FOXL2 and BMP2 act cooperatively to regulate follistatin gene expression during ovarian development

Abstract

Follistatin is a secreted glycoprotein required for female sex determination and early ovarian development, but the precise mechanisms regulating follistatin (Fst) gene expression are not known. Here, we investigate the roles of bone morphogenetic protein 2 (BMP2) and forkhead-domain transcription factor L2 (FOXL2) in the regulation of Fst expression in the developing mouse ovary. Bmp2 and Fst showed similar temporal profiles of mRNA expression, whereas FOXL2 protein and Fst mRNA were coexpressed in the same ovarian cells. In a cell culture model, both FOXL2 and BMP2 up-regulated Fst expression. In ex vivo mouse fetal gonad culture, exogenous BMP2 increased Fst expression, but this effect was counteracted by the BMP antagonist Noggin. Moreover, in Foxl2-null mice, Fst expression was reduced throughout fetal ovarian development, and Bmp2 expression was also reduced. Our data support a model in which FOXL2 and BMP2 cooperate to ensure correct expression of Fst in the developing ovary. Further, Wnt4-knockout mice showed reduced expression of Fst limited to early ovarian development, suggesting a role for WNT4 in the initiation, but not the maintenance, of Fst expression.

Figure 1

Time course of marker gene expression during mouse gonadal development. A, Expression of female marker genes and BMP receptor genes expression in fetal gonads. Data sets represent mRNA expression relative to Rps29 (mean ± sem of three biologically independent experiments performed in triplicate). The red and blue lines indicate female and male, respectively. B, Expression of female marker genes during ovarian development. Data from panel A were combined into one graph with maximum expression levels adjusted to 100%.

Figure 2

Fst and FOXL2 colocalize in fetal ovaries. In situ hybridization (Fst, purple) and immunohistochemistry (FOXL2, brown) on sagittal section of 13.5 dpc ovary. A, Low-power image of whole ovary. B, High-power image of ovary showing coexpression of Fst mRNA and FOXL2 protein in the same cells. C, Adjacent negative control tissue (mesonephros) expressing neither Fst nor FOXL2. The rectangular areas in panel A indicate the location of panels B and C. Scale bars, 200 μm (A); 40 μm (B and C).

Figure 3

FOXL2 and BMP2 cooperatively up-regulate Fst expression in KK1 cells. qRT-PCR analysis of Foxl2, Fst, Bmp2 mRNA levels in KK1 granulosa-like cells transfected with constructs expressing with Foxl2 (A, B, and F: 0, 2, and 4 μg), treated with BMP2 (C and E: 0, 10, 100 ng/ml), or treated with BMP2 (D: 100 ng/ml) and/or Foxl2 expression plasmid (D: 4 μg). Data sets represent mRNA expression relative to 18S (mean ± sem of three biologically independent experiments performed in triplicate). Asterisks indicate level of statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4

FOXL2 is also able to up-regulate Fst independently of BMP2. A, BMP antagonist Noggin suppresses the BMP2-induced up-regulation of Fst expression in KK1 cells; triangle represents increasing concentrations of recombinant Noggin from 100 to 500 ng/ml. B, Fst expression in KK1 cells transfected with constructs expressing Foxl2 (4 μg) and/or treated with Noggin (100 ng/ml). Data sets represent mRNA expression relative to 18S (mean ± sem of three biologically independent experiments performed in triplicate). Asterisks indicate level of statistical significance (*, P < 0.05).

Figure 5

BMP2 up-regulates Fst expression in ex vivo organ culture. Fst (A) and Foxl2 (B) expression in ex vivo gonadal organ culture was evaluated by using qRT-PCR. Gonads (11.5 dpc) were cultured on the medium containing BMP2 (500 ng/ml), or BMP2 (500 ng/ml) and Noggin (2 μg/ml). Data represent mRNA expression relative to Rps29 (mean ± sem of three biologically independent experiments performed in triplicate). *, P < 0.05.

Figure 6

Fst and Bmp2 expression in Foxl2^−/−, Wnt4^−/−, and Foxl2^−/−/Wnt4^−/− knockout mice. A, qRT-PCR analysis of Fst expression at 13.5 dpc and P0 in Foxl2^−/−, Wnt4^−/−, and Foxl2^−/−/Wnt4^−/− XX gonads. B, Time course of Fst expression in Foxl2^−/− and Wnt4^−/− XX gonads. C, qRT-PCR analysis of Bmp2 expression at 13.5 dpc and P0 in Foxl2^−/−, Wnt4^−/−, and Foxl2^−/−/Wnt4^−/− XX gonads. D, Time course of Bmp2 expression in Foxl2^−/− and Wnt4^−/− XX gonads. All time points analyzed are shown. Data represent mRNA expression relative to XX wild-type (WT) gonads (mean ± sem of three biologically independent experiments performed in triplicate). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7

Model for the regulation of Fst expression during ovarian development. At early stages such as 11.5 dpc, WNT4 likely acts as a major regulator for Fst expression. WNT4 also acts upstream of Bmp2 and is required for the early phase of Fst expression (this study). Later in ovarian development (15.5 dpc to P0), FOXL2 and BMP2 are together required for Fst expression. BMP2 and FOXL2 up-regulate Fst expression cooperatively and individually, and FOXL2 also up-regulates Bmp2 expression (this study).