Fig 2: Proliferation was decreased at E10.5 and E11.5 in intestine lacking GATA4. (A) EdU incorporation (E9.5–E11.5, red; E12.5–E16.5, green) was used to measure proliferation within the intestinal epithelium of Gata4loxP/+ and Gata4loxP/-ShhCre cKO embryos. At E9.5–E11.5, CDX2 (green) marked epithelial cells. At E12.5, E14.5, and E16.5, laminin (red), which marks the basement membrane separating epithelium and mesenchyme, distinguished between epithelium and mesenchyme. At all stages, 4',6-diamidino-2-phenylindole (DAPI) (blue) labeled nuclei. (B) The proportion of proliferating epithelial cells was decreased in GATA4 mutant intestine compared with controls at E10.5 and E11.5 (left). The percentage of proliferating epithelial cells was determined at E9.5–E11.5 by dividing EdU+, CDX2+ cells by total CDX2+ cells and at E12.5, E14.5, and E16.5 by dividing EdU+ cells by total DAPI+ cells located on the luminal side of laminin staining (n = 5 sections, =3 controls and mutants per stage). The proportion of proliferating mesenchymal cells was not changed in GATA4 mutants compared with controls (right). The percentage of proliferating mesenchymal cells was determined at E16.5 by dividing EdU+ cells by the total DAPI+ cells located on the basal side of the ring of laminin staining demarcating the epithelial–mesenchymal boundary. Error bars show SEM. P values were determined by 2-sample Student t test: *P = .05, **P = .01. Scale bars: 50 µm.

Fig 3: Model for Brg1/Cdx2-mediated repression of Oct4 expression in trophectoderm.Schematic diagram of Oct4 regulation in blastocysts. In the trophectoderm Brg1 is recruited to the ARE of the Oct4 promoter via Cdx2. Once at the Oct4 promoter Brg1 and Cdx2 facilitate recruitment of additional co-repressors to repress transcription.

Fig 4: Expression and localization of Cdx2 and Oct4 in Brg1 KD blastocysts.(A) ICC analysis of Oct4 and Cdx2 in Brg1 KD and control blastocysts. In control blastocysts (a–d) Oct4 expression (green) is restricted to the ICM and is largely absent in the Cdx2-positive (red) trophectoderm. In contrast, in Brg1 KD blastocysts (e–h) Oct4 is widely expressed in both the ICM and cdx2-positive (yellow) trophectoderm. Arrowheads denote co-expression of Oct4 and Cdx2. Nuclei were counter stained with DAPI (blue). (B) Quantification of the average number of cells in Brg1 KD and control blastocysts expressing Oct4, Cdx2, and Oct4 & Cdx2 (double expression). Asterisks denote statistical significance (p<0.05) between Brg1 KD and control blastocysts. A total of 25 Brg1 KD blastocysts and 15 control blastocysts were analyzed.

Fig 5: Brg1 is required for repression of Oct4 expression at the blastocyst stage.(A) qRT-PCR analysis of Brg1, Cdx2, and Oct4 transcripts in Brg1 KD 8-cell embryos, morulae, and blastocysts. Data were normalized to Ubtf (house keeping gene) and are relative to control embryos at each stage; black line = 1. Asterisk denotes significant difference between Brg1 KD and control blastocysts (p<0.05). (B) ICC analysis of Oct4 and Cdx2 expression in Brg1 KD 8-cell embryos, morulae, and blastocysts. Nuclei were counter stained with DAPI (blue). (C) Brg1 represses Oct4 expression in a dose dependent manner. One-cell embryos were injected with 0 µM (control), 0.1 µM, 1 µM, or 100 µM Brg1 siRNA and cultured to the blastocyst stage. Real-time qPCR was used to analyze the levels of Brg1 and Oct4 transcripts. Data were normalized to Ubtf and are relative to control blastocysts; dashed line = 1.