Results Wt1+/2 and Sf1+/2 B6 XYAKR mice are sex-reversed To examine the possibility that Wt1 and/or Sf1 would be haploinsufficient on the B6 genetic background and to investigate their candidacy as tda genes, we transferred alleles to the B6 and D2 backgrounds and determined if reduced dosage influenced gonadal development in XYB6 and XYAKR mice. We reasoned that if the B6 alleles of Wt1 and/or Sf1 function as hypomorphs in the testis determination pathway, then further reducing their dosage in B6 XY mice should cause sex reversal, whereas reducing their dosage in D2 XY mice should not. Sex reversal was not evident in Wt1+/2 or Sf1+/2 B6 or D2 XY weanling or adult mice, and the mice were fertile. To increase the sensitivity of the genetic background to sex reversal, we mated Wt1 +/2 and Sf1+/2 B6 XX females to B6 XYAKR males. Based on the external sexual phenotype of 39 Wt1+/2 B6 XYAKR mice examined, 20 presented as male, 11 as hermaphrodite, and 8 as female (Table 1). In contrast, of the 20 Sf1+/2 B6 XYAKR offspring, none presented as male, 4 as hermaphrodite, and 16 as female. We noted a deficiency in the expected number of B6 Sf1+/2 animals recovered, regardless of chromosomal sex. To test if these XY sex reversals were dependent on genetic background like B6-YPOS sex reversal, offspring were analyzed from mating Wt1+/2 B6 XX females to AKR/J XYAKR males and Sf1+/2 D2 XX females to B6 XYAKR males. (D2 Wt1+/2 mice were not available when these analyses were performed but similar results were obtained in the analysis of gonad morphology in E14.5–16 fetuses when mating Wt1+/2 D2 XX females to B6 XYAKR mice, Table 2.) In F1 hybrids, the heterozygous XY offspring presented as normal males and the number of Sf1+/2 (D26B6)F1 offspring recovered was Mendelian. We conclude that Wt1+/2 and Sf1+/2 B6 XYAKR mice are sex reversed, Sf1+/2 B6 mice have reduced viability, and both sex reversal and reduced viability are sensitive to genetic background. These results also indicate that the sex reversal phenotype is stronger in Sf1 mutants because more Sf1+/2 than Wt1+/2 B6 XYAKR mice presented as females and hermaphrodites. Gonad morphology in E14.5–16 fetuses was examined because assessment of external sexual phenotype does not distinguish between primary and secondary sex reversal, and thus is only an estimate of the true sex reversal frequency. At this developmental stage small amounts of ovarian tissue are visually apparent in ovotestes and the initial delay in testicular cord development has resolved in B6 XYAKR fetuses, thus preventing the misclassifica- tion of the gonad as an ovotestis. All Wt1+/2 B6 XYB6, Wt1+/2 (B66D2)F1 XYAKR and Wt1+/2 (D26B6)F1 XYAKR gonads analyzed were testes (Figure S1 and Table 2). In contrast, approximately half of the 48 Wt1+/2 B6 XYAKR gonads analyzed Modeling Dosage-Sensitive Sex Reversal in Mice were ovotestes (23) or ovaries (5), and half were normal (8) or abnormal testes (12). Similarly, all Sf1+/2 B6 XYB6 and Sf1+/2 (D26B6)F1 XYAKR gonads analyzed were testes, whereas the 33 Sf1+/2 B6 XYAKR gonads analyzed were ovotestes (3) or ovaries (31). These results are consistent with the weanling analysis and show that sex reversal is the result of primary (gonadal) sex reversal. Morphological and marker gene analysis of E13.5 Wt1+/2 and Sf1+/2 B6 XYAKR gonads To gain further insight into the mechanism of sex reversal in Wt1+/2 and Sf1+/2 B6 XYAKR gonads, whole-mount immu- nohistochemistry (WIHC, Figure 1) and whole-mount RNA in situ hybridization (WISH, Figure 2) were used to analyze morphology and marker gene expression in E13.5 gonads. Overall, the morphological analyses were consistent with the E14.5–16 analyses: Wt1+/2 B6 XYAKR gonads were classified as ovaries, ovotestes and testes and Sf1+/2 B6 XYAKR gonads were classified as ovaries and ovotestes. The Wt1+/2 and Sf1+/2 B6 XYAKR gonads that developed as ovaries or ovotestes were phenotypically indistinguishable regardless of which gene was mutant. However, some Wt1+/2 B6 XYAKR gonads appeared to be very similar in morphology and marker gene expression pattern to those observed in control +/+ B6 XYAKR gonads, whereas none of the Sf1+/2 B6 XYAKR gonads were phenotypically similar to +/+ B6 XYAKR gonads. Generally, Wt1+/2 and Sf1+/2 B6 XYAKR gonads that developed as ovaries were morphologically indistinguishable from normal XX ovaries and strongly expressed ovary-specific markers, such as FOXL2 (Figure 1F), Wnt4 (Figure 2F), Fst (data not shown) and Bmp2 (data not shown) in a pattern similar to XX ovaries. Additionally, XY ovaries generally did not express testis-specific Sertoli cell markers, such as AMH (Figure 1F) or SOX9 (data not shown), or Leydig cell markers, such as Ptch1 (Figure 2L), or markers such as GATA4, WT1 (data not shown) or FGFR2 (Figure 1L) in a testis-like pattern. However, about 20% of XY mutant gonads that had ovarian morphology contained cells that expressed Sertoli cell-specific markers, such as AMH or SOX9 (data not shown), or expressed markers, such as PDGFRa and FGFR2 in a testis-like pattern (Figure 1I). However, these cells were not organized in a pattern that resembled testicular cords. We conclude that Sertoli cell differentiation was initiated in some XY gonads that were morphologically ovarian, but was insufficient to initiate or sustain cord formation. In Wt1+/2 and Sf1+/2 B6 XYAKR ovotestes, testis markers generally were expressed in central regions containing cords whereas ovary markers were expressed at the anterior and posterior poles within regions lacking cords. For example, Figure 1C shows part of an ovotestis in which the central region Table 1. External sexual phenotype of Wt1+/2 and Sf1+/2 weaning age mice. Maternal genotype B6 Wt1+/2 B6 Wt1+/2 B6 Sf1+/2 D2 Sf1+/2 Paternal genotype B6 +/+ XYAKR AKR +/+ XYAKR B6 +/+ XYAKR B6 +/+ XYAKR aApproximately 30% of B6 Sf1+/2 pups die prior to weaning. doi:10.1371/journal.pgen.1002569.t001 XY pups +/2 Females 8 (21%) 0 (0%) 16 (80%) 0 (0%) +/2 Hermaphrodites 11 (28%) 0 (0%) 4 (20%) 0 (0%) +/2 Males 20 (51%) 20 (100%) 0 (0%) 13 (100%) Total +/2XY pups 39 20 20 13 Total XY pups 81 47 95a 18 PLoS Genetics | www.plosgenetics.org 3 April 2012 | Volume 8 | Issue 4 | e1002569