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First published online 9 April 2008
doi: 10.1242/dev.018572

Development 135, 1875-1885 (2008)
Published by The Company of Biologists 2008
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Wt1 negatively regulates β-catenin signaling during testis development

Hao Chang1,2,*, Fei Gao3,*, Florian Guillou4, Makoto M. Taketo5, Vicki Huff1,3 and Richard R. Behringer1,2,{dagger}

1 Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA.
2 Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
3 Department of Cancer Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
4 UMR 6175 Physiologie de la Reproduction Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Université de Tours, Haras Nationaux Nouzilly, France.
5 Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.


Figure 1
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  		Fig. 1. Expression pattern of β-catenin in the developing testis. (A-L) β-Catenin (red)/AMH (green) double immunostaining at different stages. Nuclei were counterstained with DAPI (blue). (A,C,E,G,I,K) β-Catenin channel only and (B,D,F,H,J,L) β-catenin and AMH channels. β-Catenin was detected in the germ cells predominantly in the plasma membrane and cytoplasm at all stages. Sertoli cells started to express β-catenin from E15.5, mainly on the Sertoli cell membrane, but not in the nucleus (G-L). SC, Sertoli cell; GC, germ cell. Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. Catnbfx/fx; AMH-Cretg/+ male mice develop normal testes. (A) Sertoli cell-specific deletion of β-catenin in testes of Catnbfx/fx; AMH-Cretg/+ male mice at E16.5. In control testes, β-catenin protein was detected in both germ cells (left panel, arrowhead) and Sertoli cells (left panel, arrows). In Catnbfx/fx; AMH-Cretg/+ testes, β-catenin protein was undetected in Sertoli cells (right panel, arrows) but its expression in germ cells remained unaffected (right panel, arrowhead). β-Catenin protein was stained by an anti-β-catenin antibody (green) and nuclei were counterstained with DAPI (blue). (B) Normal histology of Catnbfx/fx; AMH-Cretg/+ testes at P0 (e) and 7 weeks (f) compared with control testes of the same age (a and b, respectively). Adult mutant mice expressed normal Sertoli cell marker SOX9 (g) and Leydig cell marker 3β-HSD (h), when compared with control mice (c and d, respectively). Scale bars: 20 µm.

 

Figure 3
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  		Fig. 3. Stabilization of β-catenin in Sertoli cells of Catnblox(e3)/+; AMH-Cretg/+ mice. (A-H) β-Catenin protein was stained by an anti-β-catenin antibody (green) and nuclei were counterstained with DAPI (blue). The accumulation of β-catenin in Sertoli cells started from E13.5 in a few Sertoli cells (D, arrow) and expanded to more Sertoli cells at E14.5 (F, arrows). At E15.5, almost all Sertoli cells in the testicular cord were affected (H, arrows). The stabilized β-catenin was localized both in cytoplasm and nucleus in Sertoli cells. Scale bars: 20 µm.

 

Figure 4
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  		Fig. 4. Phenotype of 7-week-old Catnblox(e3)/+; AMH-Cretg/+ males. (A) Catnblox(e3)/+; AMH-Cretg/+ males developed severely reduced size of testes and had retained uterus (b, light microscopy; c, Hematoxylin and Eosin staining). ep, epididymis; sv, seminal vesicle; t, testis; u, uterus; vd, vas deferens. (B) Testis sections stained with Hematoxylin and Eosin, and anti-3β-HSD antibody showing normal tubular architecture in control testes (a-c) and disruption of tubules in mutant testes (d-f). Scale bars in B: 60 µm in a,d; 20 µm in b,c,e,f.

 

Figure 5
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  		Fig. 5. Catnblox(e3)/+; AMH-Cretg/+ testes at E13.5 and E14.5. (A-H) Normal testicular histology with normal tubules (arrowheads) observed in mutant (C,D,G,H) and control (A,B,E,F) testes at low (A,C,E,G) and high (B,D,F,H) magnification. Scale bars: 60 µm in A,C,E,G; 20 µm in B,D,F,H.

 

Figure 6
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  		Fig. 6. Disruption of testicular cords and loss of germ cells in Catnblox(e3)/+; AMH-Cretg/+ testes between E15.5 and P0. (A-L) Progressive tubule development in control testes from E15.5 to P0 (low magnification, A,C,E; high magnification, G,I,K), and progressive disruption of cord architecture (arrowheads) in Catnblox(e3)/+; AMH-Cretg/+ testes (low magnification, B,D,F; high magnification, H,J,L), shown by Hematoxylin and Eosin staining. (M-R) Germ cells (GCNA1 positive) located within tubules of control testes (M,O,Q), but located outside of the tubules in Catnblox(e3)/+; AMH-Cretg/+ testes from E15.5 (arrow, N,P), and very few germ cells remained by P0 (R). (S-X) Leydig cells (3β-HSD-positive) scattered in interstitial spaces of control testes (S,U,W); masses of Leydig cells were found in the interstitial spaces of the mutant testes (T,V,X). Scale bars: 60 µm in A-F; 20 µm in G-X.

 

Figure 7
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  		Fig. 7. Loss of SOX9 and AMH expression but unaffected WT1 expression in Catnblox(e3)/+; AMH-Cretg/+ Sertoli cells. (A-R) Control testes show normal SOX9 (A,G,M), AMH (C,I,O) and WT1 (E,K,Q) expression in Sertoli cells at E12.5, E13.5 and E14.5. SOX9 (B) and AMH (D) expression was normal in E12.5 Catnblox(e3)/+; AMH-Cretg/+ testes, but was almost absent in E14.5 testes (N,P). WT1 expression was not affected in Catnblox(e3)/+; AMH-Cretg/+ testes (F,L,R). Scale bars: 20 µm.

 

Figure 8
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  		Fig. 8. Upregulation of β-catenin after Wt1 deletion in Sertoli cells. (A) Nuclear localization of β-catenin in Sertoli cells of Wt1fx/-; AMH-Cretg/+ mice at E15.5. β-Catenin protein was stained by an anti-β-catenin antibody (green) and nuclei were counterstained with DAPI (blue). β-Catenin was normally located on Sertoli cell membrane in control testes (left panel, arrowheads). However, β-catenin was detected in Sertoli cell nuclei in testes of Wt1 conditional knockout mice (right panel, arrows). Scale bar: 20 µm. (B) Immunofluorescent images of isolated primary Sertoli cells stained with WT1 (green). Nuclei were counterstained with DAPI (blue). Over 90% of the cells were WT1 positive. (C) Real-time PCR quantification of Wt1 expression in isolated primary Sertoli cells of mutant and control mice, with or without 4OH-tamoxifen treatment. Numerical data present mean±s.e.m. of relative expression of Wt1 in three independent experiments. Column 1, Wt1fx/fx; +/+ with treatment; column 2, Wt1fx/-; CAGGCre-ERtg/+ without treatment; column 3, Wt1fx/-; CAGGCre-ERtg/+ with treatment. Wt1 expression was significantly reduced upon 4OH-tamoxifen treatment in Wt1fx/-; CAGGCre-ERtg/+ Sertoli cells. (D) Western blot to analyze the expression levels of total β-catenin and the active form of β-catenin in isolated primary Sertoli cells of mutant and control mice, with or without 4OH-tamoxifen treatment. The levels of total β-catenin and the active form of β-catenin were upregulated upon 4OH-tamoxifen treatment in Wt1fx/-; CAGGCre-ERtg/+ Sertoli cells (quantification on the right).

 

Figure 9
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  		Fig. 9. Cell proliferation and apoptosis in Catnblox(e3)/+; AMH-Cretg/+ Sertoli cells. Immunostaining with proliferative marker BrdU (A,D) and PH3 (B,E) identified proliferating Sertoli cells of both control and mutant testes at E15.5 (arrowheads). (C,F) TUNEL staining identified no apoptotic cells in either control or mutant testes at E15.5; inset in C shows apoptotic cells in the regressing Müllerian duct as a positive control. Scale bar: 20 µm.

 

Figure 10
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  		Fig. 10. Sox9flox/-; AMH-Cretg/+ male mice develop normal testes at E18.5. (A-F) Sox9flox/+; AMH-Cretg/+ male mice were crossed with Sox9flox/flox; Zp3-Cretg/+ female mice to obtain Sox9flox/-; AMH-Cretg/+ embryos. Hematoxylin and Eosin staining showing normal histology of Sox9flox/-; AMH-Cretg/+ testes (D) when compared with control testes of the same age (A). SOX9 was totally deleted in Sertoli cells of mutant testes (E). However, the expression of AMH and β-catenin in Sertoli cells was unaffected upon SOX9 deletion (E,F). Scale bar: 20 µm.

 

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© The Company of Biologists Ltd 2008