Modeling Dosage-Sensitive Sex Reversal in Mice Figure 7. Real-time qRT–PCR analysis of ovarian marker gene expression in B6 versus D2 E12.5 XX gonads. Expression of ovary-specific genes Wnt4, Adamts19, Fst, Ifgbp2, 1700106J16Rik, Irx3, Bmp2, and Rspo1 were measured in the same samples and normalized to Hprt (D2, n = 6 gonad/mesonephros complexes; B6, n = 10 gonad/mesonephros complexes). Fold change relative to mean D2 values was determined using the 22DDCt method and plotted as the average per sample group +/2 standard error. An asterisk denotes statistically significant differences in pairwise comparisons (a = 0.05). The expression levels of Wnt4 (t14 = 1.938, p,0.0365), Adamts19 (t14 = 2.13, p,0.0257), Fst (t14 = 4.865, p,0.0001), Ifgbp2 (t14 = 4.987, p,0.0001), and 1700106J16Rik (t14 = 5.002, p,0.0001) were significantly higher in B6 vs. D2 XX ovaries. Expression of Irx3 and Bmp2 did not significantly differ between the two strains, and Rspo1 expression was significantly lower in B6 vs. D2 XX ovaries (t14 = 4.612, p,0.0002). doi:10.1371/journal.pgen.1002569.g007 Why are the Wt1 and Sf1 B6 alleles haploinsufficient? To determine why the Wt1B6 and Sf1B6 alleles are haploinsuffi- cient and function as hypomorphs, we compared the coding sequences and expression levels of the B6 and D2 alleles. The results indicated that the Wt1B6 and Wt1D2 allele encode the same protein and that the 6KTS Wt1 isoform expression ratio was the same in B6 and D2 gonad/mesonephros complexes. However, we found that Wt1 expression throughout the initial stage of testis differentiation was lower in complexes from B6 fetuses, suggesting that in the B6 genetic background the Wt1B6 allele is an expression hypomorph. In contrast, we were unable to detect significantly lower Sf1 expression in complexes from B6 fetuses but we did identify one coding polymorphism in Sf1, suggesting that the Sf1B6 allele is haploinsufficient not because its expression is reduced but possibly because the SF1B6 protein variant has reduced function. It appears that two SF1 variants are present among the common inbred mouse strains: SF1A172 (present in B6) and SF1S172 (present in D2). However, we found that homozygosity for the Sf1A172 allele in F1 hybrids was not sufficient to cause sex reversal in the presence of the YPOS Chr [as in (NZB/BlNJ6B6 XYPOS) F1s] and that trans-heterozygosity for the Sf1A172 and Sf1S172 alleles was not sufficient to prevent sex reversal in the presence of the YPOS Chr [as in (BALB/cBy6B6 XYPOS) F1s]. These data show that the SF1A172 polymorphism alone does not account for sex reversal in the presence of the YPOS Chr. Therefore, if the SF1A172 protein variant does indeed have reduced function during testis determi- nation, then homozygosity for B6 alleles at other loci likely is necessary to cause Sf1B6 to function as a hypomorph. Frigeri and colleagues [41] demonstrated that the SF1A172 and SF1S172 protein variants did not consistently differ in their ability to transactivate reporter genes containing tandem SF1 binding sites, in vitro. However, further experiments are needed to determine if the SF1A172 protein variant has reduced function during gonad differentiation in vivo. The Wnt4 B6 allele is dosage-sensitive in the B6 genetic background and may contribute to hyperactivation of the ovary differentiation pathway Our genetic results suggest that the Wnt4B6 allele functions as a hypermorph in the ovary determination pathway. To ascertain if the ovary determination pathway was hyperactive in B6 vs. D2 gonads we quantified the expression of eight ovary-biased or ovary-specific genes in E12.5 B6 vs. D2 XX ovaries. We chose this developmental stage because existing data suggested that the ovary pathway would be active and that the chosen markers would be expressed at detectable levels. We chose to analyze XX and not XY gonads because an increase in ovary gene expression in XY gonads could be caused by failure to activate the testis pathway and subsequent failure to suppress the ovary pathway. We found that the expression of Wnt4, and four other genes was higher in B6 ovaries, indicating that the ovary determination pathway is activated earlier and/or to a greater degree in B6 vs. D2 ovaries. These results are consistent with those reported in an eQTL study of B6 vs. 129 gonads [53]. The B6 vs. D2 expression differences do not appear to be caused by gross differences in development between these two strains because we did not detect differences in PLoS Genetics | www.plosgenetics.org 16 April 2012 | Volume 8 | Issue 4 | e1002569