Modeling Dosage-Sensitive Sex Reversal in Mice Figure 4. Sry expression is reduced in Wt1+/2 and Sf1+/2 B6 XYAKR gonads, but increased Sry dosage did not fully restore normal cord differentiation. (A) Real-time qRT-PCR analysis of Sry expression relative to Gapdh (22DCt) showing mean Sry transcript levels +/2 standard error. The sample size for each group is shown adjacent to the corresponding data point. An asterisk denotes statistically significant effects of genotype (ANOVA, a = 0.05). There was a marginally significant effect of genotype at E11.25 (15–17 ts, ANOVA, F2,28 = 3.1670, p,0.0576) and a significant effect of genotype at E11.5 (18–20 ts, ANOVA, F2,34 = 6.999, p,0.0028) and E11.75 (21–23 ts, ANOVA, F2,7 = 15.41, p,0.0027). Pairwise comparisons revealed a significant reduction in Sry transcript levels in Wt1+/2 vs. +/+ samples at E11.25 (15–17 ts, t24 = 2.224 , p,0.0179), E11.5 (18– 20 ts, t25 = 3.653, p,0.0012), and in E11.75 (21–23 ts, t3 = 11.51, p,0.0014). Similarly, Sry expression was reduced in Sf1+/2 vs. +/+ samples at E11.5 (18–20 ts, t29 = 2.125, p,0.0422) and E11.75 (21–23 ts, t5 = 6.142, p,0.0016). (B) The amount of tissue expressing the Sertoli cell marker AMH (green) was greater in Wt1+/2 and Sf1+/2 B6 XYAKR,Sxr gonads (E and F), which have two copies of the Sry gene, compared to Wt1+/2 and Sf1+/2 B6 XYAKR gonads (B and C), which have the normal single copy of the Sry gene (top two rows, 106magnification with 0.76zoom). However, while Wt1+/2 and Sf1+/2 B6 XYAKR,Sxr gonads developed more testicular tissue than Wt1+/2 and Sf1+/2 B6 XYAKR gonads, they consistently develop less testicular tissue than +/+ B6 XYAKR,Sxr controls. Additionally, cords were less differentiated and less organized in heterozygous mutants when compared to wild- type B6 XYAKR,Sxr gonads, even in the central regions of the gonads where cord differentiation was most complete (G–I, bottom row, 206 magnification). Germ and vascular endothelial cells are labeled by PECAM staining (red). doi:10.1371/journal.pgen.1002569.g004 relative to the D2 alleles. Analysis of morphology and marker gene expression in E13.5 Wt1+/2 and Sf1+/2 B6 XYAKR gonads showed that some developed as ovaries and were indistinguishable from control XX ovaries in that they expressed ovary-specific markers, and did not express Sertoli cell or Leydig cell-specific markers. These data indicate that the testis-determining pathway is interrupted at a very early stage in these XY gonads. In addition to defects in testis differentiation, we found that double heterozygous Wt1+/2; Sf1+/2 fetuses lacked adrenal glands and that neonates die shortly after birth, presumably from adrenal insufficiency. Because the gonads and adrenal cortex share a common origin [47], because the morphological defects we observed occur early in the development of these organs, and because the lethality and sex reversal phenotypes were sensitive to genetic background in a similar way, we hypothesize that the initial defect occurs during the adrenogonadal progenitor stage and that one or more tda gene(s) plays a role in the differentiation of this progenitor into the adrenal cortex and gonads. While it has been known that Wt1, Sf1 and Wnt4 are necessary for adrenal cortex differentiation [14–17,34,35,48], our results show that Wt1 and Sf1 interact genetically during adrenal development. These results are consistent with results reported for fetuses that are double heterozygous for Sf1 and Cited2, a transcription cofactor that interacts with Wt1 [49]. Given that Wt1 is thought to directly activate Sf1 transcription in the gonadal progenitor, our results suggest that this genetic pathway is important for the differentiation of the adrenogonadal progenitor. It will be interesting to determine how Wnt4 fits into this genetic pathway in the adrenogonadal progenitor. Wt1 and Sf1 regulate Sry expression, but reduced Sry transcript levels are not solely responsible for sex reversal in Wt1+/2 or Sf1+/2 B6 XY mice Consistent with these indications of an early defect in testis differentiation, we found that Sry expression was markedly reduced in heterozygous mutant gonads. Our unpublished data indicate that reduced expression results from both a direct effect on Sry expression and an indirect effect on the differentiation of pre-Sertoli cells (manuscript in preparation). Interestingly, increasing Sry dosage by replacing the YAKR Chr in Wt1+/2 or Sf1+/2 B6 XYAKR fetuses with the YAKR,Sxr Chr dramatically increased the amount of testis tissue that developed but cord differentiation remained somewhat abnormal. Our previous experiments showed that the SryAKR and SrySxr alleles are expressed with similar profiles and at similar levels in gonads from B6 XYAKR,Sxr fetuses [29], suggesting that Sry expression is essentially doubled in these gonads. Thus, testicular cord differentiation was compromised in Wt1 and Sf1 heterozygotes, regardless of Sry dosage. These results suggest that reduced Sry transcript levels are not the sole cause of sex reversal in Wt1+/2 and Sf1+/2 B6 XYAKR mice and that the expression of critical genes other than Sry is likely to be affected. We consistently found that the Sf1 heterozygous phenotype was more severe than the Wt1 heterozygous phenotype. However, the reduction in Sry expression was not statistically different between the two heterozygotes, which supports our hypothesis that genes and cellular processes other than those controlled by SRY contribute to the phenotypic differences. Our result agree with previous reports suggesting that Sry expression levels alone do not predict whether a given Y Chr causes testis differentiation defects on the B6 genetic background [50] and that sex reversal is caused by both the presence of particular SRY protein isoforms found in M. domesticus mice and reduced Sry expression [29]. Analysis of Wt1 and Sf1 heterozygous gonads identifies potential downstream targets The analysis of marker gene expression using WISH provided insights into potential Wt1 and Sf1 target genes. For example, Wnt4 expression was reduced in Wt1+/2 and Sf1+/2 B6 XX ovaries compared to +/+ B6 XX ovaries indicating that Wt1 and Sf1 might regulate Wnt4 expression in ovaries (Figure 2). In Table 5. Increasing Sry dosage partially rescues the sex reversal phenotypes of E13.5 B6 XYAKR, Wt1+/2 B6 XYAKR, and Sf1+/2 B6XYAKR gonads. Genotype examined B6 Wt1+/2 XYAKR B6 Wt1+/2 XYAKR,Sxr B6 Sf1+/2 XYAKR B6 Sf1+/2 XYAKR,Sxr B6 +/+ XYAKR,Sxr doi:10.1371/journal.pgen.1002569.t005 Ovaries 3 (25%) 0 (0%) 21 (95%) 0 (0%) 0 (0%) Ovotestes Abnormal testes Total gonads 7 (58%) 1 (13%) 1 (5%) 5 (63%) 0 (0%) 2 (17%) 7 (88%) 0 (0%) 3 (38%) 0 (0%) Testes 0 (0%) 0 (0%) 0 (0%) 0 (0%) 17 (100%) 12 8 22 8 17 PLoS Genetics | www.plosgenetics.org 13 April 2012 | Volume 8 | Issue 4 | e1002569