Supplementary Discussion

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1 doi: /nature09513 Supplementary Discussion Time course of CNS vascular development The timing of endothelial cell entry into the rat cerebral cortex and pericyte recruitment are further demonstrated in Supplementary Figure 1. In this figure pericytes are labeled with an antibody directed against Ng2. Ng2 is expressed both in pericytes and OPCs, but here we analyze early embryonic time points from E11-E17 prior to OPC generation at E19 (Fig. 1C), therefore all Ng2 + cells are pericytes. Endothelial cells are first observed on the cortical surface at E11 in a caudal to rostral gradient (Supplementary Fig. 1A-B). Ng2 + cells are also observed on the cortical surface in a similar gradient (Supplementary Fig. 1A-B). Endothelial cells also enter the cortex in a caudal-rostral gradient starting at E12, and these initial sprouts into the CNS are not covered by pericytes (Supplementary Fig. 1C-D). Pericytes are soon recruited to the nascent vessels and are observed to be coating the vessels by E13 and this process continues as vessels continue to be generated (Supplementary Fig. 1E-F). Pericyte cell bodies are closely associated with the endothelial cells, and have processes forming all around the endothelial cells (Supplementary Fig. 1G) Time course of BBB development We further examined the time course of Glut1 and ZO-1 expression in the developing rat cerebral cortex (Supplementary Fig. 3, 4). At E11-12, when endothelial cells are associated with the cortical surface, but have yet to dive into the cortex, the developing neural tissue expresses Glut1 throughout all cells (Supplementary Fig. 3A-C). As endothelial cells enter the cortex, the expression of Glut1 is quickly restricted to the endothelial cells, and lost from the developing neural tissue (Supplementary Fig. 3D-F). Glut1 is expressed in the earliest cells that invade the CNS (Supplementary Fig. 3D). At E11-E12 the tight junction protein ZO-1 is expressed in the epithelial cells in the ependymal layer of the developing cortex (Supplementary Fig. 4A-B). At E12, endothelial cells express ZO-1 as they begin to invade the cortex (Supplementary Fig. 4B). At this early developmental time point ZO-1 staining is patchy throughout the endothelial cell, and then is observed to form complete rings around the cell borders at E15 (Supplementary Fig. 4D). This is consistent with electron microscopy studies which suggest that ultrastructural tight junctions in CNS endothelial cells gain complexity through development 1. Our data also demonstrated that genes associated with increased permeability, including Plvap (transcytosis) and Icam1 (leukocyte adhesion) are initially expressed in endothelial cells as they enter the cerebral cortex, but are down-regulated during development (Supplementary Fig. 7). This is consistent with ultrastructural work which has demonstrated that pinocytotic vesicles are significantly down-regulated during embryonic mouse development 2, and correlates with the pericyte coverage of the vessels. Interestingly, several studies suggest that there may be species specific differences in BBB development. For example, elegant studies in the opossum, a marsupial which is born prior to vascularization of the cerebral cortex, have demonstrated a tight barrier as soon as vessels enter the cortex 3. Furthermore, studies of plasma protein localization in human brain samples also have suggested that the BBB is present at early stages of brain development 4. This is in contrast with data which suggests that fenestrated vessels are present in the developing rodent brain. Our studies, however, suggest that barrier properties are present in rat and mouse vessels as soon as they enter the cerebral cortex, similar to that of humans and opossums. Perhaps a leaky BBB is not conducive to neural development. 1

2 Time Course of Glial Cell Development We have demonstrated that in the rat cerebral cortex OPC generation begins at E18 and astrocyte generation starts at birth. This corresponds with existing literature in which it has been observed that neural stem cells produce neurons and glia in a defined order: first neurons, then oligodendrocyte lineage cells, and then astrocytes 5,6. Furthermore, our work corresponds with existing literature which suggests that astrocytes extend elaborate cellular processes during the first few postnatal weeks that contact both neurons and vessels 5. The fact that we observe BBB properties prior to astrocyte generation suggests that astrocytes cannot be the cell type that induces BBB properties during embryogenesis. Interestingly, there is a striking up-regulation of Pgp that corresponds with astrocyte generation and ensheathment of vessels. Indeed, astrocytes have been demonstrated to induce Pgp expression in endothelial cells 7. Furthermore, there is an increase in trans-endothelial cell electrical resistance during postnatal development in vivo, suggesting that astrocytes may be key to the maturation of the BBB during postnatal development 8. In addition, although we observed tracer leakage in Pdgfrb F7/- and Pdgfrb F7/F7 neonatal mice, no tracer leakage was observed in the adults of these genotypes (data not shown), suggesting that astrocytes can further strengthen the barrier after they ensheath the vessels. A role for astrocytes dynamically regulating the BBB in response to blood contents, neuronal activity and disease remains very likely. Pericyte Coverage in Pdgfrb +/+ and Pdgfrb +/- mice We examined the pericyte coverage of CNS vessels in the cortex of Pdgfrb +/+ and Pdgfrb +/- mice (Supplementary Fig. 8) by Zic1 staining for pericyte nuclei, desmin staining for pericyte cytoskeleton, and Ng2 staining for pericyte cell surface. Tallquist et al 9 have identified that there was a decrease in pericyte number in Pdgfrb +/- mice. By Zic1 staining we also identified that there was a small decrease in pericyte number, however, we have identified that there is comparable coverage of the vascular tubes by Ng2+,desmin+ pericyte processes in both Pdgfrb +/+ and Pdgfrb +/- mice. This suggests that with a small decrease in pericyte number, the remaining pericytes can cover the vasculature to comparable levels. We further demonstrated that there was no difference in the vascular permeability of Pdgfrb +/+ and Pdgfrb +/- mice (Supplementary Fig. 8). Due to the comparable pericyte coverage and vascular permeability, we have combined both Pdgfrb +/+ and Pdgfrb +/- mice as littermate controls for Pdgfrb -/- mice Vascular Permeability in Pdgfrb -/- mice Here we have identified an increase in vascular permeability in Pdgfrb -/- mice that lack CNS pericytes. Hellstrom et al identified that pericyte-deficient mice have increased VEGFA expression, which suggests that the increased permeability may be due to systemic effects of VEGF or other inflammatory molecules including cytokines, and not direct signaling between endothelial cells and pericytes 10. On the other hand, our data demonstrates that co-culture of CNS pericytes with endothelial cells is sufficient to induce BBB properties in CNS endothelial cells, including increased tight junction resistance and inhibition of Icam1 and Angpt2 expression. Therefore, the increased vascular permeability observed in 2

3 the pericyte-deficient mice may be due to a combination of systemic metabolic differences and direct pericyte-endothelial cell signaling. Whereas Hellstrom et al did not analyze BBB function in the Pdgfrb -/- mice, they did demonstrate an increase in membrane folding and vesicular trafficking that was similar to our results. They further demonstrated an increase in caveolin-1 and a redistribution of occludin strands in the Pdgfrb -/- mice 10. We did not observe these defects in our analysis of Pdgfrb -/- mice, which suggests that other factors may influence vascular permeability in these mice including strain and environmental conditions. Interestingly, we observed heterogeneity in Gr1+ leukocyte infiltration in different Pdgfrb F7/F7 mice suggesting that additional environmental factors may be important for leukocyte infiltration, in addition to the increase in Icam1 expression in endothelial cells. Hellstrom et al also identified an increase in Glut1 and Pgp expression in the Pdgfb -/- mice, which lack the PDGF-B ligand and are also pericyte-deficient. They suggest that this may indicate metabolic stress 10. This paper did not analyze these genes in Pdgfrb -/- mice, and we did not observe an increase in either Glut1 or Pgp. This may also indicate strain or environmental differences, or perhaps that PDGF-B has more effects than just signaling through PDGFR-β. Identification of Pericyte-Derived Factors To determine the pericyte transcriptome we performed microarray analysis on mrna isolated from acutely purified CNS pericytes and compared these values to the microarray analysis performed on purified endothelial cells from the Pdgfrb -/- mice which completely lack pericytes. This analysis produced a dataset of pericyte-expressed molecules (Supplementary Table 2). Interestingly, pericytes express genes that are known to induce BBB properties including Angpt1 (as well as the homologous gene Angptl2), and Ace2 11,12. Ace2 is an enzyme involved in the processing of Agt to smaller peptides, which regulate BBB permeability 12. Several other interesting secreted molecules are expressed by endothelial cells, including Bmp5, nodal, netrin1, sema5a, as well as integrins and plexins. Furthermore, we have identified that pericytes express a number of extracellular matrix molecules, including collagens, laminins, vitronectin, asporin and others. We have further analyzed the protein expression of these ECM molecules in Pdgfrb -/- mice and littermate controls (Supplementary Fig. 11) and have identified that there are lower levels of collagens in Pdgfrb -/- mice, but strikingly an increase in vitronectin and MMP9. The altered composition of the endothelial ECM may be important in regulating BBB properties. Specifically, expression of MMP9 has been shown to lead to BBB breakdown in pathological conditions 13,

4 Supplementary References 1. Kniesel, U., Risau, W. & Wolburg, H. Development of blood-brain barrier tight junctions in the rat cortex. Brain Res Dev Brain Res 96, (1996). 2. Bauer, H. C. et al. Neovascularization and the appearance of morphological characteristics of the blood-brain barrier in the embryonic mouse central nervous system. Brain Res Dev Brain Res 75, (1993). 3. Ek, C. J., Dziegielewska, K. M., Stolp, H. & Saunders, N. R. Functional effectiveness of the bloodbrain barrier to small water-soluble molecules in developing and adult opossum (Monodelphis domestica). J Comp Neurol 496, (2006). 4. Mollgard, K., Dziegielewska, K. M., Saunders, N. R., Zakut, H. & Soreq, H. Synthesis and localization of plasma proteins in the developing human brain. Integrity of the fetal blood-brain barrier to endogenous proteins of hepatic origin. Dev Biol 128, (1988). 5. Bushong, E. A., Martone, M. E. & Ellisman, M. H. Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development. Int J Dev Neurosci 22, (2004). 6. 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, (2008). 7. Gaillard, P. J. et al. Astrocytes increase the functional expression of P-glycoprotein in an in vitro model of the blood-brain barrier. Pharm Res 17, (2000). 8. Butt, A. M., Jones, H. C. & Abbott, N. J. Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study. J Physiol 429, (1990). 9. Tallquist, M. D., French, W. J. & Soriano, P. Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development. PLoS Biol 1, E52 (2003). 10. Hellstrom, M. et al. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol 153, (2001). 11. Hori, S., Ohtsuki, S., Hosoya, K., Nakashima, E. & Terasaki, T. A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro. J Neurochem 89, (2004). 12. Wosik, K. et al. Angiotensin II controls occludin function and is required for blood brain barrier maintenance: relevance to multiple sclerosis. J Neurosci 27, (2007). 13. Gidday, J. M. et al. Leukocyte-derived matrix metalloproteinase-9 mediates blood-brain barrier breakdown and is proinflammatory after transient focal cerebral ischemia. Am J Physiol Heart Circ Physiol 289, H (2005). 14. He, Z. J., Huang, Z. T., Chen, X. T. & Zou, Z. J. Effects of matrix metalloproteinase 9 inhibition on the blood brain barrier and inflammation in rats following cardiopulmonary resuscitation. Chin Med J (Engl) 122, (2009). 4

5 Gene Symbol Probe Set Control PDGFRβ -/- Fold Change Pan-Endothelial cadherin 5 (VE-Cadherin) Cdh _at endothelial-specific receptor tyrosine kinase (Tie2) Tek _at Tight Junction claudin 5 Cldn _at occludin Ocln _at tight junction protein 1 (ZO-1) Tjp _a_at Influx Transporters solute carrier family 2, member 1 (Glut-1) Slc2a _a_at solute carrier family 7, member 1 (Cat1) Slc7a _at solute carrier family 7, member 5 (TA1) Slc7a _at solute carrier family 1, member 1 (EAAC1) Slc1a _a_at solute carrier family 38, member 5 (JM24) Slc38a _at solute carrier family 16, member 1 (MCT1) Slc16a _at Efflux Transporters ATP-binding cassette, sub-family B, member 1A (Pgp) Abcb1a _at ATP-binding cassette, sub-family G, member 2 Abcg _at Permeability Modulators angiopoietin 1 Angpt _at angiopoietin 2 Angpt _at plasmalemma vesicle associated protein Plvap _at Leukocyte Adhesion mucosal vascular addressin cell adhesion molecule 1 Madcam _a_at lectin, galactose binding, soluble 3 Lgals _at intercellular adhesion molecule Icam _at activated leukocyte cell adhesion molecule Alcam _at Supplementary Table 1: Gene expression in Pdgfrb -/- mice Affymetrix microarrays were used to analyze mrna expression levels in purified CD31 + vascular cells from Pdgfrb -/- mice and litter mate controls. Selected genes are listed with gene expression data from Pdgfrb -/- mice and litter-mate controls. Direction of fold change are given if >2. Pan-endothelial genes, tight junction molecules, influx transporters, efflux transporters displayed unaltered gene expression, whereas the vasculature of Pdgfrb -/- mice displayed an increase in factors that increase vascular permeability and leukocyte adhesion. 5

6 Gene Symbol Probe set Pericytes Endothelial cells Pericyte/ Endothelial Cells Pericyte Markers platelet derived growth factor receptor beta Pdgfrb _a_at chondroitin sulfate proteoglycan 4 Cspg _at desmin Des _at regulator of G-protein signaling 5 Rgs _at K+ inwardly-rectifying channel, subfamily J, member 8 Kcnj _at ATP-binding cassette, sub-family C, member 9 Abcc _s_at Extracellular Matrix procollagen, type VI, alpha 2 Col6a _a_at procollagen, type III, alpha 1 Col3a _a_at procollagen, type I, alpha 2 Col1a _a_at procollagen C-endopeptidase enhancer protein Pcolce _a_at laminin gamma 3 Lamc _at laminin, alpha 2 Lama _at vitronectin Vtn _a_at extracellular matrix protein 2 Ecm _at dentin matrix protein 1 Dmp _s_at asporin Aspn _s_at Signaling Factors angiotensin I converting enzyme 2 Ace _at angiopoietin 1 Angpt _at angiopoietin-like 2 Angptl _at bone morphogenetic protein 5 Bmp _at integrin alpha 8 Itga _at integrin beta 5 Itgb _a_at leucine rich repeat containing 16 Lrrc16a _a_at leucine rich repeat containing 4C Lrrc4c _at nodal Nodal _at Notch gene homolog 3 Notch _s_at netrin 1 Ntn _at plexin domain containing 1 Plxdc _at plexin domain containing 2 Plxdc _at retinoic acid receptor responder 2 Rarres _s_at syndecan 2 Sdc _at semaphorin 5A Sema5a _at superoxide dismutase 3, extracellular Sod _at Supplementary Table 2: Gene expression of acutely purified CNS pericytes Affymetrix microarrays were used to analyze mrna expression levels in purified PDGFR-β + pericytes from P7 mice. Selected genes are listed with gene expression data from acutely purified pericytes, and endothelial cells from Pdgfrb -/- mice for a negative control. Molecules were selected due to high ratio of expression in acutely purified pericytes to endothelial cells. Known pericyte markers, extracellular matrix proteins and signaling factors are listed. 6

7 E17 E13 E12-caudal E12-rostral E11-caudal E11-rostral Ng2 BSL Merge Ai i Bi i Ci i Di i Ei i Fi i Supplementary Figure 1 7

8 Gi Ng2 BSL E17 i Ng2 BSL Supplementary Figure 1 Cont d Supplementary Figure 1: Time course of vascular cell generation in the rat cerebral cortex A-F) Tissue sections of rat cerebral cortex at indicated ages were stained for endothelial cells with BSL (green A-F, i) and pericytes with anti-ng2 (red, A-Fi, i). Endothelial cells and pericytes are first observed at the cortical surface in a caudal to rostral gradient starting at E11, and the first endothelial cells enter the neural tissue at E12, also in a caudal to rostral gradient. Although initial endothelial sprouts entering the CNS do not have pericytes, pericytes are quickly recruited and are observed covering the endothelial cells by E13. The endothelial cells continue to vascularize the brain as the brain grows. Scale bar represents 100 µm. G) High magnification image of E17 rat CNS vessel stained for endothelial cells with BSL (green G, i) and pericytes with anti-ng2 (red, Gi, i). Pericytes extend cellular processes that cover the endothelial cells. Scale bars represent 50 µm. 8

9 P5 P1 E21 A B C BSL / Aquaporin 4 Supplementary Figure 2: Astrocyte endfeet associate with endothelial cells during postnatal development High magnification images of tissue sections of rat cerebral cortex at E21 (A), P1 (B) and P5 (C) were stained for endothelial cells with BSL (green A-C) and astrocytes with anti-aquaporin 4 (red, A-C). Astrocytes are not observed during embryogenesis, but extend processes which touch endothelial cells at P1 and start to wrap around the endothelial cells by P5. Arrows indicate astrocyte processes contacting endothelial cells. Scale bar represents 100 µm. 9

10 E17 E13 E12-caudal E12-rostral E11-caudal E11-rostral Glut-1 DAPI / BSL Merge Ai i Bi i Ci i Di i Ei i Fi i Supplementary Figure 3: Time course of Glut1 expression in the rat cerebral cortex A-F) Tissue sections of rat cerebral cortex at indicated ages were stained for endothelial cells with BSL (green A-F, i) and with anti-glut1 (red, A-Fi, i). Prior to endothelial cell invasion into the cortex, Glut1 is expressed throughout the developing neural tissue. After endothelial cells invade the cortex, Glut1 expression is quickly restricted to the endothelial cells. Scale bar represents 100 µm. 10

11 E15 E13 E12-caudal E12-rostral zo-1 BSL Merge Ai i Bi i Ci i Di i Supplementary Figure 4: Time course of ZO-1 expression in the rat cerebral cortex A-D) Tissue sections of rat cerebral cortex at indicated ages were stained for endothelial cells with BSL (green A-F, i) and with anti-zo-1 (red, A-Fi, i). Prior to endothelial cell invasion into the cortex, ZO-1 is expressed specifically in epithelial cells in the ependymal cell layer (yellow arrows), and continues to be expressed in these cells throughout development and maturity (data not shown). As endothelial cells invade the cortex at E12, ZO-1 expression is patchy within endothelial cells (B -white arrows), and then is observed to form complete junctions by E15 (D -white arrows). Scale bar represents 50 µm. 11

12 A Cereberal Cortex B Sub-cortical Forebrain e15 Supplementary Figure 5: Spatial distribution of tracer leakage in embryos E15 rats were given a transcardiac perfusion of a biotin tracer, and tissue sections were stained with streptavidin (green) and the nuclear marker DAPI (blue). Vessels restrict the movement of the biotin tracers into the CNS parenchyma (B), however leakage is observed near the pial surface (A). Scale bar represents 100 µm. 12

13 BSL DAPI Ai E10 E11 i E13 BSL PDGFRβ Bi E10 E11 i E13 BSL AQ4 Ci E18 P2 i P7 BSL zo-1 Di E11 E13 i E18 Ei E10 E11 i i E13 Glut-1 Supplementary Figure 6: Time course of cell generation and BBB properties during mouse development A-E) Tissue sections of mouse cerebral cortex at indicated ages were stained for endothelial cells with BSL (green A-D) and with DAPI (blue, A), anti-pdgfr-β (red, B), anti-aquaporin 4 (red, C), anti-zo-1 (red, D), or anti-glut1 (red, E). Endothelial cells enter the cortex starting at E11, and continue to vascularize the CNS during development. PDGFR-β + pericytes cover endothelial cells as soon as they enter the cortex, whereas astrocytes are only generated after birth. CNS endothelial cells express ZO-1 and Glut1 as they enter the cortex. Scale bars represent 100 µm (A,C,E), 50 µm (B) and 20 µm (D) 13

14 E11 E12 E18 Ai Bi Ci Icam1 BSL PLVAP Icam1 Di Ei Fi PLVAP BSL Supplementary Figure 7: Time course of Icam1 and Plvap expression during mouse development A-C) Tissue sections of mouse cerebral cortex at indicated ages were stained for endothelial cells with BSL (green A-F) and with anti-icam1 (red, A-C) or anti-plvap (red, D-F). Endothelial cells express both Icam1 and Plvap at early embryonic timepoints, but then down-regulate both during embryonic development. Scale bars represent 100 µm. 14

15 PDGFRβ +/+ PDGFRβ +/- Ai Bi Ci PDGFRβ -/- Zic1 BSL Di Ei Fi Ng2 BSL Desmin BSL Gi Hi Ii Supplementary Figure 8 Desmin Ji PDGFRβ +/+ PDGFRβ +/- Ki Ng2 PDGFRβ +/+ PDGFRβ +/- Li Mi Desmin BSL Ng2 BSL N Pericyte Number (% wild type) O Pericyte Coverage (% wild type) Streptavidin P PDGFRβ +/+ PDGFRβ +/- Q R Permeability (% wild type) Supplementary Figure 8 Cont d 15

16 Supplementary Figure 8: Pericyte coverage of CNS vessels in Pdgfrb +/+ and Pdgfrb +/- mice A-I) E15 (G-I) and E18 (A-F) mouse cerebral cortex from Pdgfrb +/+ (A,D,G), Pdgfrb +/- (B,E,H) and Pdgfrb -/- (C,F,I) mice were stained for endothelial cells with BSL (green A-I,) and pericytes with anti-zic (red, A- C), anti-desmin (red D-F), and anti-ng2 (red, G-I). Pericytes are present in the Pdgfrb +/+ and Pdgfrb +/- mice but not the Pdgfrb -/-. Note that the intense desmin staining in the cortex of Pdgfrb -/- mice is due to the fact that the anti-desmin primary antibody is a mouse monoclonal, and therefore the staining is due to the fluorescently labeled goat anti-mouse secondary antibody binding to endogenous mouse antibody that has leaked into the brain. Scale bars represent 100 µm (A-F) and 200 µm (G-I). J-M) High power magnification of pericyte coverage in mouse cerebral cortex from Pdgfrb +/+ and Pdgfrb +/- mice. E15 (L,M) and neonatal (J,K) mouse cerebral cortex from Pdgfrb +/+ (J,L), and Pdgfrb +/- (K,M) mice were stained for endothelial cells with BSL (green J-M ) and pericytes with anti-desmin (red, J,K), and anti-ng2 (red L,M). Scale bars represent 50 µm. N) Pericyte number in Pdgfrb +/+ and Pdgfrb +/- mice was quantified by analyzing number of Zic1+ nculei per length of BSL + vessels. All error bars represent s.e.m. O) Pericyte coverage of CNS vessels in Pdgfrb +/+ and Pdgfrb +/- mice was quantified by analyzing percent length of BSL + vessels opposed to desmin + pericytes. Comparable pericyte coverage was observed in both Pdgfrb +/+ and Pdgfrb +/- mice. All error bars represent s.e.m. P-R) Neonatal Pdgfrb +/+ (P) and Pdgfrb +/- (Q) mice, were given a transcardiac perfusion of a biotin tracer, and tissue sections were stained with streptavidin (green). Scale bar represents 200 µm. R) Fluorescence was quantified in Image J, and permeability relative to control was graphed. All error bars represent s.e.m. No increase in staining for tracer was observed in CNS parenchyma in the Pdgfrb +/- mice. 16

17 Ai Control PDGFRβ -/- Bi Merge BSL Glut-1 i i Ci Di Glut-1 CD31 i i Supplementary Figure 9: Glut1 Expression and Localization in Pdgfrb -/- mice E18 Pdgfrb -/- mice (B, D), and litter mate controls (A,C) were stained with endothelial marker BSL (green A,B,,i,) or anti-cd31 (C,D i,i) and anti-glut1 (red A-Di,i, E,F). A-B) low magnification images depicting overall expression of Glut1 is not affected in the vessels throughout the Pdgfrb -/- mice cerebral cortex. Scale bars represent 100 µm (A,B). (C-D) High magnification images demonstrating that the polarized abluminal localization of Glut1 is not affected in CNS vessels in Pdgfrb -/- mice. Scale bars represent 20 µm. 17

18 Plvap Plvap / BSL Ai PDGFR -/- Control Bi Ci Di Supplementary Figure 10: Plvap expression in Pdgfrb -/- mice E18 Pdgfrb -/- mice (C,D), and litter mate controls (A,B) were stained with anti-plvap (red in i,) and the endothelial marker BSL (green, ). An increase in Plvap staining intensity and the number of positive vessels was observed in the Pdgfrb -/- mice. Scale bar represents 50 µm. 18

19 A PDGFRβ N/N N/+ N/N N/+ Col III Col I Vitronectin MMP-9 PDGFRb B-actin B Relative Expression (AU) * * * * * Control Mutant Col3 Col1 Vitronectin MMP9 PDGFRb Beta actin Supplementary Figure 11: Extracellular matrix in Pdgfrb -/- mice A) Expression levels of extracellular molecules in Pdgfrb -/- mice. Western blots on brain lysates from E18 Pdgfrb -/- and litter mate heterozygote controls, probing for extracellular matrix molecules Col III, Col I, Vitronectin, MMP-9, as well as PDGFR-β and beta-actin. Collagens showed decreased protein expression whereas vitronectin and MMP-9 showed an increase in protein expression in the Pdgfrb -/- brain. B) Quantification of extracellular matrix molecules in Pdgfrb -/- mice and littermate controls. * P<0.05 by Students t-test. All error bars represent s.e.m. 19

20 Ai Endothelial cells Endothelial cells + Pericytes Bi DAPI Angpt2 Angpt2 Supplementary Figure 12: Pericytes inhibit endothelial Angpt2 expression in vitro Purified murine brain endothelial cells were grown in culture alone (A) or with a feeding layer of purified brain pericytes (B) and stained for DAPI (blue, ) and anti-angpt2 (green). Scale bar represents 100µm. 20

21 Legend Cells Endothelial cells Neural progenitors Pericytes Astrocyte endfeet Molecules Tight Junction Proteins Nutrient transporters Efflux transporters Leukocyte adhesion molecules Transcytotic vesicles Supplementary Figure 13: Model for cellular interactions that regulate blood-brain barrier formation During embryonic angiogenesis neural progenitors induce endothelial cells to express blood-brain barrier specific proteins, including tight junction molecules and specific nutrient transporters. Pericytes then strengthen the endothelial barrier during embryogenesis by regulating tight junction structure, limiting the rate of transcytosis, and inhibiting the expression of leukocyte adhesion molecules. Astrocytes further aid in regulating the function of the barrier during adulthood, injury and disease. 21