Modeling Dosage-Sensitive Sex Reversal in Mice Figure 1. WIHC analysis of morphology and marker expression in Wt1+/2 and Sf1+/2 B6 XYAKR E13.5 gonads. (A–L). Confocal images of gonad/mesonephros complexes with gonads shown above and mesonephroi below the white dotted line (206 magnification). Heterozygous XY gonads differentiated as ovotestes (C), ovaries (F, L, O), or gonads that were morphologically ovarian but expressed testicular markers (I). The Wt1+/2 XY ovotestis in (C) expressed AMH, a Sertoli cell marker, in the central region containing testicular cords but not at poles that lacked cords. FOXL2, a granulosa cell marker, was expressed at the poles. FOXL2 also was expressed in isolated cells within regions containing testicular cords in B6 XYAKR gonads (E). Heterozygous XY ovaries (F, L, O) expressed ovarian but not testicular markers. The Wt1+/2 XY ovary in (F) expressed FOXL2 but not AMH throughout. The Sf1+/2 XY ovary in (L) expressed FGFR2 on the surface of somatic cells (ovarian pattern) but did not have cells with nuclear localization of FGFR2 (testicular pattern, note localization in Sertoli cells (H, K)). The Wt1 +/2 XY gonad in (I) was ovarian morphologically but contained many cells with nuclear localization of FGFR2. These cells were not organized into testicular cords. PDGFRa was expressed at high levels in the coelomic epithelium of B6 XYAKR gonads (H, K) and at lower levels in interstitial cells of B6 XX (G, J) and XYAKR (H, K) gonads. Ovotestes, expressed PDGFRa at high levels in the coelomic epithelium of regions containing cords and at lower levels in regions without cords (data not shown). The heterozygous XY ovary in (L) expressed PDGFRa in a pattern similar to XX ovaries. The masculinized ovary in (I) expressed PDGFRa in a testis-like pattern. (M–O) Confocal images showing SF1 expression in gonads (406 magnification). At this stage, SF1 is expressed at fairly equal levels in all somatic cells in wild-type XX ovaries (M). In wild-type testes, expression is up-regulated in Sertoli cells (arrowheads) within testis cords (outlined with white dotted line) and in specific interstitial cells (double arrowheads), while it is down-regulated in most other interstitial cells (N). The expression of SF1 in Wt1+/2 XY ovaries (O) resembled that of wild-type XX ovaries as expression was uniform in all somatic cells. Note that overall SF1 expression levels were similar in normal XX (M) vs. Wt1+/2 XY (O) ovaries. doi:10.1371/journal.pgen.1002569.g001 could be sensitive to genetic background, progeny from B6 Wt1+/ 26D2 Sf1+/2 matings were analyzed. Among the 23 offspring were 4 Wt1+/2; Sf1+/2 mice, indicating that lethality was rescued in (B66D2)F1 hybrids. To determine at which develop- mental stage the lethality occurred, E14.5–15.5 fetuses from B6 Wt1+/26B6 Sf1+/2 matings were analyzed. Among the 12 offspring were 3 Wt1+/2; Sf1+/2 mice (2 XY and 1 XX), indicating that lethality occurred between E15.5 and weaning. Histological examination of the three Wt1+/2; Sf1+/2 fetuses showed that each had bilateral adrenal agenesis, which likely would cause neonatal lethality (Figure 3A and 3B). Both of the Wt1+/2; Sf1+/2 XY fetuses had bilateral testes that appeared morphologically normal, but were smaller than those in wild-type XY littermates. To extend these observations, gonad development was examined in E13.5 fetuses from B6 Wt1+/26B6 Sf1+/2 matings using WIHC (Figure 3C–3J). Of the 15 fetuses examined, 7 were Wt1+/2; Sf1+/2, which is consistent with the E14.5–15.5 survival data. Of the 9 XYB6 fetuses, 4 were Wt1+/2; Sf1+/2, 4 were Wt1+/+; Sf1+/2, and 1 was Wt1+/+; Sf1+/+. All 8 Wt1+/2; Sf1+/2 XY gonads were classified as ovotestes, with large central testicular regions (Figure 3I and 3J). (Ovotestes in E13.5 double heterozygous fetuses appear to resolve into testes at later stages because the gonads in E14.5– 15.5 double heterozygous fetuses do not contain ovarian tissue.) The 8 gonads from Wt1+/+; Sf1+/2 XY fetuses were classified as testes but their cords appeared to be less well developed and more disorganized than +/+ B6 XY gonads (Figure 3). None of the XY offspring from these intercrosses were Wt1+/2; Sf1+/+, so we examined gonad development in E13.5 Wt1+/2 B6 XYB6 fetuses generated from B6 Wt1+/26B6 +/+ XYB6 crosses. All 6 gonads from Wt1+/2 B6 XYB6 fetuses were classified as testes. However, their cords appeared to be less well-developed and more disorganized than normal B6 XY gonads: 4 displayed disorganized anterior and posterior poles and 2 had disorganized posterior poles (Figure 3E and 3F). In comparison, of the 6 gonads from +/+ B6 XYB6 fetuses, 2 had testicular cords throughout and 4 had disorganized posterior poles. In summary, the degree of testicular cord development in the E13.5 B6 XYB6 gonads could be ranked from most complete to least complete as: Sf1+/+; Wt1+/+.Sf1+/+; Wt1+/2.Sf1+/2; Wt1+/+.Sf1+/ 2; Wt1+/2. These results indicate that testis development is compromised in Wt1+/2 and Sf1+/2 B6 XY fetuses, even in the absence of the YAKR Chr, and that testis development is further compromised in mice that are double heterozygous for Wt1 and Sf1 alleles. Sry expression is reduced in Wt1+/2 and Sf1+/2 B6 XYAKR gonads and sex reversal is partially rescued by increased Sry dosage Because the morphological and marker gene expression analyses in E13.5 gonads suggested an early defect in the testis determination pathway, Sry expression in E11.5 Wt1+/2 and Sf1+/2 B6 XYAKR vs. +/+ B6 XYAKR gonad/mesonephros complexes was quantified. (Sry is not expressed in the mesoneph- ros.) During peak Sry expression (E11.75), transcript levels in both mutants were less than 50% of the controls. We found that Sry transcript levels were reduced in Wt1+/2 vs. +/+ samples at E11.25 (15–17 ts, t24 = 2.224 , p,0.0179), E11.5 (18–20 ts, t25 = 3.653, p,0.0012), and E11.75 (21–23 ts, t3 = 11.51, p,0.0014) but not significantly different at E12.0 (24–26 ts, Figure 4A). Sry transcript levels also were reduced in Sf1+/2 vs. +/ + samples at E11.5 (18–20 ts, t29 = 2.125, p,0.0422) and E11.75 (21–23 ts, t5 = 6.142, p,0.0016), but were not significantly different at E11.25 (15–17 ts) or E12.0 (24–26 ts, Figure 4A). These results suggest that sex reversal in Wt1+/2 and Sf1+/2 B6 XYAKR gonads is at least partially due to a failure to sufficiently up-regulate Sry expression and properly initiate the testis determination pathway. However, because pairwise comparisons between Wt1+/2 vs. Sf1+/2 samples did not reveal significant differences in Sry expression at any of the embryonic stages analyzed, differences in the level of Sry expression do not explain the differences in the severity of the Wt1+/2 and Sf1+/2 phenotypes. To test the hypothesis that sex reversal in Wt1+/2 and Sf1+/2 B6 XYAKR gonads is due, at least in part, to reduced Sry expression, gonad morphology was examined by WIHC in gonads from E13.5 fetuses that had increased Sry dosage due to the presence of the YAKR,Sxr Chr. [Classification was done independently by two investigators (SMC and KHA)]. This rearranged Y Chr carries two Sry alleles, one from the AKR/J inbred strain (M. domesticus) and one from the RIII inbred strain (M. musculus). Gonads in Wt1+/2 and Sf1+/2 B6 XYAKR,Sxr fetuses consistently developed more testicular tissue than gonads in Wt1+/2 and Sf1+/2 B6 XYAKR fetuses; however, it appeared that cord differentiation in the Wt1+/2 and Sf1+/2 B6 XYAKR,Sxr gonads was not completely normal (Figure 4B, Table 5). Of the 8 Wt1+/2 B6 XYAKR,Sxr gonads analyzed, 7 were classified as abnormal testes because the testicular cords appeared to be more disorganized and less well developed than those in +/+ B6 XYAKR,Sxr gonads. In fact, these cords appeared to be similar to the abnormal cords in Wt1+/2 B6 XYB6 testes. One of the Wt1+/2 B6 PLoS Genetics | www.plosgenetics.org 6 April 2012 | Volume 8 | Issue 4 | e1002569