Modeling Dosage-Sensitive Sex Reversal in Mice Table 2. Gonad phenotype in E14.5–16 Wt1+/2 and Sf1+/2 XY fetuses. Maternal genotype B6 +/+ B6 Wt1+/2 B6 Wt1+/2 D2 Wt1+/2 B6 +/+ B6 Sf1+/2 D2 Sf1+/2 Paternal genotype B6 Wt1+/2 XYB6 B6 +/+ XYAKR D2 +/+ XYAKR B6 +/+ XYAKR B6 Sf1+/2 XYB6 B6 +/+ XYAKR B6 +/+ XYAKR E14.5–16 XY gonads +/2 Ovaries 0 (0%) 5b (10%) 0 (0%) 0 (0%) 0 (0%) 31 (91%) 0 (0%) +/2 Ovotestes 0 (0%) 23 (48%) 0 (0%) 0 (0%) 0 (0%) 3 (9%) 0 (0%) +/2 Testes 28 (100%) 8 (17%) 24 (100%) 12 (100%) 20 (100%) 0 (0%) 14 (100%) +/2 Abnormal testesa 0 (0%) 12 (25%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Total +/2 XY gonads 28 48 24 12 20 34 14 aCord development is somewhat delayed at the anterior and/or posterior ends but the gonad is not an obvious ovotestis. bTwo were abnormally shaped but no testicular cords were evident. doi:10.1371/journal.pgen.1002569.t002 contained cords and strongly expressed AMH, while the adjacent posterior region lacked cords and expressed FOXL2. Figure 2J shows an ovotestis that expressed Ptch1 in the central region that contained testicular cords, but not in the ovarian regions at the anterior and posterior poles. [Ptch1 expression in ovotestes was not as strong as that observed in B6 XYAKR control gonads (compare Figure 2H and 2J)]. Conversely, Figure 2D shows an ovotestis expressing Wnt4 in the anterior and posterior regions, but not in the central testicular region. Occasionally, testis- and ovary- specific markers were expressed in the same region (Figure 1E), where FOXL2 expression was detected in specific cells within a region containing well-formed testicular cords and strongly expressing the Sertoli cell marker AMH. We noted a few cells that appeared to express both AMH and FOXL2 (Figure S2A). However, because only whole-mount tissue was examined and because AMH is localized to the cell surface and FOXL2 to the nucleus, we were not able to exclude the possibility that the expression was in adjacent cells. Nevertheless, it is possible that some supporting cell progenitors can express both granulosa and Sertoli cell markers under certain conditions. Because Wt1 is functionally upstream of Sf1 and is necessary for the initiation of Sf1 expression [30], we examined SF1 expression in Wt1+/2 B6 XYAKR gonads using WIHC and found that expression levels appeared normal (Figure 1N–1O). Additionally, quantitative real-time RT-PCR (qRT-PCR) analysis did not detect a significant difference in Sf1 expression in E12 gonads from Wt1+/2 B6 XYAKR vs. +/+ B6 XYAKR fetuses (data not shown). On the other hand, Wnt4 expression appeared to be reduced in Wt1+/2 and Sf1+/2 B6 XX ovaries compared to +/+ B6 XX ovaries, and Ptch1 expression was strongly reduced in Wt1+/2 B6 XX and B6 XY mesonephroi (Figure 2). Overall, the morpholog- ical and marker gene expression analyses showed that the testis determination pathway is aberrant at an early stage, that the ovarian pathway is activated in Wt1+/2 and Sf1+/2 B6 XYAKR gonads, and that gene expression is affected in both XX and XY heterozygous gonad/mesonephros complexes. A multigene qRT-PCR strategy was used to quantify gene expression changes in E12 +/2 vs. +/+ B6 XYAKR gonads, and E14 +/2 ovaries vs. +/+ B6 XYAKR testes (Table 3). These quantitative results were consistent with the morphological and marker gene analyses. For example, Adamts19, an ovary-specific gene, was significantly up-regulated in E14 Wt1+/2 and Sf1+/2 B6 XYAKR ovaries vs. +/+ B6 XYAKR control testes, and Cyp11a1 and Cyp17a1, both Leydig cell specific genes, were expressed at significantly lower levels in Wt1+/2 and Sf1+/2 B6 XYAKR ovaries. Ovary-specific markers are expressed at the anterior and posterior of B6 XYAKR gonads During analysis of marker gene expression in E13.5 Wt1+/2 and Sf1+/2 B6 XYAKR gonads using WIHC and WISH we observed the expression of ovary-specific somatic cell markers in gonads from +/+ B6 XYAKR fetuses. As expected, testis-specific markers, such as SOX9, AMH and Ptch1, were expressed in the central regions that contained testicular cords (Figure 1, Figure 2, and Figure S2). However, FOXL2, Wnt4, and Fst often were expressed in the anterior and posterior regions that lacked cords (Figure 1, Figure 2, Figure S2, and data not shown), indicating that the ovary differentiation pathway was activated in these regions. WISH analysis of the meiotic germ cell markers Stra8 and Rec8 [37] and the ovary-specific somatic cell marker Irx3 [38] was used to determine if activation of the ovary differentiation pathway extended to the germ cells and to quantify the occurrence of ovary-specific marker gene expression (Figure S2B). We found that 11 of 13 B6 XYAKR gonads clearly expressed either Stra8 or Rec8 at one or both poles and that 7 of 7 gonads expressed Irx3 at both poles. In the 9 gonads where Stra8 or Rec8 were expressed at both poles, expression was higher at the anterior pole than the posterior pole, suggesting that the anterior- to-posterior wave of meiotic germ cell gene expression is maintained despite the presence of intervening testicular tissue [37]. It is important to note that testicular cords are present throughout B6 XYAKR gonads after about E14.5. Therefore, these results are consistent with the idea that B6 XYAKR gonads develop as transient ovotestes [39,40]. Testis and adrenal gland development is abnormal in B6 XYB6 fetuses that are double-heterozygous for Wt1 and Sf1 Because the Wt1+/2 and Sf1+/2 B6 XYAKR sex reversal phenotypes appear to be very similar and because Wt1 is necessary for activation of Sf1 expression in fetal gonads [30], we examined if B6 XYB6 mice were sex reversed when double heterozygous for Wt1 and Sf1 (Wt1+/2; Sf1+/2). We first attempted to ascertain the external sexual phenotype of the double heterozygotes at weaning. However, no Wt1+/2; Sf1+/2 mice were recovered among the 84 progeny from B6 Wt1+/26B6 Sf1+/2 matings (Table 4). Because our previous results suggested that lethality PLoS Genetics | www.plosgenetics.org 4 April 2012 | Volume 8 | Issue 4 | e1002569