Fig 1.
Schematic representation of TES and TESCO.
TES (3161 bp, blue box) is located 10–13 kb upstream of the Sox9 transcription start site. TESCO is located within TES (1293 bp, purple box). The location of binding sites for SF1, SRY and SOX9 transcription factors are indicated. Nine putative sites for SF1 (Green triangular), eight for SRY (blue circle) and three for SOX9 (Pink square) are present.
Table 1.
Primers for CRISPR genome editing.
Fig 2.
Histological and immunofluorescence analysis of TESCO deleted XY gonads.
(A) Haematoxylin and eosin staining of 12.5 dpc, 14.5 dpc and 6 week postnatal XY testis of wild type and TESCO-/- mice. (B) Immunostaining of 12.5 and 14.5 dpc XY testis of wild type and TESCO-/- embryos. Testes were stained for AMH (green), FOXL2 (cyan) and DAPI (blue). (C) Immunostaining of 6 week-old XY testes of wild type and TESCO-/- mice. Testes were stained for SOX9 (green), FOXL2 (cyan) and DAPI (blue).
Fig 3.
Immunofluorescence analysis of C57BL/6J TES deleted XY gonads.
(A) Bright field images of 8-week old wild type, TES+/- and TES-/- testes. (B) Immunostaining of 13.5 dpc XY testis of wild type and TES-/- embryos. (C) Immunostaining of 8-week old XY testes of wild type and TES-/- mice. Testes were stained for SOX9 (green), FOXL2 (red) and DAPI (blue).
Fig 4.
Real-time quantitative RT-PCR of genes involved in male and female sex determination in XY TESCO and TES deleted mice.
(A) Gene expression in XY TESCO deleted gonads at 14.5 dpc (B) Gene expression in XY TESCO deleted gonads at 6 weeks (C) Gene expression in XY C57BL/6J TES deleted gonads at 13.5 dpc (D) Gene expression in XY C57BL/6J TES deleted gonads at 8 weeks. Data are presented as mean 2-ΔΔCt values, normalized to Tbp/ Hprt at the embryonic stages and Hprt at the adult stages. Sample size represents number of individuals and is indicated below each genotype. Error bars show SEM of 2-ΔΔCt values. P value is presented above the relevant bars (unpaired, two-tailed t-test on 2-ΔΔCt values). Dark grey bars: XY; light grey bars: XX.
Fig 5.
Immunofluorescence and real-time quantitative RT-PCR analysis of mice carrying TES deletion on Sox8-null background.
(A) Immunostaining of 14.5 dpc XY testis of wild type and TES-/- embryos on a Sox8-null background. (B) Immunostaining of 8 week-old XY testes of wild type and TES-/- mice on a Sox8-null background. Testes were stained for SOX9 (green), FOXL2 (red) and DAPI (blue). (C) Gene expression in XY TES deleted gonads with Sox8-null background at 14.5 dpc. Data are presented as mean 2-ΔΔCt values normalized to Hprt. Sample size represents number of individuals and is indicated below each genotype. Error bars show SEM of 2-ΔΔCt values. P value is presented above the relevant bars (unpaired, two-tailed t-test on 2-ΔΔCt values). Dark grey bars: XY; light grey bars: XX.
Fig 6.
Comparison of Sox9fl/ΔTES and Sox9-/ΔTES gonads.
(A) Immunostaining of 13.5 dpc XY gonads of Sox9fl/ΔTES and Sox9-/ΔTES; β-Actin:Cre embryos. Gonads were stained for SOX9 (green), FOXL2 (red) and DAPI (blue). The pole of the gonad is indicated in the inset box. (B) Gene expression in XY gonads with or without conditional Sox9-null and TES deletions at 13.5 dpc. Data are presented as mean 2-ΔΔCt values normalized to Hprt. Sample size represents number of individuals and is indicated below each genotype. Error bars show SEM of 2-ΔΔCt values. P value is presented above the relevant bars (unpaired, two-tailed t-test on 2-ΔΔCt values).
Table 2.
Primers for genotyping TES/TESCO deleted mice.
Table 3.
Primers for Real-time quantitative RT-PCR.