Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination

Abstract

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.

Figure 1. Stage- and Cell-Specific Expression of FGF9 in Embryonic Gonads

(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 No signal was detected in XY Fgf9 ^−/− gonads (unpublished data). (G–J) Serial sections of wild-type XX and compound heterozygous Kit ^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 Kit ^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. g, gonad; m, mesonephroi.

Figure 2. Epistatic Relationship of Sry, Fgf9, and Sox9

(A–D) Sry expression is not dependent on Fgf9. Fgf9 ^+/− (A) and Fgf9 ^−/− (B) XY gonads at 11.5 dpc expressing GFP (green) from the Sry promoter (polygonal cells, arrows). Blood cells show background fluorescence (doughnut-shaped cells). Fgf9 ^+/− (C) and Fgf9 ^−/− (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. (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.

Figure 3. Interdependent Relationship between Fgf9 and Sox9

(A–F) Immunostaining of SOX9 (red) in Fgf9 ^+/− and Fgf9 ^−/− XY gonads shows that Fgf9 is required for maintenance of SOX9. The up-regulation of SOX9 in Sertoli precursor cells appears normal in Fgf9 ^−/− 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). (G–J) mRNA whole-mount in situ hybridization for Sry and Fgf9 in Sox9 ^flox/Δ and Sox9 ^Δ/Δ XY gonads shows that Sox9 is required for Fgf9 expression. Sry expression is detected in both Sox9 ^flox/Δ and Sox9 ^Δ/Δ gonads at 11.5 dpc (G and H), whereas Fgf9 expression is markedly decreased or absent in Sox9 ^Δ/Δ gonads at 11.5 dpc (I and J). (K–O) Comparison of cell proliferation in Sox9 ^Δ/Δ versus Sox9 ^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 Sox9 (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 Sox9 ^flox/Δ gonads. n = 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.

Figure 4. Sertoli Cell Precursors Switch from Expression of Male to Female Pathway Genes

(A–F) Whole-mount in situ hybridization for genes in the male pathway downstream of Sox9, Dhh, and Amh. Dhh expression is disrupted in XY Fgf9 ^−/− gonads (g) at 11.5 dpc (A and B). Amh expression is severely reduced in XY Fgf9 ^−/− gonads at 12.5 dpc (E and F). (G and H) Analysis of cell death in Fgf9 ^−/− XY gonads using an apoptotic marker, active caspase-3 (red). No increased apoptosis is observed in XY Fgf9 ^−/− 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). (I–K) Whole-mount in situ hybridization for Wnt4, an ovary marker. Wnt4 is expressed in Fgf9 ^−/− XY gonads at 12.5 dpc (K) similar to the level in XX Fgf9 ^+/− controls (I) but not in XY controls (J). The scale bars represent 50 μm. g, gonad; m, mesonephros.

Figure 5. Mutual Antagonism between Fgf9 and Wnt4

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

Figure 6. Ectopic Expression of Male Factors, SOX9 and FGF9, in XX Wnt4 ^−/− Gonads

(A–C) FGF9 (red) immunostaining shows that FGF9 is expressed in XX Wnt4 ^−/− gonads at 12.5 dpc (C) relative to littermate controls (A and B). (D–L) SOX9 (red) immunostaining shows that SOX9 is transiently up-regulated in XX Wnt4 ^−/− gonads. SOX9 expression is detected in Wnt4 ^−/− XX gonads at 11.5–12.0 dpc (F and I), albeit at reduced levels compared with XY gonad controls (E-H). SOX9 is not detected in control XX Wnt4 ^+/− gonads (D-J) or Wnt4 ^−/− XX gonads at 12.5 dpc (L). The ectopic coelomic vessel in XX Wnt4 ^−/− gonads [ 28] is indicated by arrowheads. PECAM, green. The scale bars represent 50 μm.

Figure 7. Opposing Signals Regulate Sex Determination in the Bipotential Gonad

In both XX and XY gonads at 11.25 dpc (15 tail-somite stage), Fgf9 transcripts (white arrows) are detected near the gonad surface (A and B), whereas Wnt4 transcripts are detected near the gonad mesonephric boundary (C and D). We propose a model in which the fate of the gonad is balanced between these competing signals. A genetic or environmental switch initiates the male pathway by creating an imbalance between these signals. In mammals, this imbalance occurs through the up-regulation of Sox9. Sox9 up-regulates Fgf9, and Fgf9 maintains Sox9, forming a positive feed-forward loop in XY gonads. In this situation, the balance between FGF9 and WNT4 signals is shifted in favor of FGF9, and the dominance of the male pathway is established. In the absence of a feed-forward loop between SOX9 and FGF9 (e.g., in XX gonads), WNT4 blocks Fgf9, initiating the female pathway.