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. 2013 May 20;2(7):647-59.
doi: 10.1242/bio.20135009. Print 2013 Jul 15.

Foxc1 Is Required by Pericytes During Fetal Brain Angiogenesis

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Free PMC article

Foxc1 Is Required by Pericytes During Fetal Brain Angiogenesis

Julie A Siegenthaler et al. Biol Open. .
Free PMC article

Abstract

Brain pericytes play a critical role in blood vessel stability and blood-brain barrier maturation. Despite this, how brain pericytes function in these different capacities is only beginning to be understood. Here we show that the forkhead transcription factor Foxc1 is expressed by brain pericytes during development and is critical for pericyte regulation of vascular development in the fetal brain. Conditional deletion of Foxc1 from pericytes and vascular smooth muscle cells leads to late-gestation cerebral micro-hemorrhages as well as pericyte and endothelial cell hyperplasia due to increased proliferation of both cell types. Conditional Foxc1 mutants do not have widespread defects in BBB maturation, though focal breakdown of BBB integrity is observed in large, dysplastic vessels. qPCR profiling of brain microvessels isolated from conditional mutants showed alterations in pericyte-expressed proteoglycans while other genes previously implicated in pericyte-endothelial cell interactions were unchanged. Collectively these data point towards an important role for Foxc1 in certain brain pericyte functions (e.g. vessel morphogenesis) but not others (e.g. barriergenesis).

Keywords: Angiogenesis; Blood brain barrier; Neurovascular development; Pericyte; foxc1.

Conflict of interest statement

Competing Interests: The authors have no competing interests to declare.

Figures

Fig. 1.
Fig. 1.. Foxc1 is expressed by brain pericytes.
(A) Foxc1 (green) and PECAM (red) co-immunolabeling of a cerebral vessel at E14.5. Arrowhead indicates Foxc1+ cells immediately adjacent to the PECAM+ vessel. Arrows indicate Foxc1+ cell in the inner lumen of the PECAM+ vessel. (B) Arrowheads indicate Foxc1 (green) and PDGFrβ (red; B) or SMA-α (red; C) co-labeled pericytes around the outside of cerebral blood vessels. Arrows indicate Foxc1+ cell in the inner lumen of the vessel cross-section. (D,E) Foxc1 (D) and Foxc2 (E) in the meninges and adjacent cortex at E14.5. Laminin (red) labels cerebral vessels. Scale bars: 25 µm (A–C); 50 µm (D,E).
Fig. 2.
Fig. 2.. Altered brain pericyte proliferation in Foxc1 mutants.
(A–C) PDGFrβ (green) and IB4 (red) immunofluorescence highlight pericyte cell bodies in the cortices of E14.5 WT, Foxc1h/h and Foxc1l/l. (D–F) Triple immunofluorescence for Zic1 (pericyte nuclei; green), BrdU (red) and IB4 (blue) highlight proliferating (Zic1+/BrdU+; arrows) pericytes in the E14.5 cerebral vasculature of WT, Foxc1h/h and Foxc1l/l embryos. (G) Graph depicting quantification of Zic1+ pericyte nuclei/vessel length in all three genotypes at E14.5. (H) Graph depicting quantification of pericyte proliferation (labeling index) at E14.5. Scale bars: 100 µm. Means represent analysis of three independent litters for each mutant genotype (n = 3). Asterisks indicate a statistically significance difference from WT (P<0.05). Error bars depict ±SEM.
Fig. 3.
Fig. 3.. Vascular defects and brain micro-hemorrhage in pericyte conditional Foxc1 mutants.
(A) RT-PCR of Foxc1 transcript from three RNA sources: (1) E18.5 PDGFrb-cre; Foxc1fl-+ meninges (2) E18.5 microvessels from PDGFrb-cre; Foxc1fl-+ brain and (3) E18.5 microvessels from PDGFrb-cre; Foxc1fl-fl brain. Housekeeping gene GAPDH serves as internal control. (B) Whole embryo images of E18.5 PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrb-cre; Foxc1fl-+ littermate. (C–E) Dorsal view of E18.5 PDGFrβ-cre; Foxc1fl-+ (C) and PDGFrβ-cre; Foxc1fl-fl mutant (D,E) brains. Arrows indicate hemorrhage within the cerebral cortex. (F,G) Glut-1 (green), Ib4 (red) and DAPI (blue) stained cortical sections of E18.5 PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrb-cre; Foxc1fl-+ animals. (H) Graphs depicting quantification of average cerebral vessel diameter (top) and cerebral vessel density (bottom) in E18.5 PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrβ-cre; Foxc1fl-+ animals. Means represent analysis from three separate litters (n = 3). (I,J) ICAM-1 and IB4 immunofluorescence of E18.5 PDGFrβ-cre; Foxc1fl-+ and PDGFrβ-cre; Foxc1fl-fl cortices. Arrowheads indicate superficial ICAM-1+ cerebral vessels descending from the perineural vascular plexus (PNVP). Arrows indicate deeper, ICAM-1+ vessels in PDGFrβ-cre; Foxc1fl-fl mutant cortices. (K) Quantification of percentage of ICAM+ cerebral vessels in control and mutant samples analyzed from three independent litters (n = 3). (L,M) Sagittal view of midbrain and cerebellum of E18.5 PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrb-cre; Foxc1fl-+ brains. Arrows indicate hemorrhage sites in PDGFrβ-cre; Foxc1fl-fl sample. (N,O) Glut-1 (green), Ib4 (red), DAPI (blue) immunofluorescence at the level of the midbrain (N) and cerebellum (O) in E18.5 PDGFrβ-cre; Foxc1fl-fl mutant brains. In G,N,O, arrowheads indicate amoeboid morphology of Ib4+ activated microglia; arrows indicate dilated/dysplastic vessels; asterisks indicate red blood cells in the neural parenchyma. Scale bars: 100 µm. Asterisks in graph indicate a statistically significant difference from PDGFrβ-cre; Foxc1fl-+ samples (P<0.05). Error bars depict ±SEM.
Fig. 4.
Fig. 4.. Pericyte conditional Foxc1 mutants have increased pericyte proliferation and density in the cerebral vasculature.
(A,C) Zic1 (green) and IB4 (red) co-immunofluorescence in the cortices of PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrβ-cre; Foxc1fl-+ brains at E14.5. (B,D) PDGFrβ (green) and IB4 (red) co-immunofluorescence in the cortices of PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrβ-cre; Foxc1fl-+ brains at E14.5. Arrowheads indicate PDGFrβ+ pericytes. (E,F) Zic1 (green), BrdU (red), and IB4 (blue) triple immunofluorescence of E14.5 PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrβ-cre; Foxc1fl-+ cerebral cortex. Arrows indicate Zic1+/BrdU+ pericytes. (G) Graph depicts quantification of Zic1+ pericyte proliferation (labeling index) in the cerebral vasculature at E14.5. (H,J) Zic1 (green) and IB4 (red) co-immunofluorescence in the cortices of PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrβ-cre; Foxc1fl-+ brains at E18.5. (I,K) PDGFrβ (green) and IB4 (red) co-immunofluorescence in the cortices of PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrβ-cre; Foxc1fl-+ brains at E18.5. Arrowheads indicate PDGFrβ+ pericytes. (L) Graph depicts pericyte density (Zic1+ pericyte nuclei/vessel length) in the cerebral vasculature at E14.5 and E18.5 in PDGFrβ-cre; Foxc1fl-fl mutant and PDGFrβ-cre; Foxc1fl-+ animals. Asterisks indicate a statistically significance difference from PDGFrβ-cre; Foxc1fl-+ samples (P<0.05). Analysis of pericyte proliferation and density was performed on control and mutant brains from three separate litters at each time point (n = 3). Error bars depict ±SEM. Scale bars in A,C,E,F,H,J: 100 µm. Scale bars in B,D,I,K: 50 µm.
Fig. 5.
Fig. 5.. Ultrastructure analysis of pericyte–EC interactions reveals EC hyperplasia and increased EC cell proliferation in conditional Foxc1 mutants.
(A–D) High magnification image of contact points between EC and pericyte cell membranes highlight pericyte–EC interactions – “peg and socket” (A,B) and adhesion plaques (arrows in C,D) in cerebral vessels of Foxc1fl-fl and PDGFrβ-cre; Foxc1fl-fl mutant animals. (E) Graph depicting analysis of pericyte coverage of abluminal surface of cerebral blood vessels in E18.5 control and mutant samples. (F,G) EM images of cerebral vessel cross-sections highlighting increased EC nuclei and tight-juntions (red arrows) in PDGFrβ-cre; Foxc1fl-fl mutant cortices. (H) Graph depicting quantification of tight junction density (left) and EC nuclei density (right) from EM images of Foxc1fl-fl and PDGFrβ-cre; Foxc1fl-fl mutant animals. (I,J) Triple immunofluoresence for Lef-1 (green: ECs), BrdU (red) and IB4 (blue) in E16.5 PDGFrβ-cre; Foxc1fl-+ and PDGFrβ-cre; Foxc1fl-fl cortices. Arrows indicates Lef-1+/BrdU+ ECs. (K) Graph depicts quantification of Lef-1+ EC proliferation (labeling index) in the cerebral vasculature at E16.5. PC: pericyte. EC: endothelial cell. TJ:tight junctions. Scale bars: 0.5 µm (A–D); 2 µm (F,G); 100 µm (I,J). Asterisks indicate a statistically significance difference between control and mutant samples (P<0.05). EM analysis was performed on images from three PDGFrβ-cre; Foxc1fl-fl samples (n = 3) and two control Foxc1fl-fl samples (n = 2). EC proliferation analysis was performed on control and mutant brains from two independent litters (n = 2). Error bars depict ±SEM.
Fig. 6.
Fig. 6.. BBB integrity in pericyte conditional Foxc1 mutants.
(A,B) Foxc1fl-fl and PDGFrβ-cre; Foxc1fl-fl mutants injected with fluorophore-linked cadaverine tracer show strong labeling of bone and skin tissues where the tracer leaked out of fenestrated (non-barrier) vessels, but no significant leakage in brain tissue except for adjacent to a hemorrhage site in the PDGFrβ-cre; Foxc1fl-fl mutant brain (arrow). Magnified inset in (B) highlights tracer leak out of a vessel (arrow) and an adjacent activated, IB4+ microglia with amoeboid morphology (arrowhead). (C–F) Representative images of biotin-conjugated 3kDa (C,D) and 70 kDa dextran tracers (E,F) from E18.5 Foxc1fl-fl and PDGFrβ-cre; Foxc1fl-fl mutants cortices. Note tracer signal trapped in choroid plexus (CP). In F, magnified inset is of cerebral vessel with leakage of 70 kDa dextran tracer. (G) Graph depicts quantification of BBB permeability to 3 kDa and 70 kDa dextran tracers as measured by fluorescent intensity of cerebral tissue relative to PBS control. (H,H′) Representative large diameter cerebral vessel (Ib4+, red) in PDGFrβ-cre; Foxc1fl-fl mutant brain displaying vascular leak of 70 kDa tracer (green). (I) Graph depicting mean cerebral vessel diameter of vessels without and with 70 kDa tracer extravasation in control and mutant samples. Scale bars: 100 µm. Asterisks indicate a statistically significance difference from control samples (P<0.05). Analysis of BBB permeability was performed on three control and three mutant brains (n = 3). Error bars depict ±SEM.
Fig. 7.
Fig. 7.. qPCR profiling of pericyte conditional Foxc1 mutant brain microvessels.
(A) Schematic of microvessel isolation from E18.5 brain and both brightfield (top) and fluorescence (bottom) image of microvessels following magnetic bead isolation. Arrow indicates recombined, GFP+ pericyte associated with IB4+ microvessel isolated from PDGFrb-Cre; Rosa26-YFP brain (B,C). Graphs depicting expression analysis of EC-expressed genes. (D) Immunoblots of Par1 and MMP9 on whole cortex lysate from E18.5 PDGFrβ-cre; Foxc1fl-+ or Foxc1fl-fl (control or “C”) and PDGFrβ-cre; Foxc1fl-fl (mutant or “M”). Graphs depict relative intensity of the Par1 or MMP9 band to control samples. (E–I) Graphs depicting expression analysis of pericyte-enriched genes (E), cell cycle genes and TGFβ signaling components (F), pericyte-enriched ECM genes (G), and pericyte-enriched proteoglycans (H). (I) Immunoblots of glypican-3 and asporin on whole cortex lysate from E18.5 PDGFrβ-cre; Foxc1fl-+ or Foxc1fl-fl (control) and PDGFrβ-cre; Foxc1fl-fl (mutant). Graphs depict relative intensity of the glypican-3 or asporin band to control samples. Asterisks indicate a statistically significance difference from control samples (P<0.05). qPCR analysis was performed on RNA isolated from six mutant (n = 6) and five control (n = 5) E18.5 brains. Immunoblot analysis was performed on lysates from three control and three mutant cortices (n = 3). Error bars depict ±SEM.

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