Data presented here suggest a role for Foxd3 in maintenance of ES cell self-renewal and survival, and inhibition of differentiation into multiple lineages.

(A): qRT-PCR analysis of endoderm marker Foxa2 mRNA levels in D2-D8 EBs cultured in the presence (red) or absence (blue) of 2μM TM. (B): Sections of untreated and TM-treated D4 and D8 EBs that have undergone whole mount immunocychemistry for Foxa2 (red) and counter-staining with DAPI (blue). Scale bar: 100μm. (C): Percent of Foxa2-positive cells in D4 and D8 EBs. (D-I): qRT-PCR analysis of transcript levels of endoderm markers Sox17, AFP and Alb1, trophectoderm marker Cdx2, mesendoderm marker Gsc and neurectoderm marker Sox1 in D2-D8 EBs. Graphs in (A), (C)-(I) were plotted in logarithmic scale. mRNA levels of each gene are compared between control and TM-treated cells at each stage by Student's t-test. Data shown are Mean±SEM (*** p<0.001; ** p< 0.01; * p<0.05).

(A): Cell number analysis of Foxd3^fl/fl; Cre-ER cell lines and Foxd3^fl/fl control cell lines treated with 0, 2 or 4μM TM. Surviving cells were counted with a hemocytometer after 2, 3 and 4 days of TM treatment. (B): Alkaline phosphatase staining and Foxd3 immunolocalization in colonies generated from the self-renewal analysis of Foxd3^fl/fl; Cre-ER cell lines. Scale bar: 50μm. (C): Colony formation efficiency as shown by the number of AP-positive colonies normalized against total number of cells plated. (D): Number of cells per colony in control and mutant self-renewal cultures.
Data shown are Mean±SEM (*** p<0.001).

(A): Immunocytochemistry shows nuclear staining of Oct4, Sox2 and Nanog (red) in Foxd3^fl/fl; Cre-ER ES cells with or without 2μM TM treatment for 3 days. ES cells were counter-stained with DAPI (blue). Oct4 (red) and Foxd3 (green) double staining (leftmost panel) reveals that most Foxd3-deficient ES cells are Oct4-positive in mutant cultures and very little Foxd3 protein is observed in treated cells. Scale bar: 50μm. (B): qRT-PCR analysis of stem cell marker gene expression in 2μM TM (red line) or no TM (blue line) treated cells for 1-3 days. mRNA levels of each gene are compared between control and TM-treated cells by Student's t-test (***: p<0.001; ** p< 0.01; * p< 0.05).

(A): Diagram of the Foxd3 wild type, conditional (“floxed”) and recombined alleles. Arrows indicate PCR primers that flank the 5′ LoxP site. (B): Genomic DNA PCR of wild type ES cells, Foxd3^fl/fl; Cre-ER and Foxd3^fl/fl ES cells cultured in the presence or absence of 2μM TM for 3 days using flox and Cre primers. HPRT serves as an internal control. (C): Foxd3, Cre and Hprt RT-PCR of cDNA from multiple lines of ES cells treated with or without 2μM TM for 3 days. (D): qRT-PCR showing significant reduction of Foxd3 mRNA in Foxd3^fl/fl; Cre-ER ES cells treated with 2μM TM. Levels of Foxd3 transcript are compared by Student's t-test (***: p<0.001). (E): Immunocytochemistry of Foxd3 (red) and DAPI (blue) showing absence of nuclear Foxd3 in most TM-treated Foxd3^fl/fl; Cre-ER ES cells with rare retention of Foxd3 in the nuclei of some cells (arrowheads). Scale bar: 50μm.

(A): TUNEL labeling (red) and Foxd3 staining (green) of Foxd3^fl/fl; Cre-ER and Foxd3^fl/fl cell lines with or without 2μM TM treatment revealing increased apoptosis of Foxd3-deficient ES cells (arrows). (B): Quantification of TUNEL-positive cells normalized against total number of DAPI-stained cells in Foxd3^fl/fl; Cre-ER cultures after 2 or 3 days of TM treatment. (C): BrdU incorporation as a measure of cells in S phase (red) and Foxd3 (green) staining. Many Foxd3-deficient ES cells are BrdU-positive (arrows). (D): Quantification of percentage of BrdU-positive cells in Foxd3^fl/fl; Cre-ER cell cultures. (E): Phospho-histone H3 (red) staining for cells in G2-M phase. (F): Quantification of percentage of pH3-positive cells in Foxd3^fl/fl; Cre-ER cell cultures. ES cells in (A), (C) and (E) were counter-stained with DAPI (blue). Arrowheads in (A) and (C) point to a few Foxd3-positive cells in mutant cultures. Scale bar: 50μm. Data in (B), (D) and (F) were obtained from three independent experiments with more than 7000 DAPI-positive cells counted for each treatment, and are shown as Mean±SEM (*** p<0.001).

(A): qRT-PCR analysis of transcript levels of primitive endoderm markers Foxa2, AFP and Sox17 in ES cells cultured with or without TM for 3 days. Bars represent transcript levels for each gene in 2μM TM treated cells, and horizontal lines indicate the mRNA levels in untreated ES cells arbitrarily designated as 1. (B): qRT-PCR analysis of mRNA levels of trophectoderm markers Cdx2, Fgfr2 and Csh1/PL1. (C): qRT-PCR analysis of mRNA levels of epiblast marker Fgf5, mesendoderm markers T and Gsc. (D): qRT-PCR analysis of neurectoderm markers Sox1 Nestin, and Pax6. (E): Immunocytochemistry of Foxa2 (red) and either Sox2 or Oct4 (green) in Foxd3^fl/fl; Cre-ER ES cells treated with 2μM TM for 3 days. Arrowheads: Sox2/Foxa2 double-positive cells. Scale bar: 50μm. (F): Percent of Oct4- or Foxa2-positive cells in control and 2μM TM-treated cultures. Data were collected from three independent experiments, and > 2000 cells were counted for each treatment group. (G): TUNEL labeling (green) and immunocytochemistry for either Oct4 or Foxa2 (red) in Foxd3^fl/fl; Cre-ER ES cells treated with 2μM TM for 3 days. Arrowheads: TUNEL/Foxa2 double-positive cells that are also DAPI-positive. Arrows: TUNEL/Foxa2 double-positive cells that are DAPI-negative indicating they have lost their nuclear integrity. Scale bar: 25 μm. (H): Percent of TUNEL-positive cells in Oct4-positive or Foxa2-positive populations in control and mutant cultures. More than 2000 cells were counted for each treatment group. Data shown are Mean±SEM (*** p<0.001; ** p< 0.01; * p<0.05).