Author Summary It has been proposed that mice do not adequately model human disorders of sex development because testis determination is gene dosage-sensitive in humans, but initial studies suggested it is gene dosage-insensitive in mice. For example, XY humans with reduced functional WT1 or SF1 gene-dosage or increased WNT4 gene-dosage can be sex reversed, whereas the equivalent XY mice were not sex-reversed on the genetic backgrounds previously reported. However, because testis determination in C57BL/ 6J mice is very sensitive to disruption, we tested the hypothesis that these genes are dosage-sensitive in C57BL/6J XY mice. We found that C57BL/6J-YAKR mice with reduced Wt1 or Sf1 gene-dosage were sex-reversed, whereas decreased Wnt4 gene-dosage partially rescued testis development in four genetic systems where C57BL/ 6J XY mice develop ovarian tissue. Our results demonstrate that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, that genetic background affects this sensitivity (as we suspect is true in humans), and that the mouse models described here are good models for human dosage-sensitive sex reversal. The results from these and other experiments lead to the hypothesis that the Wt1 and/or Sf1 alleles found in C57BL/6J mice are relatively ‘‘weak’’ testis- determining genes and their Wnt4 allele is a hyperactive ovary-determining gene. transferred to B6. For example, testis differentiation in B6 XY mice containing the BUB/BnJ Y Chr appears to be normal whereas testis differentiation in B6 XY mice containing the AKR/ J Y Chr is transiently delayed [28]. B6 XYAKR mice do not develop permanent ovaries or ovotestes like B6 XYPOS mice. However, if B6 XYAKR mice are heterozygous for either the TOrl or Thp deletion on Chr 17 they develop bilateral ovaries. In contrast, B6 XYB6 or B6 XYBUB mice heterozygous for the TOrl deletion develop testes. Thus, the B6-YAKR consomic strain is more sensitive to XY sex reversal than the B6 strain. We previously showed that the transient delay in testicular cord differentiation is caused by the presence of particular SRY protein isoforms found in M. domesticus mice and that the reduced expression of some of these variants, such as the SryPOS allele, causes permanent sex reversal [29]. Further investigations revealed that B6-YPOS sex reversal occurs because the Sry testis-determining gene present on the YPOS Chr does not properly induce testis development if specific interacting genes, designated tda (testis determining autosomal) genes, are homozygous for B6 alleles. Two tda loci that differ between the B6 and D2 genomes and play a role in B6-YPOS sex reversal were mapped to Chrs 2 (tda2) and 4 (tda1) [24]. Intriguingly, the Wt1 (Wilms’ tumor 1) and Sf1 (steroidogenic factor 1, officially Nr5a1; nuclear receptor subfamily 5, group A, member 1, also known as Ad4BP, adrenal 4-binding protein) map to the tda2 chromosomal region and Wnt4 (wingless-related MMTV integration site 4) to the tda1 region. Thus, we investigated Wt1 and Sf1 as candidates for tda2, and Wnt4 as a candidate for tda1. The Wt1 and Sf1 genes share a number of characteristics in addition to those noted above, some of which result from the fact that Wt1 is a direct activator of Sf1 expression [30] including: 1) both encode zinc finger transcription factors [31– 33]; 2) both are expressed in XX and XY mouse urogenital ridges from about embryonic day (E) 9.5 until E12.5, when expression becomes sexually dimorphic and cell-type restricted; and 3) the gonad progenitor (i.e., genital ridge) is initially present in fetal mice that are homozygous for a allele of Modeling Dosage-Sensitive Sex Reversal in Mice either gene, but differentiation of the genital ridge does not progress and it regresses via apoptosis [14–17]. The Wnt4 gene encodes a member of the WNT family of secreted signaling molecules that regulate cell-cell interactions during develop- ment. Wnt4 is initially expressed in XX and XY genital ridges; thereafter expression is down-regulated in XY gonads but maintained in XX gonads [34]. Homozygosity for a Wnt4 mutation causes partial sex reversal of XX gonads resulting in the development of a testis-like coelomic vessel and the presence of cells expressing steroidogenic enzymes [34,35]. In contrast, the gonadal phenotype in homozygous Wnt4 XY mice is relatively less severe; the gonads develop as testes, but the cords are initially fewer in number and disorganized [36]. To our knowledge, Wnt4 heterozygotes have no reported gonad differentiation defects. In XY mice, transgenic Wnt4 over- expression also causes testis differentiation defects but not sex reversal [13,35]. To examine the possibility that the Wt1, Sf1, and Wnt4 genes would be haploinsufficient on the B6 genetic background, we transferred alleles to the B6 and D2 backgrounds and determined if reduced dosage influenced gonadal development in XYB6 and XYAKR mice. Gonad morphology and marker gene expression in mutant versus normal fetal mice was analyzed using immunohistochemistry and confocal microscopy, RNA in situ hybridization, and quantitative RT-PCR. The results of these experiments indicated that testis differentiation was compromised at a very early stage and that Sry expression was significantly reduced in Wt1+/2 or Sf1+/2 B6 XYAKR but not in Wt1+/2 or Sf1+/2 (D26B6)F1 XYAKR fetal gonads. We also found that testis development was mostly rescued in Wt1+/2 and Sf1+/2 B6 XYAKR fetuses that contained two Sry alleles. These results are consistent with the hypothesis that the Wt1B6 and Sf1B6 alleles behave as hypomorphic alleles of testis-determining pathway genes in B6 XY mice if other genes in the genome are homozygous for the B6 allele. In contrast, reduced Wnt4 dosage did not exacerbate the transient delay in testis differentiation in B6 XYAKR gonads but rather appeared to ameliorate the phenotype, which suggests that the B6-derived Wnt4 allele (Wnt4B6) is a hypermorphic allele of a gene in the ovary determination pathway. We also found that B6 Wt1+/2 XYAKR, B6 Sf1+/2 XYAKR, and B6 XYPOS fetuses with reduced Wnt4 dosage developed more testicular tissue than Wnt4+/+ fetuses. These data are consistent with the hypothesis that Wnt4B6 is an early functioning allele of an ovary-determining gene that antagonizes testis differentiation in XY mice. Expression of Wnt4 and four other ovary-biased or ovary-specific genes was found to be elevated in E12.5 B6 vs. D2 XX ovaries. Together, our results suggest that B6 mice are sensitive to XY sex reversal compared to D2 mice because genes in the ovary determination pathway function earlier in the B6 genome than they do in the D2 genome. The results reported here show that the Wt1, Sf1, and Wnt4 genes are dosage-sensitive during mouse primary sex determina- tion, as they are in humans, and that this effect is modulated by genetic background in the mouse, as is probably the case in humans. We propose that the dosage-sensitive C57BL/6 XY mouse strains described here are powerful models for further investigation of human dosage-sensitive XY sex reversals. In addition, our results support the candidacy of Wt1, Sf1, and Wnt4 as tda genes, and suggest the testable hypothesis that B6 XYPOS sex reversal is cause by the additive effects of: 1) hypomorphic Wt1B6 and Sf1B6 alleles, and reduced activation of the testis differentiation pathway, 2) a hypermorphic Wnt4B6 allele and hyperactivation of the ovary differentiation pathway, and 3) reduced function and expression of the SryPOS allele. PLoS Genetics | www.plosgenetics.org 2 April 2012 | Volume 8 | Issue 4 | e1002569