The Wilms tumor gene, Wt1, is required for Sox9 expression and maintenance of tubular architecture in the developing testis

Abstract

Mutation of the transcription factor and tumor suppressor gene WT1 results in a range of genitourinary anomalies in humans, including 46,XY gonadal dysgenesis, indicating that WT1 plays a critical role in sex determination. However, because knockout of Wt1 in mice results in apoptosis of the genital ridge, it is unknown whether WT1 is required for testis development after the initial steps of sex determination. To address this question, we generated a mouse strain carrying a Wt1 conditional knockout allele and ablated Wt1 function specifically in Sertoli cells by embryonic day 14.5, several days after testis determination. Wt1 knockout resulted in disruption of developing seminiferous tubules and subsequent progressive loss of Sertoli cells and germ cells such that postnatal mutant testes were almost completely devoid of these cell types and were severely hypoplastic. Thus, Wt1 is essential for the maintenance of Sertoli cells and seminiferous tubules in the developing testes. Of particular note, expression of the testis-determining gene Sox9 in mutant Sertoli cells was turned off at embryonic day 14.5 after Wt1 ablation, suggesting that WT1 regulates Sox9, either directly or indirectly, after Sry expression ceases. Our data, along with previous work demonstrating the role of Wt1 at early stages of gonadal development, thus indicate that Wt1 is essential at multiple steps in testicular development.

Fig. 1.

Generation of the Wt1^flox mouse strain. (A) Scheme for generating animals carrying the conditional knockout allele (Wt1^flox) and the recombined allele (Wt1^Δ). (B) Southern blot of EcoRV restricted ES cell DNA probed with 5′ probe yielded a 7.14-kb fragment in recombinant animals in addition to the wild-type 5.34-kb fragment, confirming insertion of the targeting construct into the Wt1 locus. (C) PCR genotyping of wild-type and Wt1^flox allele using primers loxpF and loxpR. (D) PCR genotyping of wild-type and Wt1^flox allele using primers 1.75 and 1.55. (E) PCR detection of recombined Wt1^Δ allele using primers ckodelF and 1.55.

Fig. 2.

Expression of Cre-recombinase and recombination of Wt1^flox allele. (A) IHC demonstrating expression of Cre-recombinase specifically in Sertoli cells of E14.5 Wt1^−/flox; AMH-Cre testes (arrows) but not in mutant testes at E13.5 or in controls (Wt1^−/flox). (B) Detection of recombined Wt1^Δ allele specifically in testes of E14.5 Wt1^−/flox; AMH-Cre embryos.

Fig. 3.

Phenotype of 7-week-old Wt1^−/flox; AMH-Cre males. (A) Normal external genitalia of mutant male (Aa), severely reduced size of testes from three different Wt1^−/flox; AMH-Cre males (Ab), and reproductive tracts from control male and Wt1^−/flox; AMH-Cre male displaying reduced size and the development of both a vas deferens and uterus in mutant (Ac and Ad). (B) Testis sections stained with H&E or with anti-3β-HSD antibody and showing normal tubular architecture (asterisk) in control testes (Ba and Bb), lack of tubules in mutant testes (arrowhead) (Bd and Be), and presence of Leydig cells (lc) in both control (Bc) and Wt1^−/flox; AMH-Cre (Bf) testes. Ep, epididymis; t, testis; vd, vas deferens; sv, seminal vesicle; u, uterus.

Fig. 4.

Mutant testes at E13.5 and E14.5. (d, e, j, and k) Normal testicular histology with normal tubules (arrowheads, arrows) observed in mutant testes at low and high magnification. (c, f, j, and l) WT1 immunostaining of Sertoli cells.

Fig. 5.

Disruption of seminiferous tubule architecture and loss of Sertoli cells and germ cells in Wt1^−/flox; AMH-Cre testes between E15.5 and P7. (a–d) Progressive tubule development and organization in control testes from E15.5 to P7 shown by H&E staining. (e–h) Germ cells (GCNA-1-positive) and Sertoli cells (WT1-positive) located within tubules of control testes. (i and j) Progressive disruption of tubule architecture in Wt1^−/flox; AMH-Cre testes (low magnification). (k and m) Predominant histology of E15.5 and P7 mutant testes (high magnification). (l and n) Histology of occasional aberrant tubules in E15.5 and P7 mutant testes (high magnification). (o–r) Sertoli cells in mutant testes identified by WT1 IHC. (s–v) Germ cells in mutant testes identified by GCNA-1 IHC. sc, Sertoli cells; gc, germ cells; lc, Leydig cell.

Fig. 6.

Loss of SOX9, SOX8, and AMH expression by E14.5 in Wt1^−/flox; AMH-Cre Sertoli cells. Control testes show normal SOX9 (a and g), SOX8 (b and h), and AMH (c and i) expression in Sertoli cells at E13.5 and E14.5. SOX9 (d), SOX8 (e), and AMH (f) expression was normal in E13.5 Wt1^−/flox; AMH-Cre testes but was virtually absent in E14.5 testes, even in those Sertoli cells associated with tubules (asterisks, j–l). sc, Sertoli cells.