Modeling Dosage-Sensitive Sex Reversal in Mice Figure 3. Histological and WIHC analysis of Wt1+/2; Sf1+/2 B6 XYB6 fetuses. (A and B, 106magnification) Hematoxylin and eosin staining of E14.5–15.5 fetal sagittal sections showed the absence of adrenal glands in Wt1+/2; Sf1+/2 B6 XYB6 and XX (data not shown) fetuses. The inset panels in A and B are a magnified view showing that normal testicular cords were present in both Wt1+/+; Sf1+/+ and Wt1+/2; Sf1+/2 B6 XYB6 fetuses (white dotted line outlines a section of a single cord). (C–J) WIHC analysis of E13.5 B6 XYB6 gonad/mesonephros complexes stained with SOX9 (Sertoli cells, green) and PECAM (germ cells and vascular endothelial cells, red). Panels C, E, G and I show 106 images taken with 0.76 zoom. Panels D, F, H, and J show 206 images. The gonads in all wild-type and single heterozygous E13.5 B6 XYB6 gonads were classified as testes, but testis cords in Wt1+/2; Sf1+/+ (E and F) and Wt1+/+; Sf1+/2 (G and H), B6 XYB6 gonads were less developed and more disorganized than cords in Wt1+/+; Sf1+/+ (C and D) B6 XYB6 gonads. B6 Wt1+/2; Sf1+/+ gonads, however, were more similar to wild-type gonads than were Wt1+/+; Sf1+/2 gonads. Testis differentiation was most affected in Wt1+/2; Sf1+/2 B6 XYB6 gonads and these were classified as ovotestes: Sertoli cells in the anterior and posterior poles often expressed SOX9 but were not organized into cords (I and J). doi:10.1371/journal.pgen.1002569.g003 ascertainment of the fetal genotype and was done by a single investigator (SMC) for consistency. A subset of complexes of each genotype was processed for WIHC and examined by two investigators (SMC and KHA) to confirm the initial classification (Figure 6). In all four experimental groups, reducing Wnt4 dosage increased the area containing testicular cords and reduced the area without obvious structure in XY gonads. In B6 XYPOS fetuses, 5 of the 12 gonads analyzed from Wnt4+/+ fetuses were classified as ovaries and none were classified as testes, whereas 7 of the 28 gonads analyzed from Wnt4+/2 fetuses were classified as ovaries and 4 were classified as testes. These data indicate that reducing Wnt4 dosage in B6 XYPOS fetuses decreased the percentage of ovaries from 42% to 25% and increased the percentage of testes from 0% to 14%. In B6 XYAKR fetuses, 12 of the 16 gonads analyzed from Wnt4+/+ fetuses were classified as ovotestes and 4 were classified as testes, whereas 6 of the 18 gonads analyzed from Wnt4+/2fetuses were classified as ovotestes and 12 were classified as testes. Thus, reducing Wnt4 dosage in B6 XYAKR mice decreased the percentage of ovotestes from 75% to 33% and increased the percentage of testes from 25% to 67%. In B6 Wt1+/2 XYAKR fetuses, 3 of the 12 gonads analyzed from Wnt4+/+ fetuses were classified as ovaries, 5 as ovotestes, and 4 as testes, whereas none of the 6 gonads analyzed in Wnt4+/2 fetuses were classified as ovaries, 1 was an ovotestis and 5 were testes. Thus, reducing Wnt4 dosage in Wt1+/2 B6 XYAKR mice decreased the percentage of ovaries from 25% to 0%, decreased the percentage of ovotestes from 42% to 17%, and increased the percentage of testes from 33% to 83%. In Sf1+/2 B6 XYAKR fetuses, all 8 gonads analyzed from Wnt4+/+ fetuses were classified as ovaries, whereas 2 of the 12 gonads analyzed from Wnt4+/2 fetuses were classified as ovaries, 3 as ovotestes, and 7 as testes. Thus reducing Wnt4 dosage in B6 Sf1+/2 XYAKR mice decreased the percentage of ovaries from 100% to 17%, increased the percentage of ovotestes from 0% to 25%, and increased the percentage of testes from 0% to 58%. The WIHC analyses confirmed these results (Figure 6). These data indicate that Wnt4B6 acts like an anti-testis gene. Using qRT-PCR we found that Sry expression in Wnt4+/+ vs. Wnt4+/2 B6 XYAKR was not significantly different during peak Sry expression in E11.75 (21–23 ts) gonad/mesonephros complex- es (data not shown), suggesting that Wnt4B6 compromises the testis pathway downstream of Sry [36]. Together, these data suggest that Wnt4B6 behaves as a hypermorphic allele of a gene in the ovary determination pathway, rather than a hypomorphic allele of a gene in the testis determination pathway. Elevated expression of ovary-biased genes in B6 versus D2 XX ovaries The Wnt4 results presented above suggested the possibility that the ovary differentiation pathway is prematurely- or hyper- activated in B6 gonads, thereby suppressing the testis determina- tion pathway. As with Wt1 and Sf1, the B6 and D2 Wnt4 alleles could encode different protein variants or have different expression patterns. We determined the DNA sequence of the Wnt4 ORF present in the D2 genome and found that it did not differ from that in B6, which was confirmed by data present in dbSNP Build 128 (data not shown). We used qRT-PCR to compare the expression of Wnt4 and seven other genes in the ovary differentiation pathway in E12.5 B6 vs. D2 XX ovaries to test the hypothesis that Wnt4B6 is an expression hypermorph and to investigate the possibility that the ovary differentiation pathway is prematurely- or hyper-activated in B6 gonads (Figure 7). The seven genes were chosen because their expression normally is ovary-biased at E12.5 (i.e., their expression is either ovary-specific or higher in ovaries vs. testes) [34,38,44–46]. We found that the expression of Wnt4 (p,0.0004), Adamts19 (p,0.026), Fst (p,0.0001), Igfbp2 (p,0.0001), and 1700106J16Rik (p,0.0001) was significantly higher in B6 vs. D2 ovaries; that expression of Irx3 (p,0.177) and Bmp2 (p,0.323) was not significantly different; and that expression of Rspo1 (p,0.0002) was significantly lower. These results demonstrate that Wnt4 and other genes known, or thought to be, in the ovary differentiation pathway are hyperactivated in E12.5 B6 vs. D2 XX gonads. Discussion It has been suggested that some genetic events leading to testis determination differ between humans and mice because several genes that are haploinsufficient in humans appeared to be haplosufficient in mice [18]. However, based on our studies of B6-YPOS sex reversal we hypothesized that genetic background would have a significant influence and that some of these genes would be haploinsufficient on the B6 genetic background. We chose to analyze the Wt1, Sf1, and Wnt4 genes for two main reasons: 1) they are dosage-sensitive in humans but initial studies suggested they were dosage-insensitive in mice, and 2) they are good candidates for tda genes based on genetic location and because gonad differentiation is abnormal in homozygous knockout mice. Our analyses show that these three genes can be dosage-sensitive in mice, depending on the genetic background, and provide insights into the mechanisms of B6 XY sex reversal. The Wt1 and Sf1 genes are dosage-sensitive on the B6 genetic background Our results show that the Wt1 and Sf1 B6 alleles are haploinsufficient for normal testis differentiation when present on the B6 genetic background. We found that Wt1+/2 and Sf1+/2 B6 XYB6 mice developed as males; however, testis differentiation was transiently delayed and was further compromised in double heterozygous Wt1+/2; Sf1+/2 B6 XYB6 fetuses. These data are consistent with evidence that Wt1 activates Sf1 transcription [30]. When the genetic background was further sensitized by substituting the YB6 Chr with the YAKR Chr, testis differentiation was more severely affected and Wt1+/2 and Sf1+/2 B6 XYAKR mice developed ovotestes and ovaries. In contrast, all Wt1+/2 and Sf1+/2 (D26B6)F1 XYAKR mice developed testes. Taken together, these results show that the Wt1 and Sf1 B6 alleles are hypomorphic PLoS Genetics | www.plosgenetics.org 11 April 2012 | Volume 8 | Issue 4 | e1002569