DEV029587 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Anti-SCP3 immunohistochemical staining of the control (4-day cultured) explants of XX and XY wild-type genital ridges at 13 ts (11.1 dpc). In the XX explants (left), SCP3-positive meiotic germ cells are observed through the gonadal area, while, in XY explants (right), most of the germ cells are negative for anti-SCP3 staining (note that some SCP3-positive cells remain in XY explants). Scale bar: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. Anti-SRY immunohistochemical staining of XX Tg genital ridges HS-treated at 13 (11.1 dpc) and 18 (11.5 dpc) ts. All procedures for HS treatment, fixation and staining were performed as carefully as possible under the same conditions. At both 9 and 12 hours after HS treatment, no appreciable differences in SRY-positive signal intensities are detectable between the SRY-induced explants initiated at 13 ts and those initiated at 18 ts. Inset shows higher magnification, with anti-SRY positive signals in the presumptive supporting cells being directly associated with germ cells (asterisk). Scale bar: 100 µm.

• Supplemental Figure S3 -

  Fig. S3. Addition of FGF9 and sFRP2 together does not cause any appreciable changes in the SOX9 expression pattern in wild-type XX and XY gonads in vitro. Wild-type XX and XY gonads were isolated at 18-19 ts (approximately 11.5 dpc), and then cultured for 24 hours in the presence or absence of FGF9 (100 ng/ml) and sFRP2 (WNT antagonist; 1.5 µg/ml). Sagittal sections of the FGF9/sFRP2-treated and non-treated explants were comparatively analyzed by anti-SOX9 immunostaining (brown staining). Scale bar: 100 µm.

• Supplemental Figure S4 -

  Fig. S4. Effects of exogenous FGF9 and/or sFRP2 on the maintenance of SOX9 expression in XX Tg gonads with delayed Sry induction. A pair of XX Tg gonads was isolated at 18-19 ts (approximately 11.5 dpc) and then heat shock treated for Sry induction. One gonad of each pair was cultured in the presence of sFRP2 (1.5 µg/ml) alone, FGF9 (100 ng/ml) alone, or FGF9 (100 ng/ml)/sFRP2 (1.5 µg/ml) for 24 hours. The other gonad was cultured in absence of these two additives (None). Sagittal sections of the XX Tg explants were comparatively analyzed by anti-SOX9 immunostaining. (A) Anti-SOX9 immunostaining (brown), showing almost no SOX9-positive cells in non-treated (None) and sFRP2-treated (sFRP2) XX Tg explants, and only a small number of SOX9-positive cells in FGF9-treated (FGF9) explants at 24 hours after Sry induction. By contrast, XX Tg explants treated with FGF9 and sFRP2 together (FGF9/sFRP2) clearly show increased numbers of SOX9-positive cells at 24 hours. (B) Quantification of the relative number per gonadal area of SOX9-positive cells in the immunostained sections at 24 hours after Sry induction (mean values of cell number ±s.e.m.; n=4). XX and XY wild-type explants of the same littermates (FGF9/sFRP2 treatment) are also shown (solid bars). The value in the XY wild-type explants at 72 hours is set as 100%, as shown in Fig. 5D. Asterisks indicate a significant difference (*P<0.05 or **P<0.01), as compared with the data in the FGF9/sFRP2-treated XX Tg explants. Scale bar: 100 µm.