When ESCs are cultured in the presence of mitotically inactivated embryonic fibroblast feeders, serum, and LIF, the overwhelming majority of the cells maintain an undifferentiated phenotype. To examine the differentiation status of Foxd3 mutant ESCs cultured under conditions that normally inhibit differentiation, we used qRT-PCR to monitor expression of differentiation markers for several lineages. A two- to fourfold increase in mRNA levels of primitive endoderm markers Foxa2, AFP, and Sox17 in TM treated cultures was observed (Fig. 5A), indicating increased differentiation to primitive endoderm of Foxd3-deficient ESCs. Trophectoderm is an extraembryonic lineage that contributes to the placenta, and normally ESCs do not contribute to trophectoderm in chimeras or in vitro . Despite this lineage restriction, Foxd3 mutant ESCs showed 12-, 3.4-, and 4.4-fold increases in mRNAs for trophectoderm markers Cdx2, Fgfr2, and Csh1/PL1, respectively (Fig. 5B). After implantation, the ICM gives rise to primitive ectoderm/epiblast cells, which then contribute to cells in the three primary germ layers. One of the first differentiation markers expressed in epiblast cells in vivo, Fgf5 , was increased twofold in Foxd3 mutant ESCs (Fig. 5C). Goosecoid (Gsc) and Brachyury (T), molecular markers for mesendoderm , increased 5.4- and 12-fold, respectively (Fig. 5C). Together, these results suggest that Foxd3 normally functions to maintain ESCs in an undifferentiated state by repressing differentiation toward extraembryonic and embryonic lineages. Interestingly, expression levels of neuroectoderm markers Nestin, Pax6, and Sox1 either decreased or remained the same in mutant cells (Fig. 5D), suggesting that Foxd3 selectively represses commitment of epiblast cells into mesoderm and endoderm lineages, but not ectoderm.
Figure Figure 5.. Foxd3 represses embryonic stem cell (ESC) differentiation toward multiple lineages. (A): Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of transcript levels of primitive endoderm markers Foxa2, AFP, and Sox17 in ESCs cultured with or without TM for 3 days. Bars represent transcript levels for each gene in 2 μM TM-treated cells, and horizontal lines indicate the mRNA levels in untreated ESCs, 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 and mesendoderm markers T and Gsc. (D): qRT-PCR analysis of neuroectoderm markers Sox1 Nestin, and Pax6. (E): Immunocytochemistry of Foxa2 (red) and either Sox2 or Oct4 (green) in Foxd3fl/fl;Cre-ER ESCs treated with 2 μM TM for 3 days. Arrowheads: Sox2/Foxa2 double-positive cells. Scale bar = 50 μm. (F): Percentage of Oct4- or Foxa2-positive cells in control and 2 μM TM-treated cultures. Data were collected from three independent experiments, and >2,000 cells were counted for each treatment group. (G): TUNEL labeling (green) and immunocytochemistry for either Oct4 or Foxa2 (red) in Foxd3fl/fl;Cre-ER ESCs treated with 2 μM TM for 3 days. Arrowheads: TUNEL/Foxa2 double-positive cells that were also DAPI-positive. Arrows: TUNEL/Foxa2 double-positive cells that were DAPI-negative, indicating they had lost their nuclear integrity. Scale bar = 25 μm. (H): Percentage of TUNEL-positive cells in Oct4-positive or Foxa2-positive populations in control and mutant cultures. More than 2,000 cells were counted for each treatment group. Data shown are mean ± SEM (***, p < .001; **, p < .01; *, p < .05). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; TM, 4-hydroxytamoxifen; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.
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During lineage diversification ESCs upregulate lineage-specific genes while downregulating self-renewal genes. One possible mechanism for precocious and aberrant differentiation is the disruption of this coordinated regulation, with the induction of lineage-specific gene networks at the same time that self-renewal gene expression is maintained. To test this possibility in Foxd3 mutant ESCs, we performed double immunocytochemistry for the endoderm marker Foxa2 and stem cell markers Oct4 and Sox2 (Fig. 5E). Both TM-treated Foxd3fl/fl;Cre-ER cultures (Fig. 5E) and untreated cultures (data not shown) contained very few cells that were double-positive for Foxa2 and Oct4 or Sox2 (Fig. 5E, arrowheads; 4%–5% cells in both mutant and control cultures), suggesting the coordination between differentiation and self-renewal was not disrupted in Foxd3 mutant ESCs. Surprisingly, the percentages of Oct4-positive and Foxa2-positive cells in control and mutant cultures were similar (Fig. 5F). Because we observed increased apoptosis in Foxd3 mutant cultures, we performed TUNEL analysis and immunolocalization for either Oct4 or Foxa2 to examine which population of cells preferentially undergoes apoptosis. Oct4 cells are mostly in the internal portion of the colonies, whereas Foxa2 cells are at the periphery (Fig. 5E, 5G). In control cultures, TUNEL/Oct4 double-positive cells were rarely observed (Fig. 5H; 1.5% of total Oct4-positive cells); in mutant cultures, the percentage of TUNEL/Oct4 double-positive cells was similar (Fig. 5G, 5H; 2%). In contrast, we measured 6.6% TUNEL/Foxa2 double-positive cells in control cultures versus a significantly higher 28.7% TUNEL/Foxa2 double-positive cells in mutant cultures (Fig. 5G, 5H). Furthermore, in mutant cultures, not only did we observe TUNEL/Foxa2 double-positive cells that were DAPI-positive (Fig. 5G, arrowheads), we also observed many cells at the edge of colonies that were TUNEL/Foxa2 double-positive but DAPI-negative, suggesting that these cells had lost their nuclear integrity (Fig. 5G, arrows). Together, these data indicate that Foxd3 mutant ESCs undergo aberrant differentiation, and these differentiated cells have a higher tendency for apoptosis.