Wnt signaling is down-regulated, and β-catenin protein is degraded in Brg1 mutants. (A and B) Embryos were crossed onto the BAT-gal Wnt reporter line for detection of Wnt signaling activity. E10.5 X-gal–stained yolk sacs were cryosectioned and counterstained with eosin. Blue arrowhead, reporter-positive endothelium; white arrowhead, reporter-negative endothelium. (Scale bars: 50 μm.) (C and D) Endothelial cells from littermate control and mutant yolk sacs were isolated, RNA was purified, and cDNA was synthesized. (C) Samples were processed for qPCR using a custom-designed array containing 28 Wnt target genes (Fig. S5A). Data from four independent experiments were compiled using SABiosciences Excel-based data analysis. (D) qPCR was carried out for five Wnt target genes (Axin2, T, CyclinD1, Wisp1, and Myc). Errors represent ±SEM from six independent experiments, and significant differences between littermate controls and mutants were calculated using a two-tailed Student's t test (**P < 0.005). (E and F) C166 cells were transfected with nonspecific (NS) or Brg1-specific siRNA for 48 h. (E) Protein samples were subjected to Western blot analysis with antibodies that recognize BRG1, β-catenin, or GAPDH. (F) RNA was isolated, cDNA was synthesized, and qPCR for Brg1 or β-catenin was carried out. Errors represent ±SEM from four independent experiments, and significant differences between NS or Brg1-specific siRNA-transfected cells were calculated using a two-tailed Student's t test (**P < 0.005).

BRG1 mediates expression of multiple Fzd receptors. (A) C166 cells were transfected with nonspecific (NS) or Brg1-specific siRNA for 48 h. RNA was isolated, cDNA was synthesized, and qPCR for Fzd1-8 was performed. Errors represent SEM, and significant differences between NS or Brg1-specific siRNA-transfected cells were calculated using a two-tailed Student's t test (**P < 0.005). (B) Endogenous chromatin from C166 cells was immunoprecipitated using isotype-matched antibodies against BRG1, acetylated histone H3 (AcH3) as a positive control or a polyhistidine epitope tag (His) as a negative control. DNA was isolated and amplified by qPCR to determine whether BRG1 and AcH3 were associated with the promoter regions of Fzd1, -4, -5, -6, or -8. A region upstream of the Fzd5 promoter was used as a negative control, and the ADAMTS1 promoter acted as a positive control gene region (17). Data from four independent experiments were combined and are presented as the percentage (%) input ±SEM; significant differences were calculated using a two-tailed Student's t test (*P < 0.05).

Brg1 deletion results in vascular abnormalities in extraembryonic yolk sacs. (A and B) E10.5 littermate control Brg1^fl/fl (A) and mutant Brg1^fl/fl;Tie2-Cre^+/0 embryos (B). (Scale bars: 1 mm.) (C and D) Anti-PECAM1 staining on histological sections of E10.5 yolk sacs reveal thinner vessels in mutants (D) compared with controls (C). (Scale bars: 100 μm.) (E and F) Hematoxylin and eosin (H&E)-stained paraffin sections of E9.5 littermate control Brg1^fl/fl (E) and mutant Brg1^fl/fl;Tie2-Cre^+/0 (F) yolk sac vessels. Vessels are flattened in mutants (F) compared with controls (E). Arrows indicate embryonic blood cells. (Scale bars: 50 μm.) (G–J) Transmission electron micrographs (TEM) of control Brg1^fl/fl (G and I) versus mutant Brg1^fl/fl;Tie2-Cre^+/0 (H and J) yolk sac vessels reveal discontinuities in the mutant endothelial lining (arrow in J). MC, mural cell; VE, visceral endoderm; BC, blood cell; EC, endothelial cell. (Scale bars: 5 μm.)

Inhibition of β-catenin degradation with LiCl treatment rescues vascular anomalies and Wnt signaling in Brg1 mutant yolk sacs. (A–H) Pregnant mice were injected with 400 mg/kg NaCl (A and B, E and F) or LiCl (C and D, G and H) on E8.5 and E9.5. Embryos were dissected at E10.5, and littermate control Brg1^fl/fl (A, C, E, G) and mutant Brg1^fl/fl;Tie2-Cre^+/0 (B, D, F, H) yolk sacs were whole-mount immunostained with antibodies against PECAM1. (A–D) (Scale bars: 50 μm.) (E–H) (Scale bars: 100 μm.) (I) Mean vitelline vessel (V. V.) diameter measurements from 10 control and 3 mutant yolk sacs treated with NaCl or from 12 control and 4 mutant yolk sacs treated with LiCl. Errors were calculated as ±SEM, and a two-tailed Student's t test was used to detect statistical differences in controls versus mutants or between NaCl- and LiCl-treated mutants (*P < 0.05; **P < 0.005). (J and K) Embryos carrying the BAT-gal Wnt reporter transgene were treated in utero with LiCl for 2 d before dissection at E10.5. X-gal–stained yolk sacs were cryosectioned and counterstained with eosin. Blue arrowheads, reporter-positive endothelium. (Scale bars: 50 μm.) (L) Endothelial cells from LiCl-treated littermate control and mutant yolk sacs were isolated, RNA was purified, cDNA was synthesized, and qPCR was carried out for Wnt target genes (Axin2, T, CyclinD1, Wisp1, and Myc). Errors represent ±SEM from six independent experiments, and significant differences between littermate controls and mutants were calculated using a two-tailed Student's t test (**P < 0.005). (M) Chromatin immunoprecipitation assays were carried out using isotype-matched antibodies against BRG1, acetylated histone H3 (AcH3) as a positive control, or a polyhistidine epitope tag (His) as a negative control. DNA was isolated and amplified by qPCR to determine whether BRG1 and AcH3 were associated with the promoter region of Wisp1. Data from four independent experiments were combined and are presented as the percentage (%) of input ±SEM; significant differences were calculated using a two-tailed Student's t test (*P < 0.05).

Model for how BRG1 impacts Wnt signaling during yolk sac vascular development. BRG1 epigenetically modifies Wnt signaling at two levels—through transcriptional regulation of multiple Fzd receptor genes and through subsequent association with β-catenin for regulation of certain downstream Wnt target genes. The dashed line indicates that BRG1 is not required for coregulation of all Wnt target genes in endothelial cells.