Epiblast ground state is controlled by canonical Wnt/β-catenin signaling in the postimplantation mouse embryo and epiblast stem cells

Abstract

Epiblast stem cells (EpiSCs) are primed pluripotent stem cells and can be derived from postimplantation mouse embryos. We now show that the absence of canonical Wnt/β-catenin signaling is essential for maintenance of the undifferentiated state in mouse EpiSCs and in the epiblast of mouse embryos. Attenuation of Wnt signaling with the small-molecule inhibitor XAV939 or deletion of the β-catenin gene blocked spontaneous differentiation of EpiSCs toward mesoderm and enhanced the expression of pluripotency factor genes, allowing propagation of EpiSCs as a homogeneous population. EpiSCs were efficiently established and propagated from single epiblast cells in the presence of both XAV939 and the Rho kinase (ROCK) inhibitor Y27632. Cell transplantation revealed that EpiSCs were able to contribute to primordial germ cells and descendants of all three germ layers in a host embryo, suggesting that they maintained pluripotency, even after prolonged culture with XAV939. Such an improvement in the homogeneity of pluripotency achieved with the use of a Wnt inhibitor should prove advantageous for manipulation of primed pluripotent stem cells.

Figure 1. Canonical Wnt/β-catenin signaling down-regulates the pluripotency of EpiSCs and perigastrulation embryos.

(A) In situ hybridization analysis of EpiSCs cultured without Activin and Fgf2 as well as in the absence (control) or presence of 20 µM CHIR99021 for 6 hours. Probes are indicated in each pair of panels. Scale bar, 200 µm. (B) Quantitative reverse transcription and polymerase chain reaction (RT-PCR) analysis of EpiSCs cultured without Activin and Fgf2 as well as in the presence of 20 µM CHIR99021 for the indicated times. The amounts of Nanog, Sox2, Oct4, and Foxd3 mRNAs are shown relative to those in untreated EpiSCs. (C) In situ hybridization analysis of E6.5 mouse embryos cultured in the absence (control) or presence of 40 µM CHIR99021 or 100 µM XAV939 for 6 hours. Lateral views of embryos are shown with anterior to the left. The PS region is marked with a line in the T panels. Asterisks in the Sox2 panels indicate expression in the extraembryonic ectoderm. Scale bar, 100 µm.

Figure 2. Inhibition of canonical Wnt signaling promotes the propagation of EpiSCs in the undifferentiated state.

(A) In situ hybridization analysis of EpiSCs cultured with Activin and Fgf2 as well as in the absence or presence of 10 µM XAV939 for 2 days. Scale bar, 200 µm. (B) Immunofluorescence analysis of Nanog and β-catenin in EpiSCs cultured as in (A). Scale bar, 50 µm. (C) Quantitative RT-PCR analysis of EpiSCs cultured as in (A). The amounts of Nanog, Sox2, Oct4, and Foxd3 transcripts in XAV939-treated cells are shown relative to those in untreated cells.

Figure 3. Efficient establishment of EpiSC lines from single cells.

(A) Establishment of EpiSCs from E6.5 mouse epiblasts cultured with the indicated combinations of Activin, Fgf2, and XAV939 (25 µM) for 3 days. The cells were observed by phase-contrast microscopy, stained for alkaline phosphatase (ALP) activity (blue), or subjected to immunochemical staining for Oct4 (brown). Scale bars, 200 µm. (B) Culture of dissociated epiblast cells on a feeder layer with Activin and Fgf2 as well as in the absence or presence of 25 µM XAV939 or 10 µM Y27632. Epiblast cells from one embryo were seeded in each well of a four-well plate. The cells were stained for alkaline phosphatase activity after culture for 5 days. (C) Enlarged view of each well in (B) showing EpiSC colonies. Scale bar, 200 µm. (D) Number of alkaline phosphatase–positive (black bar) and –negative (gray bar) colonies for cultures similar to those in (B). Data are means ± SEM for three independent cultures per condition.

Figure 4. β-Catenin is dispensable for the propagation of undifferentiated EpiSCs.

(A) Two independent EpiSC lines (#3 and #4) derived from β-catenin ^fl/fl epiblasts harboring the UBC-CreER transgene were incubated in the absence or presence of 100 nM tamoxifen (4OHT) for four days, after which β-catenin and Cre genotype was determined by PCR analysis. The positions of amplification products corresponding to the floxed (fl/fl) and null (Δ/Δ) alleles of β-catenin as well as to the Cre transgene are indicated. (B) Immunoblot analysis of β-catenin, E-cadherin, Oct4, and proliferating cell nuclear antigen (PCNA) in EpiSCs of line #3 in (A). (C) Quantitative RT-PCR analysis of the indicated transcripts in EpiSC lines as in (A). The amounts of Nanog, Sox2, Oct4, and Foxd3 transcripts are shown relative to those in β-catenin ^fl/fl cells of line #3. (D) Phase-contrast images and in situ hybridization analysis of the epiblast markers Fgf5 and Fgf8 in β-catenin ^fl/fl or β-catenin ^Δ/Δ EpiSC colonies of line #3. Scale bars, 200 µm. (E) In situ hybridization analysis of T and Pax6 expression in β-catenin ^fl/fl or β-catenin ^Δ/Δ EpiSC colonies of line #3 cultured with or without 20 µM CHIR99021 for 6 h or 100 nM PD0325901 for 2 days. Scale bar, 200 µm. (F) Immunofluorescence analysis of E-cadherin, Oct4, Sox2, Nanog, and β-catenin in β-catenin ^fl/fl or β-catenin ^Δ/Δ EpiSC colonies of line #3. Scale bar, 200 µm.

Figure 5. EpiSCs cultured with XAV939 contribute to chimeric embryos.

Mouse embryos at E6.5 were injected with EpiSCs that harbor a lacZ transgene (ROSA26-lacZ) and which had been cultured in the presence of 10 µM XAV939 for at least 10 passages and exposed to 10 µM Y27632 for 1 hours before dissociation into single cells. The embryos were cultured until the early somite stage and then subjected to X-gal staining (green). The embryo in (H) was also stained for alkaline phosphatase activity (purple). LacZ-positive descendants of EpiSCs were detected in the allantoic bud [asterisks in (A) and (C)], yolk sac [arrowhead in (A)], amnion [asterisk in (B)], hindgut endoderm [arrowheads in (C)], somitic mesoderm [arrowheads in (D)], surface ectoderm [arrowheads in (E)], neural plate [arrowheads in (F)], myocardium [arrowhead in (G)], and migrating PGCs [arrowheads in (H)]. Embryos are oriented with anterior to the left (A, B, and E), posterior to the top (C, D, and H), or anterior to the top (F and G). A distal view of somites (D), enlarged view of the headfold (E), dorsal view of the neural plate (F), and ventral view of the heart primordium (G) are shown. Scale bars, 100 µm.