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First published online 16 October 2008
doi: 10.1242/dev.024653

Development 135, 3731-3743 (2008)
Published by The Company of Biologists 2008
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Ovarian development in mice requires the GATA4-FOG2 transcription complex

Nikolay L. Manuylov, Fatima O. Smagulova*, Lyndsay Leach and Sergei G. Tevosian{dagger}

Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA.


Figure 1
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  		Fig. 1. Normal ovarian differentiation requires GATA4-FOG2 Interaction. (A-K) Whole-mount in situ hybridization (ISH) was performed with the indicated RNA probes on XX gonads from wild-type, Gata4ki/ki and Fog2-/- E12.5-13.5 mouse embryos. In all images, samples are oriented with anterior towards the right, and a gonad (g) on top of a mesonephros (m) as indicated in A. In images in which gonadal expression is not apparent, a black dashed line is used to show the gonad-mesonephric border. (L) Real-time PCR analysis of Fst and Bmp2 gene expression in wild-type, Fog2-/- and Gata4ki/ki E12.5 XX gonads. The y-axis shows values for both genes normalized to Gapdh RNA copy number.

 

Figure 2
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  		Fig. 2. The GATA4-FOG2 complex is essential for the activation of dimorphically expressed genes. (A-G,J-M) Whole-mount ISH was performed with Sprr2d (A-D), Foxl2 (E-G), Gng13 (J,K) and Sf1 (Nr5a1; L,M) RNA probes on XX gonads from wild-type, Gata4ki/ki and Fog2-/- E12.5-13.5 mouse embryos as indicated. (H,I) Immunofluorescent staining of frozen gonadal sections from Fog2+/- (control) or Fog2-/- embryos with an anti-FOXL2 antibody (red). Embryonic germ cells are detected by the anti-PECAM1 antibody (e.g. Yao et al., 2003Go) (green). Magnification: 200x.

 

Figure 3
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  		Fig. 3. GATA4/FOG2 loss affects multiple aspects of early ovarian differentiation without activating Sertoli cell differentiation. (A,B) Whole-mount ISH was performed on XX E12.5 control or Gata4ki/ki gonads (A) or XY E13.5 control or Fog2-/- gonads (B) with the indicated probes. Note that the expression of Sertoli cell markers (Sox9, Mis and Dhh) is not increased in E12.5-13.5 XX gonads upon GATA4/FOG2 loss, whereas expression of genes encoding steroidogenic enzymes (e.g. Hsd3b1) is relaxed. Scale bar: 1 mm. (C) ISH was performed with an Inha probe on XX and XY wild-type and Fog2-/- gonads. Expression of Inha is derepressed in the XX Fog2-/- gonad. (D) The coelomic blood vessel does not form in Fog2-/- gonads. XX and XY gonads from E12.5 mouse embryos carrying Flk1-lacZ with wild-type or homozygous mutant Fog2 were stained with X-Gal, which marks the Flk1-lacZ-positive endothelial cells. The coelomic blood vessel in the testis is indicated by arrows.

 

Figure 4
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  		Fig. 4. Dkk1 is a downstream target of the GATA4-FOG2 transcription complex. (A,B) qRT-PCR analysis of Dkk1 expression was performed with wild-type (A) or wild-type and Fog2-/- (B) gonad-mesonephros samples. (C-L) ISH was performed with a Dkk1 RNA probe on XX and XY gonads from wild-type, Gata4ki/ki and Fog2-/- E11.5-13.5 mouse embryos as indicated.

 

Figure 5
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  		Fig. 5. Comparative expression of Wnt pathway genes in Gata4/Fog2 and Wnt4 mutants. (A-D) The GATA4-FOG2 complex is essential for canonical β-catenin signaling. Gonads from E12.5 mouse embryos carrying Axin2lacZ and either wild-type (A,B) or homozygous mutant (C,D) Fog2 were stained with X-Gal, which labels Axin2lacZ-positive cells. Only the ovary (B) is positive for Axin2lacZ expression. (E-L) ISH was performed with the indicated probes on XX gonads from wild-type, Gata4ki/ki and Fog2-/- E12.5-13.5 embryos. (M) qRT-PCR analysis of Sp5, Irx3 and Rspo1 gene expression in wild-type and Gata4ki/ki E12.5 XX gonads. (N) qRT-PCR analysis of gene expression in wild-type and Wnt4-/- E12.5 gonads.

 

Figure 6
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  		Fig. 6. Dkk1 acts cell-autonomously in the somatic cells of the developing ovary. (A-E) Immunofluorescent staining of frozen sections with an anti-DKK1 antibody (red). Embryonic germ cells are detected by the anti-PECAM1 antibody (green). Note the DKK1 staining in the wild-type testis (A, arrowheads) and mutant gonads (C,D), but not in wild-type ovaries (B) or Dkk1-null testis (E). Magnification: 200x. (F-O) ISH was performed with Oct4 (F,G), Dkk1 (H-M) and Lrp6 (N,O) RNA probes on gonads from wild-type, Gata4ki/ki, Fog2-/- and W/Wv E12.5-13.5 mouse embryos as indicated. (G,I,K,M) Samples were derived from in vivo busulfan-treated embryos.

 

Figure 7
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  		Fig. 7. GATA4/FOG2 loss does not affect the initiation of germ cell sexual differentiation. (A) ISH was performed with an Oct4 RNA probe on XX gonads from wild-type or Fog2-/- mouse embryos as indicated. (B-D) Immunofluorescent staining of frozen sections with an anti-{gamma}H2AX antibody (red). Embryonic germ cells are detected by the anti-PECAM1 antibody. Note the anti-{gamma}H2AX staining in the normal ovaries (C) and mutant gonads (D), but not in the control testis (B). (E) qRT-PCR analysis of Stra8 and Scp1 gene expression in wild-type and Fog2-/- E13.5-14.5 XX and XY gonads. The y-axis shows values for both genes normalized to Gapdh RNA copy number.

 

Figure 8
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  		Fig. 8. Analysis of sexual differentiation in Dkk1 mutant mice. (A,B) ISH was performed with the indicated RNA probes on XX gonads from wild-type and Dkk1-/- (A) or Dkk1-/-;Fog2-/- (B) E12.5 embryos. (C) qRT-PCR analysis of gene expression in E12.5 wild-type, Fog2-/-, Dkk1-/- and Dkk1-/-; Fog2-/- XX gonads.

 

Figure 9
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  		Fig. 9. Analysis of the sexual differentiation phenotype in mutants with somatic cell-restricted loss of β-catenin. (A,B) Whole-mount ISH with the indicated RNA probes was performed with XX E12.5 gonads from wild-type and Sf1-Cre/β-catflox/flox (where β-cat is Ctnnb1) mouse embryos (A) or with E13.5 XY gonads from wild-type and XX Sf1-Cre/β-catflox/flox embryos (B) to examine the status of the female (A) and the male (B) pathway in XX gonads upon β-catenin loss. (C) qRT-PCR analysis of gene expression in the XX E12.5 wild-type and Sf1-Cre/β-catflox/flox gonads. The data are shown as the ratio of normalized expression (gene/Gapdh RNA copy number) in wild-type over mutant samples. The combined *Wnt4 level in the gonad-mesonephros block does not change; see the corresponding panel in A for gonadal Wnt4 expression. (D,E) Double immunofluorescent staining of E18.5 gonadal sections for either {gamma}H2AX (D; red) or SYN/COR (E; red) and PECAM1 (green). The wild-type E18.5 ovary contains numerous germ cells with {gamma}H2AX (D, top panel) or SYN1 (synapsin I) staining (E, top left). In the XX Sf1-Cre/β-catflox/flox XX gonad, germ cells are largely lost; no SYN1-positive cells are observed in the control or Sf1-Cre/β-catflox/flox testis.

 

Figure 10
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  		Fig. 10. Formation of the male-specific coelomic blood vessel in control and mutant gonads. (A-H) Confocal microsopy images of whole-mount anti-PECAM1 immunostaining of E12.5 XY (top row) and E12.5 XX (bottom row) gonads isolated from control mouse embryos (A,B) or Dkk1-/- (C,D), Fog2-/- (E,F) and Sf1-Cre/β-catflox/flox (G,H) mutants. The male-specific coelomic vessel (white arrowheads) normally develops in the control (A), Dkk1-/- (C) and Sf1-Cre/β-catflox/flox testis (G). An ectopic vessel is visible (especially in the posterior region) in the XX Sf1-Cre/β-catflox/flox gonad (H). The vessel lacks the branches normally descending between the testis cords in XY gonads. No coelomic vessel could be seen in the Fog2-null samples (E,F; compare with Fig. 3D). (I-K) Light microscopy images of E12.5 gonads. A clear vesel (arrowheads) is seen on the coelomic surface of Sf1-Cre/β-catflox/flox XY (I) and Sf1-Cre/β-catflox/flox XX gonads (K and the higher magnification inset), but not on the Sf1-Cre/β-cat+/flox ovary (J). Scale bars: 100 µm in A-H; 200 µm in I-K.

 

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