Supplemental Table 1. Genotyping primers. Primer Sequence (5-3 ) Zfy. Sf1(Nr5a1)-Cre. Gata4. Gata6. Supplemental Table 2. Quantitative RT-PCR primers
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1 Supplemental Table 1. Genotyping primers Zfy Sf1(Nr5a1)-Cre Gata4 Gata6 Primer Sequence (5-3 ) Forward: CTA-TTG-CAT-GGA-CAG-CAG-TCT-TAT-G Reverse: ACT-AGA-CAT-GTC-TTA-ACA-TCT-GTC-C Forward: GAG-TGA-ACG-AAC-CTG-GTC-GAA-ATC Reverse: GCA-TTA-CCG-GTC-GAT-GCA-ACG-AGT-G Forward: CCC-AGT-AAA-GAA-GTC-AGC-ACA-AGG Reverse: AGA-CTA-TTG-ATC-CCG-GAG-TGA-ACA Forward: GTG-GTT-GTA-AGG-CGG-TTT-GT Reverse: ACG-CGA-GCT-CCA-GAA-AAA-GT Supplemental Table 2. Quantitative RT-PCR primers Gapdh Amh Cyp11a1 Cyp11b1 Cyp11b2 Cyp21a1 Dhh Dmrt1 Foxl2 Hsd3b1 Hsd3b6 Hsd17b3 Gata1 Primer Sequence (5-3 ) Forward: GCT-CAC-TGG-CAT-GGC-CTT-CCG-TG Reverse: TGG-AAG-AGT-GGG-AGT-TGCTGT-TGA Forward: GCA-GTT-GCT-AGT-CCT-ACA-TCT-GGC-T Reverse: TGG-AGG-CTC-TTG-GAA-CTT-CAG-CAA Forward: AAG-TAT-GGC-CCC-ATT-TAC-AGG Reverse: TGG-GGT-CCA-CGA-TGT-AAA-CT Forward: GCT-TCA-CCA-TGT-GCT-GAA-ATC-C Reverse: AGA-AGA-GAG-GGC-AAT-GTG-TCA Forward: GCA-CCA-GGT-GGA-GAG-TAT-GC Reverse: CCA-TTC-TGG-CCC-ATT-TAG-C Forward: CGC-TTG-GGG-ATG-CAA-GAT-GTG-G Reverse: AGC-ATC-AGG-GCT-GAG-CGA-GAG-A Forward: ATC-CAC-GTA-TCG-GTC-AAA-GC Reverse: GTA-GTT-CCC-TCA-GCC-CCT-TC Forward: TGG-GTT-CTG-GAA-GCA-AGA-AG Reverse: CTG-TCT-TCT-CAG-GGC-CAC-CT Forward: GCA-AGG-GAG-GCG-GGA-CAA-CAC Reverse: GAA-CGG-GAA-CTT-GGC-TAT-GAT-GT Forward: CTT-GTC-AAC-TGG-GAG-GAA-GC Reverse: TTT-CCA-TCA-CTG-GCA-CTT-TG Forward: TCC-CCA-TTC-AGA-GCA-TGT-ATA-GC Reverse: TTT-TTT-TGA-GGT-ATT-GAC-AAG-TAT-TTA-TTG Forward: ATG-GAG-TCA-AGG-AGG-AAA-GGC Reverse: GGC-TGT-AAA-GAG-GCC-AGG-G Forward: TGT-CCT-CAC-CAT-CAG-ATT-CCA Reverse: TCC-CTC-CAT-ACT-GTT-GAG-CAG
2 Gata4 Gata6 Insl3 Lhr Mc2r Mvh Sf1(Nr5a1) Sox9 Star Forward: AAA-CGG-AAG-CCC-AAG-AAC-CTG-AAT Reverse: GAG-CTG-GCC-TGC-GAT-GTC-TGA-GTG Forward: AGT-TTT-CCG-GCA-GAG-CAG-TA Reverse: AGT-CAA-GGC-CAT-CCA-CTG-TC Forward: CAT-GCG-CGC-GCC-GCT-GCT-AC Reverse: TCA-GTG-GGG-ACA-CAG-ACC-C Forward: GAG-ACG-CTT-TAT-TCT-GCC-ATC-T Reverse: CAG-GGA-TTG-AAA-GCA-TCT-GG Forward: TGG-AAA-AGT-TCT-CAG-CAC-CAC Reverse: TCT-TTG-TGT-GGA-AGG-ATC-TGG Forward: CCA-AGA-TCA-GGG-GAC-ACA-GC Reverse: CTT-TGG-TAA-GTG-TCA-CCA-TTG-C Forward: CTC-CCT-CTG-GTC-CTC-TTC-CT Reverse: TCG-TGG-TGG-TAG-TCG-TCG-TA Forward: GAG-CCG-GAT-CTG-AAG-AGG-GA Reverse: GCT-TGA-CGT-GTG-GCT-TGT-TC Forward: GCA-GCA-GGC-AAC-CTG-GTG Reverse: TGA-TTG-TCT-TCG-GCA-GCC
3 Supplemental Figure 1. Assessment of cell proliferation using BrdU in conditional double mutant embryonic testes. Testicular sections from controls (A-E) and Sf1Cre; Gata4 flox/flox Gata6 flox/flox mice (F-J) at embryonic days (E)15.5 (A-C; E-G) and 17.5 (D, E, I, J) were stained for bromodeoxyuridine (BrdU; green) (A-J); mouse vasa homologue (MVH; red) (B, G, E, J) and Wilms Tumor 1 (WT1; red) (C and H). Nuclei are stained with DAPI (blue). (K) Changes in somatic cell proliferation in Sf1Cre; Gata4 flox/flox Gata6 flox/flox testes at E17.5. The results are shown as the percentage of BrdU-positive cells relative to the number of DAPI-positive cells from three different males (n = 3) of each genotype. Supplemental Figure 2. TUNEL assay in embryonic testes. Sections of control (A, D) and Sf1Cre; Gata4 flox/flox Gata6 flox/flox (B, C, E, F) testes at embryonic days (E) 15.5 (A-C) and 17.5 (D-F). Cell death was assessed by TUNEL using TMR Red conjugated dutp to identify apoptotic nuclei (yellow) and MVH (green) to identify germ cells. Nuclei were counterstained with DAPI (blue). Panels C and F are higher magnifications of B and E, respectively. Arrows in panels B and E point to TUNEL-positive cells localized close to the coelomic epithelium. Arrowheads in panels C and F show TUNEL- and MVH-positive cells. Scale bars are 100 μm (A, B, D, E) and 50 μm (C, F). Supplemental Figure 3. Normal testis development in transgene-rescued Gata1 null males (Gata1 - /Tr, (35)). Sectioned adult testes from the control (A, B, E-G) or Gata1 - /Tr (C, D, H-J) males were stained with antibodies against GATA1 (A, C), H2AX (B, D), GATA4 (E, H), GATA6 (F, I) and AMH (G, J). All proteins (with notable exception of GATA1) are expressed normally in the double mutants. Scale bars represent 100µm (A-D); 50µm (E-J).
4 Supplemental Figure 4. Whole-mount in-situ hybridization (WISH) comparing Cyp11a1 (A and D), Cyp17a1 (B and E) and Hsd17b3 (C and F) in controls (A-C) and Sf1Cre; Gata4 flox/flox Gata6 flox/flox (D-E) male gonads at embryonic day (E) The dark purple staining represents specific binding of the RNA antisense probes in the gonad. In all panels, the gonad (g) and mesonephros (m) are as indicated in panel A. Supplemental Figure 5. Ventral view of testicular position in control (A) and Sf1Cre; Gata4 flox/flox Gata6 flox/flox (B) males at postnatal day (PND) 9. In panels A and B, arrows point to testes. K, kidney; B, bladder. (C) Gross appearance of the seminal vesicles from control (asterisks) and Sf1Cre; Gata4 flox/flox Gata6 flox/flox (arrowheads) males at PND 90. (D) Seminal vesicle weights in 90 days-old control and Sf1Cre; Gata4 flox/flox Gata6 flox/flox males. The data were analyzed using Student s t-test, with significance considered at **P < (E) Submaxillary gland weights in 90 days-old control males (black bar), control females (light grey bar) and Sf1Cre; Gata4 flox/flox Gata6 flox/flox males (dark grey bar). The data were analyzed using ANOVA (one-way) followed by the Sidak's multiple comparisons test. Bars with different superscripts differ significantly (***P < 0.001). The results are graphed as the means ± s.e.m. from n = 3 of each genotype. Supplemental Figure 6. Testicular function is not altered in Sf1Cre; Gata6 flox/flox animals. Sections from controls (A-C) and Sf1Cre; Gata6 flox/flox testis (D-O) at postnatal day (PND) 180 were stained for GATA4 (green) (A, D) and GATA6 (red) (B, E); sex determining region Y-box 9 (SOX9; red) (G) and 3β-hydroxysteroid dehydrogenase (3βHSD; green) (H); Wilms Tumor 1
5 (WT1; red) (J) and anti-müllerian hormone (AMH; green) (K); mouse vasa homolog (MVH; red) (M) and GATA1 (N). Panels C, F, I, L and O are merged images. Nuclei are stained with DAPI (blue; inset in F; I, L and O). Insets in panels F and O are scaled high-magnification (400x) images. Notice that Sertoli cells lost GATA6 staining, but retained GATA4 (inset in F, arrows, compare to C). Scale bars: 100µm.
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