Arrest of WNT/β-catenin signaling enables the transition from pluripotent to differentiated germ cells in mouse ovaries

Abstract

Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell-intrinsic β-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in β-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of β-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3, a negative regulator of WNT/β-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that β-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.

Keywords: POU5F1/OCT4; WNT/β-catenin; differentiation; germ cells; ovary.

Fig. 1.

Ctnnb1 deletion in the somatic progenitor cells indirectly impairs germ cell licensing. (A, Upper) Immunodetection of POU5F1 (red) and DDX4 (germ cells, green) in 13.5 dpc control (Ctnnb1^flox/flox) and Wt1-CreERT2; Ctnnb1^flox/flox ovaries. (Lower) Immunodetection of DAZL (red) and SSEA1 (white) in 13.5 dpc control (Ctnnb1^flox/flox) and Wt1-CreERT2; Ctnnb1^flox/flox ovaries. DAPI (blue): nuclei. (Scale bars: 50 or 20 μm.) (B) In situ hybridization using Bmp2, Stra8, and Nanos2 riboprobes at 13.5 dpc in control (Ctnnb1^flox/flox, Left) and Wt1-CreERT2; Ctnnb1^flox/flox (mutant, Right) ovaries (dotted circles). (Scale bars: 50 μm.) (Inset) Bmp2 expression in the developing bone of !the same Wt1-CreERT2; Ctnnb1^flox/flox mutant animal as positive control. (Inset) Nanos2 expression in sex cords from 13.5 dpc testis as positive control. Histograms: qRT-PCR analysis of Bmp2, Stra8, and Nanos2 (expression in 13.5 dpc control (Ctnnb1^flox/flox, gray) and Wt1-CreERT2; Ctnnb1^flox/flox (pale gray) ovaries. Student's t test, unpaired. Bars represent mean + SEM, n = 6 individual gonads. ***P < 0.001.

Fig. 2.

Ctnnb1 deletion in the primordial germ cells does not affect the size of the animals nor the somatic cell differentiation but impairs germ cell differentiation. (A) Schematic representation of the strategy used to delete Lox-P–flanked Ctnnb1 (encoding β-catenin) in the primordial germ cells in an inducible manner using the Sox2-CreERT2 line specifically expressed in germ cells. (B) Protocol of induction of Sox2-CreERT2 (9.5 and 10.5 dpc onward). TAM: Tamoxifen. (C) Macroscopic view of a 13.5 dpc litter containing control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox (black asterisks) embryos displaying any major physical abnormalities and normal size. Histograms: quantification of the percentage of both MKI67-positive somatic cells (i.e., proliferating somatic cells) in 12.5 dpc control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries. Student’s t test, unpaired. Bars represent mean + SEM, ns: not significant. (D) Immunodetection of β-catenin (red) and TRA98 (germ cells, green) (Upper) and CDH1 (red) and LEF1 (green) (Lower) in 13.5 dpc control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries. DAPI (blue): nuclei. (Scale bars: 50 or 20 μm.) Immunodetection of CDH1 (germ cells, red) and FOXL2 (pregranulosa cells, green) (Upper) and SOX9 (male somatic cells, red), AMH (male somatic cells, white), and TRA98 (germ cells, green) (Lower) in 13.5 dpc control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries. (Inset) The 13.5 dpc XY embryonic gonad used as a positive control for SOX9 and AMH expression. DAPI (blue): nuclei. (Scale bars: 50 or 20 μm.) (E, Left) qRT-PCR analysis of Axin2 expression in purified germ cells from 12.5 dpc control (Ctnnb1^flox/flox) (dark gray) and Sox2-CreERT2; Ctnnb1^flox/flox (pale gray) ovaries. Student’s t test, unpaired. Bars represent mean + SEM, n = ∼30,000 germ cells FACS-purified from 20 individual gonads. ***P < 0.001. (Right) qRT-PCR analysis of Pou5f1, Sox2, Nanog, Ddx4, and Piwil2 expression in purified germ cells from 12.5 dpc control (Ctnnb1^flox/flox) (dark gray) and Sox2-CreERT2; Ctnnb1^flox/flox (pale gray) ovaries. Student’s t test, unpaired. Bars represent mean + SEM, n = ∼30,000 germ cells FACS-purified from 20 individual gonads. ***P < 0.001.

Fig. 3.

Germinal ablation of Ctnnb1 impairs gonocyte proliferation and differentiation. (A, Left) Immunodetection of MKI67 (proliferating cells, red) and TRA98 (germ cells, green) in 12.5 and 13.5 dpc control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries. DAPI (blue): nuclei. (Scale bars: 50 or 20 μm.) Histograms: quantification of the percentage of total germ cells (TRA98-positive cells) versus total gonadal cells in 12.5 dpc control (Ctnnb1^flox/flox, dark gray) and Sox2-CreERT2; Ctnnb1^flox/flox (dotted gray) ovaries. Graphs (Right): quantification of both MKI67- and TRA98-positive cells (i.e., proliferating germ cells) in 11.5, 12.5, and 13.5 dpc control (Ctnnb1^flox/flox, dark gray) and Sox2-CreERT2; Ctnnb1^flox/flox (dotted pale gray) ovaries. Student’s t test, unpaired. Bars represent mean + SEM; **P < 0.01 and ***P < 0.001. (B, Upper) Immunodetection of POU5F1 (red) and DDX4 (germ cells, green) in 12.5 and 13.5 dpc control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries. (Lower) Immunodetection of DAZL (red) and SSEA1 (white) in 13.5 dpc control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries. DAPI (blue): nuclei. (Scale bars: 50 or 20 μm.) (Right) Histograms: quantification of DDX4-positive cells (dark gray) and POU5F1-DDX4 double positive cells (dotted gray) (%) in control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries from 12.5 (Upper) and 13.5 dpc (Middle) embryos after immunodetection. Quantification of DAZL-positive cells (dark gray) and SSEA1-DAZL double positive cells (dotted gray) (%) (Lower) in control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries from 13.5 dpc embryos after immunodetection. Student’s t test, unpaired. Bars represent mean + SEM. ns: not significant; ***P < 0.001.

Fig. 4.

Germinal ablation of Ctnnb1 triggers POU5F1 precocious exit from germ cell nucleus. (A) Gene Ontology terms associated with chromatin regions that are differentially opened/closed between control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox isolated germ cells from 12.5 dpc ovaries based on PANTHER classification software (log fold change ≥ 0.5) and names of the corresponding genes. Adjusted P values are indicated. n = 50,000 germ cells in duplicate for each genotype. (B) Motif enrichment analysis of differentially open chromatin regions (with adjusted P value and percentage of target sequences with motif). POU5F1 fixation motif was found at first rank among genomic regions being down-regulated between control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox isolated germ cells. (C) Immunodetection of POU5F1 (red) in 12.5 and 13.5 dpc control (Ctnnb1^flox/flox) and Sox2-CreERT2; Ctnnb1^flox/flox ovaries. DAPI (blue): nuclei. (Scale bars: 50 or 20 μm.) (D and E) Histograms: quantification of the percentage of nuclear (gray) and cytoplasmic (dotted gray) POU5F1-positive cells in 12.5 and 13.5 dpc control (Ctnnb1^flox/flox, gray) and Sox2-CreERT2; Ctnnb1^flox/flox (dotted gray) ovaries. Student’s t test, unpaired. Bars represent mean + SEM. *P < 0.05 and ***P < 0.001. (F) qRT-PCR analysis of Pou5f1 and Dppa3 (Stella) expression in 13.5 dpc control (Ctnnb1^flox/flox, gray) and Sox2-CreERT2; Ctnnb1^flox/flox (dotted gray) ovaries. Student’s t test, unpaired. Bars represent mean + SEM; n = 10 individual gonads. **P < 0.01; ***P < 0.001.

Fig. 5.

POU5F1 and β-catenin physically interact during gonocyte differentiation. (A) Immunodetection of POU5F1 (red) and β-catenin (green) in 12.5 and 14.5 dpc wild-type ovaries. DAPI (blue): nuclei. (B) High magnification of immunodetection of POU5F1 (red) and β-catenin (green) in 12.5 and 14.5 dpc wild-type ovaries. DAPI (blue): nuclei. (C) Immunodetection by Western blot of β-catenin, POU5F1, CDH1, and GAPDH directly in protein lysate (“input”) or in pellets after immunoprecipitation with control IgG (“control IP”) or with POU5F1-raised specific antibody (“POU5F1 IP”) or in immunoprecipitation supernatant (“supernatant”) at 12.5 (Left) and 14.5 (Right) dpc ovaries. (D) Immunodetection by Western blot of β-catenin, POU5F1, RNA Polymerase II and Histone H3 (as controls for nuclear fraction), CDH1, and GAPDH (as controls for cytoplasmic/membranous fraction) either on whole 14.5 dpc ovaries (Left) or after subcellular fractionation (Middle and Right) in total cell lysate, nuclear and cytoplasmic fractions, before (Middle) and after immunoprecipitation with control IgG (“control IP”) or with specific anti-POU5F1 antibody (“POU5F1 IP”) (Right). Histograms: quantification of the expression of β-catenin (green) and POU5F1 (red) after Western blot in nuclear and cytoplasmic fractions (nuclear or cytoplasmic expression normalized with the total expression for each protein).

Fig. 6.

Genetic ablation of Ctnnb1 in gonocytes triggers precocious meiosis initiation. (A) In situ hybridization using Bmp2 and Stra8 riboprobes at 12.5 and 13.5 dpc in control (Ctnnb1^flox/flox, left) and Sox2-CreERT2; Ctnnb1^flox/flox (mutant, Right) ovaries (dotted circles). (Scale bars: 50 μm.) (B) qRT-PCR analysis of levels of Bmp2 and Stra8 expression in 12.5 and 13.5 dpc control (Ctnnb1^flox/flox, dark gray) and Sox2-CreERT2; Ctnnb1^flox/flox (dotted gray) ovaries. Student’s t test, unpaired. Bars represent mean + SEM; n = 6 pairs of ovaries for each genotype and each age. ***P < 0.001; ns not significant.

Fig. 7.

β-catenin stabilization through GSK3β ablation leads to the maintenance of pluripotency in germ cells. (A) Immunodetection of GSK3β (red) and TRA98 (germ cells, green) and CDH1 (red) and LEF1 (green) in 13.5 dpc control and Gsk3β^−/− ovaries. DAPI (blue): nuclei. (Scale bars: 50 or 20 μm.) (B) Immunodetection of DDX4 (germ cells, red) and BrdU (proliferating cells, green) in 13.5 dpc control and Gsk3β^−/− ovaries. DAPI (blue): nuclei. (Scale bars: 50 or 20 μm.) (C) qRT-PCR analysis of Gsk3b expression in 12.5 and 14.5 dpc control FAC-sorted germ cells. Student’s t test, unpaired. Bars represent mean + SEM; n = 10 individual ovaries. **P < 0.01. (D) Histograms: quantification of DDX4-positive cells (germ cells, dark gray) and BrdU-DDX4 double positive cells (proliferating germ cells, dotted gray) (%) in control and Gsk3β^−/− ovaries from 13.5 dpc embryos. Student’s t test, unpaired. Bars represent mean + SEM. *P < 0.05; **P < 0.01. (E) qRT-PCR analysis of Axin2, Pou5f1, Sox2, and Nanog expression in 13.5 dpc control (dark gray) and Gsk3β^−/− (dotted gray) ovaries. Student’s t test, unpaired. Bars represent mean + SEM; n = 10 individual gonads. *P < 0.05; ***P < 0.001. (F) Immunodetection of POU5F1 (red) and DDX4 (green) and DAZL (red) and SSEA1 (FUT4, white) in 13.5 dpc control and Gsk3β^−/− ovaries. (Scale bars: 50 or 20 μm.) Histograms: quantification of DDX4-positive cells (pale gray) and POU5F1-DDX4 double positive cells (dotted gray) (%) and DAZL-positive cells (pale gray) and SSEA1-DAZL double positive cells (dotted gray) (%) in control and Gsk3β^−/− ovaries from 13.5 dpc embryos after immunodetection. Student’s t test, unpaired. Bars represent mean + SEM. ***P < 0.001.

Fig. 8.

ZNRF3, an inhibitor of WNT/β-catenin activity, positively regulates gonocyte differentiation. (A) RNAscope in situ hybridization using Znrf3 riboprobe (red) and immunodetection of DAZL (green) in 12.5 and 14.5 dpc wild-type ovaries. (B) Histograms: quantification of the percentage of Znrf3-DAZL double positive cells versus DAZL-positive cells in 12.5 (gray) and 14.5 (dotted gray) dpc ovaries. Student’s t test, unpaired. Bars represent mean + SEM. ***P < 0.001. (C) Immunodetection of SOX9 (red) and FOXL2 (green) in 13.5 dpc XY, XX control, and Znrf3^−/− gonads. DAPI (blue): nuclei. (Scale bars: 50 or 25 μm.) (D) Immunodetection of POU5F1 (red) and SCP3 (green) in 13.5 dpc XY, XX control, and Znrf3^−/− gonads. DAPI (blue): nuclei. (Scale bars: 50 or 25 μm.)