Fgf9 and Wnt4 Act as Antagonistic Signals to Regulate Mammalian Sex Determination

The genes encoding members of the wingless-related MMTV integration site (WNT) and fibroblast growth factor (FGF) families coordinate growth, morphogenesis, and differentiation in many fields of cells during development. In the mouse, Fgf9 and Wnt4 are expressed in gonads of both sexes prior to sex determination. Loss of Fgf9 leads to XY sex reversal, whereas loss of Wnt4 results in partial testis development in XX gonads. However, the relationship between these signals and the male sex-determining gene, Sry, was unknown. We show through gain- and loss-of-function experiments that fibroblast growth factor 9 (FGF9) and WNT4 act as opposing signals to regulate sex determination. In the mouse XY gonad, Sry normally initiates a feed-forward loop between Sox9 and Fgf9, which up-regulates Fgf9 and represses Wnt4 to establish the testis pathway. Surprisingly, loss of Wnt4 in XX gonads is sufficient to up-regulate Fgf9 and Sox9 in the absence of Sry. These data suggest that the fate of the gonad is controlled by antagonism between Fgf9 and Wnt4. The role of the male sex-determining switch— Sry in the case of mammals—is to tip the balance between these underlying patterning signals. In principle, sex determination in other vertebrates may operate through any switch that introduces an imbalance between these two signaling pathways

Stage- and Cell-Specific Expression of FGF9 in Embryonic Gonads

<div><p>(A–F) Detection of FGF9 protein (red) at different stages of gonad development. FGF9 is up-regulated in XY gonads at 11.5 (B), 12.5 (D), and 13.5 dpc (F) while it is down-regulated in XX after 11.5 dpc (A, C, and E). No signal was detected in XY <i>Fgf9</i>^−/− gonads (unpublished data). </p> <p>(G–J) Serial sections of wild-type XX and compound heterozygous <i>Kit</i>^W/Wv XY gonads stained for alkaline phosphatase (purple; G and I) and FGF9 (red; H and J). Testis cords are formed in the absence of germ cells in XY <i>Kit</i>^W/Wv mutant gonads at 12.5 dpc (arrowhead in J). Expression of FGF9 is present in the mutant gonads where Sertoli cells are the only remaining cell type in the cords (J). Semitransparent dotted line indicates the boundary between gonad and mesonephroi. PECAM (green) marks germ cells and vascular endothelial cells (C–F, H, and J). The scale bars represent 25 μm. </p> <p>g, gonad; m, mesonephroi.</p></div

Sertoli Cell Precursors Switch from Expression of Male to Female Pathway Genes

<div><p>(A–F) Whole-mount in situ hybridization for genes in the male pathway downstream of <i>Sox9, Dhh,</i> and <i>Amh</i>. <i>Dhh</i> expression is disrupted in XY <i>Fgf9</i>^−/− gonads (g) at 11.5 dpc (A and B). <i>Amh</i> expression is severely reduced in XY <i>Fgf9</i>^−/− gonads at 12.5 dpc (E and F). </p> <p>(G and H) Analysis of cell death in <i>Fgf9</i>^−/− XY gonads using an apoptotic marker, active caspase-3 (red). No increased apoptosis is observed in XY <i>Fgf9</i>^−/− gonads (g) compared with control XY gonads, although apoptotic cells are increased around mesonephric tubules (m) of the mutant gonads (arrow in H). Semitransparent dotted line indicates boundary between mesonephros and gonad. (PECAM, green). </p> <p>(I–K) Whole-mount in situ hybridization for <i>Wnt4,</i> an ovary marker. <i>Wnt4</i> is expressed in <i>Fgf9</i>^−/− XY gonads at 12.5 dpc (K) similar to the level in XX <i>Fgf9</i>^+/− controls (I) but not in XY controls (J). The scale bars represent 50 μm. </p> <p>g, gonad; m, mesonephros.</p></div

Epistatic Relationship of <i>Sry, Fgf9,</i> and <i>Sox9</i>

<div><p>(A–D) <i>Sry</i> expression is not dependent on <i>Fgf9</i>. <i>Fgf9</i>^+/− (A) and <i>Fgf9</i>^−/− (B) XY gonads at 11.5 dpc expressing GFP (green) from the <i>Sry</i> promoter (polygonal cells, arrows). Blood cells show background fluorescence (doughnut-shaped cells). <i>Fgf9</i>^+/− (C) and <i>Fgf9</i>^−/− (D) XY gonads at 11.5 dpc expressing SRY^MYC protein (red, arrowheads). Inset shows nuclear counterstain (green, Syto13) colocalizing with SRY^MYC. PECAM (blue) marks endothelial and germ cells. Scale bars represent 25 μm. </p> <p>(E–K) Exogenous FGF9 can up-regulate SOX9 expression in XX gonads. Immunostaining of SOX9 (green) in primary cultures of gonadal cells. XX cells (E) and XY cells (G) cultured with exogenous FGF9 show induction of SOX9 expression (F and H, respectively). Cells were counterstained using the nuclear marker, Syto13 (red). Immunostaining of SOX9 (red) in gonad explants cultured with BSA- or FGF9-coated beads. SOX9 is expressed in XY gonads and cells contacting FGF9-coated beads (dotted circle labeled “F”) in XX gonads (I and K) but not in XX cells contacting BSA-coated control beads (“B”) (J). PECAM (blue) marks endothelial and germ cells. Scale bars (I–K) represent 50 μm.</p></div

Interdependent Relationship between <i>Fgf9</i> and <i>Sox9</i>

<div><p>(A–F) Immunostaining of SOX9 (red) in <i>Fgf9</i>^+/− and <i>Fgf9</i>^−/− XY gonads shows that <i>Fgf9</i> is required for maintenance of SOX9. The up-regulation of SOX9 in Sertoli precursor cells appears normal in <i>Fgf9</i>^−/− gonads at 11.5 dpc (D) compared with heterozygous littermate controls (A). However, SOX9 is detected in fewer cells in mutant gonads at 12.0 dpc (B and E), and is lost by 12.5 dpc (C and F). </p> <p>(G–J) mRNA whole-mount in situ hybridization for <i>Sry</i> and <i>Fgf9</i> in <i>Sox9</i>^flox/Δ and <i>Sox9</i>^Δ/Δ XY gonads shows that <i>Sox9</i> is required for <i>Fgf9</i> expression. <i>Sry</i> expression is detected in both <i>Sox9</i>^flox/Δ and <i>Sox9</i>^Δ/Δ gonads at 11.5 dpc (G and H), whereas <i>Fgf9</i> expression is markedly decreased or absent in <i>Sox9</i>^Δ/Δ gonads at 11.5 dpc (I and J). </p> <p>(K–O) Comparison of cell proliferation in <i>Sox9</i>^Δ/Δ versus <i>Sox9</i>^flox/Δ gonads at 11.5 dpc using immunostaining for phosphorylated histone H3. XY-specific proliferation at the gonad surface (K) is reduced in the absence of <i>Sox9</i> (L). Bar graph (O) shows quantitation of proliferation obtained by counting positive cells in the cortical region of each gonad (right brace) and normalizing to the number obtained from XY <i>Sox9</i>^flox/Δ gonads. <i>n</i> = 30, with five sections of each gonad and three pairs of gonads for each genotype. PECAM, green (A–F and K–N). The scale bars represent 25 μm. </p></div

Mutual Antagonism between <i>Fgf9</i> and <i>Wnt4</i>

<div><p>(A–C) <i>Wnt4</i> whole-mount in situ hybridization on gonad cultures. Adding exogenous FGF9 in gonad cultures results in the down-regulation of <i>Wnt4</i> expression in cultured XX gonads (C). Controls (A and B) were cultured without FGF9 peptide. </p> <p>(D–G) Reduction in the dose of <i>Wnt4</i> allows FGF9 to induce SOX9 in XX gonads. Immunostaining of SOX9 (red) shows that addition of FGF9 up-regulates SOX9 expression in heterozygous <i>Wnt4</i>^+/− XX gonads (G), but not in <i>Wnt4</i>^+/+ XX gonads (F). PECAM, green. The scale bars represent 50 μm. </p></div

Stabilization of β-catenin in XY gonads causes male-to-female sex-reversal

During mammalian sex determination, expression of the Y-linked gene Sry shifts the bipotential gonad toward a testicular fate by upregulating a feed-forward loop between FGF9 and SOX9 to establish SOX9 expression in somatic cells. We previously proposed that these signals are mutually antagonistic with counteracting signals in XX gonads and that a shift in the balance of these factors leads to either male or female development. Evidence in mice and humans suggests that the male pathway is opposed by the expression of two signals, WNT4 and R-SPONDIN-1 (RSPO1), that promote the ovarian fate and block testis development. Both of these ligands can activate the canonical Wnt signaling pathway. Duplication of the distal portion of chromosome 1p, which includes both WNT4 and RSPO1, overrides the male program and causes male-to-female sex reversal in XY patients. To determine whether activation of β-catenin is sufficient to block the testis pathway, we have ectopically expressed a stabilized form of β-catenin in the somatic cells of XY gonads. Our results show that activation of β-catenin in otherwise normal XY mice effectively disrupts the male program and results in male-to-female sex-reversal. The identification of β-catenin as a key pro-ovarian and anti-testis signaling molecule will further our understanding of the mechanisms controlling sex determination and the molecular mechanisms that lead to sex-reversal

SRY and the Standoff in Sex Determination

SRY was identified as the mammalian sex-determining gene more than 15 yr ago and has been extensively studied since. Although many of the pathways regulating sexual differentiation have been elucidated, direct downstream targets of SRY are still unclear, making a top down approach difficult. However, recent work has demonstrated that the fate of the gonad is actively contested by both male-promoting and female-promoting signals. Sox9 and Fgf9 push gonads towards testis differentiation. These two genes are opposed by Wnt4, and possibly RSPO1, which push gonads toward ovary differentiation. In this review, we will discuss the history of the field, current findings, and exciting new directions in vertebrate sex determination

The Mammalian Ovary from Genesis to Revelation

Two major functions of the mammalian ovary are the production of germ cells (oocytes), which allow continuation of the species, and the generation of bioactive molecules, primarily steroids (mainly estrogens and progestins) and peptide growth factors, which are critical for ovarian function, regulation of the hypothalamic-pituitary-ovarian axis, and development of secondary sex characteristics. The female germline is created during embryogenesis when the precursors of primordial germ cells differentiate from somatic lineages of the embryo and take a unique route to reach the urogenital ridge. This undifferentiated gonad will differentiate along a female pathway, and the newly formed oocytes will proliferate and subsequently enter meiosis. At this point, the oocyte has two alternative fates: die, a common destiny of millions of oocytes, or be fertilized, a fate of at most approximately 100 oocytes, depending on the species. At every step from germline development and ovary formation to oogenesis and ovarian development and differentiation, there are coordinated interactions of hundreds of proteins and small RNAs. These studies have helped reproductive biologists to understand not only the normal functioning of the ovary but also the pathophysiology and genetics of diseases such as infertility and ovarian cancer. Over the last two decades, parallel progress has been made in the assisted reproductive technology clinic including better hormonal preparations, prenatal genetic testing, and optimal oocyte and embryo analysis and cryopreservation. Clearly, we have learned much about the mammalian ovary and manipulating its most important cargo, the oocyte, since the birth of Louise Brown over 30 yr ago