DEV024653 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig S1. A histological analysis of the E14.5 Fog2 null gonads. Representative E14.5 wild-type (A,B) and Fog2^−/− (C,D) XX and XY gonadal sections stained with Hematoxylin and Eosin. tc, testis cords; s, Sertoli cell; g, germ cell. Scale bar: 100 µm.

• Supplemental Figure S2 -

  Fig S2. Real-time PCR analysis of gene expression in E12.5 wild-type and Gata4^ki/ki gonads. The data are shown as ratios of normalized expression (gene/Gapdh RNA copy number) in wild-type over mutant samples; expression in the control XY gonad (or in the control XX gonad for Fst) is designated as 100%. Note that expression of some genes encoding steroidogenic enzymes is relaxed in the mutants (e.g. Cyp11a1 and Hsd3b1), whereas other male-specific genes (Cyp17a1 and Hsd17b1) are only active in the control testis sample.

• Supplemental Figure S3 -

  Fig S3. Cre expression in somatic cells of control gonads and gonads with the conditional deletion of β-catenin. (A-C) Double immunostaining for a nuclear marker of somatic cells (GATA4, green) of a Sf1-Cre⁺ gonad at E14.5 shows robust Cre expression (red) in a subset of gonadal somatic cells in the control Sf1-Cre/β-cat^+/flox (A) and Sf1-Cre/β-cat^flox/flox testis (B) but not Sf1-Cre/β-cat^flox/flox XX gonads (C). (D-I) Double immunostaining for PECAM1 (green) of a Sf1-Cre⁺ gonad at E13.5 (D,E) and E14.5 (F-I) shows Cre expression (red) in numerous gonadal somatic cells in the control ovaries (D) and Sf1-Cre/β-cat^flox/flox testis (H,I) but not Sf1-Cre/β-cat^flox/flox XX gonads (E-G). G and I are higher magnifications of samples in F and H, respectively. One of the very few surviving Cre-positive cells in the Sf1-Cre/β-cat^flox/flox XX sample is shown (arrowhead). Germ (PECAM1⁺) cells do not express Cre.

• Supplemental Figure S4 -

  Fig S4. β-catenin expression in wild-type and Sf1-Cre/β-cat^flox/flox gonads. Double immunostaining of XX (A,B) and XY (C,D) gonads that were either wild-type (A,C) or Sf1-Cre/β-cat^flox/flox (B,D) with an anti-CTNNB1 antibody (red). Embryonic germ cells are detected by the anti-PECAM1 antibody (green). Somatic cell staining of CTNNB1 (white arrowheads) is apparent in the control (A,C) but not in β-catenin mutant samples (B,D). Magnification: 200×.

• Supplemental Figure S5 -

  Fig S5. The loss of FOXL2 expression and increased apoptosis upon Sf1-Cre-mediated β-catenin deletion in XX gonads. PECAM1 is green throughout. The gonad (top) and mesonephros are separated by a dotted line. (A-D) Double immunostaining of XX gonads for a nuclear marker of ovarian granulosa cells (FOXL2, red) at E13.5-14.5 shows numerous FOXL2-expressing cells in the control (A,C) but not in the Sf1-Cre/β-cat^flox/flox XX gonads (B,D). (E,F) A TUNEL assay (red) of XX E14.5 gonads demonstrates a greater number of cells undergoing apoptosis in the Sf1-Cre/β-cat^flox/flox gonad (F) as compared with a control sample (E). Note that TUNEL-positive cells do not generally overlap with PECAM1-positive cells. (G,H) The loss of β-catenin in the XY gonad (H) does not activate the female differentiation program as defined by FOXL2 expression.

• Supplemental Figure S6 -

  Fig S6. The loss of β-catenin does not activate the male pathway in E18.5 XX gonads. (A-D). Histological staining of the control (A,C) and Sf1-Cre/β-cat^flox/flox mutant gonads (B,D). (E-L) Double immunostaining of the XY (E-G) and XX (I-L) gonads at E18.5 for SOX9 (E,G,I,K) and MIS (F,H,G,L). Male-specific protein staining is observed only in the XY but not XX samples irrespective of their β-catenin status.