completely lack CNS pericytes14,15, exhibit endothelial cell
hyperplasia, increased vessel diameter and morphological signs of
increased vascular permeability16. These mice die at birth; therefore,
we examined BBB function in dissected embryos. Indeed, Pdgfrb2/2
mice show an increased vascular permeability to biotin (0.5kDa), as
observed by an increase in tracer staining throughout the CNS par-
enchyma (Fig. 2a, b). Decreasing permeability was observed with
increasing size of tracer (Fig. 2c).
CNS vessels have the highest pericyte coverage of any vessels, and
correlates with the relative permeability of these vessels17. To deter-
important regulator of BBB permeability, we measured CNS vascular
permeability in mice with different combinations of null, hypo-
morphic and wild-type Pdgfrb alleles. One study15generated an allelic
series of Pdgfrb hypomorphs, showing that varying the strength of
PDGFR-b signalling leads to different pericyte:endothelial cell ratios.
1UCSF Department of Anatomy, 513 Parnassus Avenue, HSW1301, San Francisco, California 94143-0452, USA.2Stanford University School of Medicine, Department of Neurobiology, Fairchild Science
Building D200, Stanford, California 94305-5125, USA.
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and raw image analysis with Affymetrix GCOS 1.3 software was performed as
Western blots. Cerebral cortex from E18 Pdgfrb2/2and littermate controls were
homogenized and re-suspended and lysed in RIPA buffer (50mM Tris pH7.4,
was quantified using BCA protein assay (Pierce). Samples were analysed by SDS–
PAGE as previously described33. Briefly, samples were resolved on SDS–PAGE and
transferred to PVDF membranes. Membranes were blocked with 5% milk solution
(Abcam), Col III (Abcam), vitronectin (Abcam), MMP9 (Abcam), PDGFR-b
(eBiosciences) or b-actin (Sigma) followed by incubation with an appropriate
secondary conjugated to HRP (Jackson 1:10,000) and visualized using a chemi-
luminescent ECL substrate for HRP (GE), and either exposed on film and quan-
tified with ImageJ or imaged with a Fuji-Film LAS 4000 and analysed with Multi
Gauge V3.0 software (n52–5 for mutant and n54–8 for littermate controls).
regulated by c-secretase and astrocytes in a rapidly myelinating CNS coculture
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32. Cahoy, J. D. et al. A transcriptome database for astrocytes, neurons, and
oligodendrocytes: a new resource for understanding brain development and
function. J. Neurosci. 28, 264–278 (2008).
oligodendrocytes. Neuron 43, 183–191 (2004).
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