limb morphology or in the size of the gonads or mesonephroi at the 27–28 ts stage used for these analyses (data not shown). However, not all eight genes were up-regulated in B6 ovaries: two were not significantly different, and one was down-regulated. We find it intriguing that Rspo1, which maps close to Wnt4 within the tda1 region, had reduced expression in B6 ovaries. However, because the Rspo1 and Wnt4 knockout phenotypes are very similar in that ovary and not testis differentiation is primarily affected, and because Wnt4 expression is greatly reduced in Rspo12/2 XX gonads [54,55], we think it is unlikely that Rspo1 is tda1 because we would expect that a strain with reduced Rspo1 expression like B6 would be less sensitive to XY sex reversal, whereas B6 is, in fact, more sensitive. We note that for Rspo1 no SNPs that differ between the B6 and D2 strains are present in dbSNP (Build 128), indicating that the RSPO1 protein does not differ between these strains. Furthermore, we suggest that the interaction between Rspo1 and Wnt4 may be indirect because Rspo1 expression in decreased in B6 mice while Wnt4 expression is increased. Nevertheless, these data do not formally exclude Rspo1 as a tda1 candidate. Are Wt1, Sf1, and Wnt4 tda genes? More than twenty-five years ago, two of the authors hypothesized that the B6-derived tda genes could function later than normal in the testis determination pathway and/or, because the testis and ovary determination pathways antagonize each other, could function earlier than normal in the ovary determi- nation pathway [25,56]. Thus, if a tda gene functions in the testis- determining pathway, then reducing its dosage in B6 XY mice should cause sex reversal, whereas reducing its dosage in D2 XY mice should not. Similarly, if a tda gene functions in the ovary- determining pathway and antagonizes testis differentiation, then reducing its dosage should ameliorate B6 XY sex reversal. The Wt1 and Sf1 genes fulfill the criteria as late functioning testis- determining genes and Wnt4 fulfills the criteria as an early function ovary-determining gene that antagonizes testis differentiation. Furthermore, our results show that B6 XYPOS, Wt1+/2 B6 XYAKR, and Sf1+/2 B6 XYAKR sex reversal share similar gonadal phenotypes and that these three sex reversals are sensitive to genetic background in a similar way. Additionally, we have recently determined that a major modifier of Sf1+/2 B6 XYAKR sex reversal that differs between the B6 and D2 genomes, maps to the distal region of Chr 4, which suggests that the tda1 modifier gene plays a prominent role in both Sf1+/2 B6 XYAKR and B6 XYPOS sex reversal (manuscript in preparation). However, while our data are consistent with the hypothesis that tda2 is comprised of Wt1 and Sf1 and that tda1 is Wnt4, the genetic intervals defined by the linkage analyses are fairly broad, and further experiments are needed to directly test this hypothesis and to exclude the possibility of other candidate genes in the tda1 and tda2 intervals. Undoubtedly, tda genes in addition to tda1 and tda2 contribute to B6 XYPOS sex reversal because, as shown in the original linkage analysis, some XYPOS fetuses that were homozygous for B6 alleles at the tda1 and tda2 loci contained normal appearing testes [24]. Nevertheless, it appears that tda1 and tda2 play an important role in many cases of B6 XY sex reversal because we have mapped a major modifier of Dax1 2/Y sex reversal that differs between the B6 and D2 genomes to the same chromosome region as tda1 [22], and have recently determined that major modifiers of Gata4 +/2 B6 XYAKR sex reversal [23] map to the same chromosome regions as tda1 and tda2 (manuscript in preparation). An additive model to explain B6 XY sex reversal Overall, our data suggest that the B6 inbred strain is sensitive to XY sex reversal, at least in part, due to hypomorphic alleles of Wt1 Modeling Dosage-Sensitive Sex Reversal in Mice and/or Sf1 that compromise the testis determination pathway, as well as a hypermorphic allele of Wnt4 that antagonizes testis development. We suggest that in XY gonads of most inbred strains, such as DBA/2J, the testis determination pathway reaches a critical threshold that is sufficient to inactivate the ovary determination pathway prior to the developmental stage when the ovary determination pathway reaches a threshold that is sufficient to inactivate the testis determination pathway. In contrast, in a few inbred strains such as B6, the testis determination pathway is compromised and the ovary determination pathway is hyperacti- vated, thus the interval between the developmental stages at which these two thresholds are reached is greatly reduced. We propose that this interval is even further reduced or eliminated in B6 mice carrying certain M. domesticus-derived Sry alleles that do not efficiently activate the testis determination pathway, thus allowing ovotestes to develop transiently in B6 XYAKR mice and allowing stable ovotestes and ovaries to develop in B6 XYPOS mice, for example. In summary, we propose that sex reversal in B6 XYPOS gonads is the result of the additive effects of 1) hypomorphic Wt1B6 and/or Sf1B6 alleles, 2) a hypermorphic Wnt4B6 allele and hyperactivation of the ovary pathway, 3) reduced function of M. domesticus-derived SRY proteins, 4) reduced expression of the SryPOS allele, and 5) the presence of other unidentified tda genes. Mouse models of human dosage-sensitive sex reversal These studies demonstrate that Wt1, Sf1, and Wnt4 are dosage- sensitive in mice, as they are in humans, and show that this effect is modulated by genetic background, as we suspect it is in humans. They also demonstrate that the Wt1+/2 and Sf1+/2 B6-YAKR mice are good models for the corresponding human XY sex reversals involving heterozygous mutations in these genes. We suggest that these heterozygous mice will be very useful for the elucidation of Wt1 and Sf1 functions during gonad differentiation because the heterozygous phenotype is far less severe than the homozygous phenotype, which is a complete abrogation of gonad differentiation and subsequent apoptosis at the genital ridge stage. Thus, we think it is likely that these strains will be particularly useful in providing further insights into the roles of Wt1 and Sf1 in organogenesis and human disease. Materials and Methods Animals and genotyping Null (knockout) alleles of Wt1 (Wt1tm1Jae) [14], Sf1 (Nr5a1tm1Klp) [16], and Wnt4 (Wnt4tm1Amc) [34] were transferred to the B6 and D2 inbred strain backgrounds using standard backcrossing. No sex ratio disturbances were noted in heterozygous progeny during construc- tion of these congenic strains. Experimental results reported here were conducted after backcross generation N10 or greater was achieved. The B6-YPOS, B6-YAKR, B6-YAKR,Sxr and D2-YAKR consomic strains were previously described [19,20,23,29,57]. This study was carried out in strict accordance with the recommenda- tions in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Jackson Laboratory and Boston University Laboratory Animal Science Center are AALAS accredited and all animal procedures were approved by The Jackson Laboratory Animal Care and Use Committee (protocols LAH:93-26, LAH:95-01, LAH:95-02) or the Boston University Animal Care and Use Committee (protocol AN-13935). The presence of a Wt1, Sf1, or Wnt4 knockout allele was determined by multiplex PCR using primers to detect the neomycin cassette and the myogenin gene as a control [23]. In crosses involving multiple knockout alleles, gene-specific PCR assays were used to amplify both the normal and the targeted PLoS Genetics | www.plosgenetics.org 17 April 2012 | Volume 8 | Issue 4 | e1002569