Table 4. Survival of Wt1+/2; Sf1+/2 offspring. Stage Weaning E14.5–15.5 Maternal genotype B6 Wt1+/2 B6 Wt1+/2 B6 Wt1+/2 Paternal Genotype B6 Sf1+/2 D2 Sf1+/2 B6 Sf1+/2 Offspring genotypes Sf1+/+; Wt1+/+ 43 3 6 Sf1+/+; Wt1+/2 34 4 3 Modeling Dosage-Sensitive Sex Reversal in Mice Sf1+/2; Wt1+/+ 7 2 10 Sf1+/2; Wt1+/2 0 3 4 Total 84 12 23 doi:10.1371/journal.pgen.1002569.t004 patterns. A PCR primer-walking strategy using cDNA and genomic DNA as template was employed to determine the DNA sequence of the Wt1 and Sf1 genes present in the D2 genome and these sequences were compared to the mouse B6 reference sequence (data not shown). For Wt1, the ORF, 59 and 39 UTRs, and about 1 kb of the upstream flanking region were sequenced. One synonymous difference was identified in the ORF: a T (B6) vs. C (D2) substitution at the third position of codon 339, which was confirmed by its presence in dbSNP Build 128 (NCBI, rs27444886). There were no differences in the 59 UTR and upstream flanking region. Four differences were identified in the 39 UTR, two of which were confirmed by their presence in dbSNP (rs33129887 and rs13467742). Two single base pair differences in homopolymer length were not confirmed in dbSNP. The functional significance, if any, of the 39 UTR polymorphisms is not readily apparent. For Sf1, the ORF, 59 and 39 UTRs, and ,1.5 kb of the upstream and 500 bp of the downstream flanking regions were sequenced. One synonymous and one non-synonymous difference were identified in the ORF. The synonymous polymorphism was a T (B6) vs. C (D2) substitution at the third position of the third codon. This polymorphism is represented in dbSNP (rs3142930) for certain inbred strains, but not for DBA/2J. The non- synonymous polymorphism was a G (B6) vs. T (D2) substitution in the first position of codon 172 resulting in an alanine (B6) vs. serine (D2) amino acid substitution and was confirmed by its presence in dbSNP (rs3142929). This polymorphism was inde- pendently discovered by Frigeri and colleagues who first identified it in ACTH resistant derivatives of the mouse Y1 adrenocortical tumor cell line [41]. There were no differences in the 59 and 39 UTRs. The upstream and downstream regions each had one difference, both in CA repeat units: The upstream CAAA repeat had 6 units in B6 vs. 5 units in D2, while the downstream CA repeat had 17 units in B6 vs. 22 units in D2. Neither polymorphism is present in dbSNP and their functional signifi- cance is not readily apparent. To gain insight into whether the presence of the Sf1A172 (B6) vs. Sf1S172 (D2) allele was correlated with B6 XY sex reversal, and thus of potential functional significance, we took advantage of the fact that when females from different inbred strains are mated to B6 XYPOS hermaphrodites the F1 XY offspring are either sex reversed or normal, and thus the inbred strains can be classified as B6-like or D2-like, respectively (Table 6). Using this assay we identified two strains (C57BL/10J and NZB/BlNJ) that are B6-like and five strains (129S1/SvImJ, C3H/HeSnJ, SM/J C58/J and BALB/cBy) that are D2-like. A PCR-RFLP genotyping assay that was developed by Frigeri and colleagues [41] was used to determine the strain distribution pattern of the Sf1A172 and Sf1S172 alleles among these seven strains. As shown in Table 6, two strains were phenotypically and genotypically discordant. NZB/ BlNJ was B6-like phenotypically but it has an Sf1S172 allele, and BALB/cBy was D2-like phenotypically but it has an Sf1A172 allele. We thus conclude that the presence of an Sf1A172 vs. Sf1S172 allele alone is not strictly correlated with B6 XY sex reversal. The Wt1 gene encodes multiple splice isoforms. Of particular interest here, alternative splicing of exon nine gives rise to two protein isoforms that differ by three amino acids and are referred to as the +KTS and 2KTS isoforms. Both isoforms and their correct relative expression are necessary for normal gonad development [42]. For example, Frasier syndrome in humans is associated with XY sex reversal and caused by reduced expression of the Wt1 +KTS isoform [43]. Thus, it was possible that B6 XYAKR Wt1+/2 sex reversal could be caused by altered +KTS/ 2KTS isoform ratios and qRT-PCR using primers that span the exon nine splice junction was employed to examine if the ratio of +KTS/2KTS splice isoforms is different in E10.5–14.5 B6 vs. D2 XX and XY gonad/mesonephros complexes. We found that the relative expression of the Wt1 +KTS and 2KTS isoforms did not differ between B6 and D2 gonad/mesonephros complexes (data not shown). To test the hypothesis that the Wt1B6 and Sf1B6 alleles function as hypomorphs because they are expressed at lower levels or with different temporal profiles than the Wt1D2 and Sf1D2 alleles, respectively, transcript levels in B6 vs. D2 XY gonad/mesoneph- ros complexes from E10.5–13.5 fetuses were quantified using qRT-PCR (Figure 5). Wt1 expression was significantly lower in B6 vs. D2 samples at all stages. Sf1 expression also was lower in B6 vs. D2 gonad/mesonephros complexes from E11.0–12.0 fetuses, but these differences were not statistically significant. These Wt1 and Sf1 expression results were corroborated in XX samples from similar stages, and the Sf1 expression results were further confirmed by using an independent SYBR green qRT-PCR assay to analyze a subset of the samples (data not shown). In summary, these molecular analyses suggest that Wt1B6 is a hypomorph due to reduced expression and that Sf1B6 is a hypomorph possibly due to altered protein structure and/or the presence of other modifier B6 loci. Reducing Wnt4 dosage increases the amount of testis tissue that develops in multiple cases of B6 XY sex reversal To examine the possibility that Wnt4 would be haploinsufficient on the B6 genetic background and to investigate its candidacy as a tda gene, we transferred a Wnt4 allele onto the B6 genetic background and assessed if Wnt4 heterozygosity exacerbated XY sex reversal in B6 XYPOS, B6 XYAKR, Sf1+/2 B6 XYAKR, and Wt1+/2 B6 XYAKR fetuses. WIHC was used to examine gonad morphology and marker gene expression at E13.5. Each gonad/ mesonephros complex was assessed at dissection for the presence of cords and male-specific vasculature, and classified as an ovary, ovotestis, or testis (Table 7). Classification was done prior to PLoS Genetics | www.plosgenetics.org 9 April 2012 | Volume 8 | Issue 4 | e1002569