alleles [46,58]. The genetic sex of fetuses was determined by PCR using Y Chr specific primers and myogenin control primers [59]. The B6-YAKR,Sxr strain carries a rearranged Y Chr containing two Sry alleles: the M. domesticus-derived SryAKR allele and the M. musculus-derived SrySxr allele. The presence of the M. domesticus and/or M. musculus Sry alleles was determined by PCR followed by digestion with NlaIV restriction enzyme using primers that flank a polymorphism within the Sry gene [29]. Embryo isolation and staging, and determination of sexual phenotype Timed matings were used for all embryonic experiments. Noon on the day of vaginal plug detection was designated as E0.5. Embryos between E10.5 and 12.5 were precisely staged by counting the number of tail somites (E10.5,8 ts, E11.5,18 ts, E12.5,30 ts) [60] and those between E12.5 and E16 were staged by limb morphology and the extent of cranial vessel formation [61]. To classify the gonads of individual E14.5–16 fetuses, the gonads were removed from the body cavity and immediately examined with the aid of an inverted microscope using transmitted light, and then photographed [62]. Testes were identified as having distinctive cords and vasculature, and an incipient tunica albuginea. Ovaries were identified as having a reticular appearance and lacking the structures noted above in the testes. Ovotestes contained a region with a reticular appearance at one or both ends of the gonad with the remainder containing cords. The sexual phenotype of weaning age (,3 weeks postpartum) mice was determined by examining the external genitalia, noting the anal-genital distance, and observing whether mammary gland-associated yellow-pigmented hairs were present [63]. Gonads from E13.5 fetuses were classified at the time of dissection, when the observer was blind to the fetal genotype. Gonads without any testicular cords were classified as ovaries, those with testicular cords only in the central region of the gonad were classified as ovotestes, and those with testicular cords throughout were classified as testes. Those with disorganized testicular cords throughout the gonad were classified as abnormal testes. Whole-mount immunohistochemistry and RNA in situ hybridization Gonad/mesonephros complexes were fixed overnight in 4% paraformaldehyde in PBS at 4uC. Whole-mount indirect fluores- cent immunohistochemistry (WIHC) was performed essentially as previously described [64], except that a blocking buffer containing 3% BSA, 10% donkey serum, 0.1% Triton-X, 0.02% sodium azide in PBS was used when the anti-PDGFRa or anti-MIS (AMH) antibodies were employed. Primary antibodies and dilutions are listed in Table S1. Secondary antibodies were donkey anti-IgG conjugated with Cy3 or Cy5 (Jackson Immu- noResearch, 1:500) or AF488 (Molecular Probes, 1:750). Samples were imaged using a Zeiss LSM510 confocal microscope. Standard protocols for whole-mount in situ hybridization (WISH) were used with minor changes [46,65]. Gene-specific probes are listed in Table S1. The Rec8 probe was synthesized from a full-length cDNA clone (IMAGE ID:6335959, GenBank: BC052155.1). Antisense and sense probes were tested in parallel, and only signal specific to the antisense probes was reported as gene-specific expression. Each result reported was replicated in a minimum of four samples. RNA extraction, reverse transcription, and quantitative RT–PCR Total RNA was isolated using the RNeasy mini kit (Qiagen). Samples were treated with Turbo DNase (Ambion) and subse- Modeling Dosage-Sensitive Sex Reversal in Mice quently treated with DNase inactivation reagent using the DNA- free protocol (Ambion). RNA Samples were determined to be free of DNA by PCR using primers specific for the Myog or Lhx1 genes [29]. cDNA was synthesized using oligo-dT primers and reverse transcriptase (RT). The RT reaction was tested for the presence of intact cDNA by PCR amplification with primers specific for the Hprt transcript [46]. Relative Sry expression in mutant vs. normal gonad/mesonephros complexes was determined using TaqMan Universal PCR Master Mix, and Sry (Mm00441712_s1) and Gapdh (Mm99999915_g1) Assays-on-Demand following the manufacturer’s standard protocol (Applied Biosystems). Relative Wt1 and Sf1 expression in B6 vs. D2 gonad/mesonephros complexes was determined using Wt1 (Mm00460570_m1), Sf1(Mm00446826_m1) and Gapdh TaqMan Assays-on-Demand. Relative expression of ovary pathway genes in B6 vs. D2 ovaries was determined using Power SYBR Green PCR Master Mix (Applied Biosystems) and the following primers: Adamts19 (59-CCAGATGCCTCCTGCTTTTA and 59-GGTG- CGGGTGACCTATGAT: 165 bp product), Wnt4 (59-AGGA- TGCTCGGACAACATCG and 59-CGCATGTGTGTCAAGA- TGGC: 149 bp product), Igfbp2 and 1700106J16Rik as in [46], Fst, Bmp2, and Hprt as in [44], and Irx3 as in [38]. Quantitative RT-PCR (qRT-PCR) assays were performed on an ABI PRISM 7900HT Sequence Detection System using SDS 2.3 software to determine the average cycle threshold (Ct) value from three technical replicates for each sample. Average Ct values for Hprt or Gapdh were subtracted from the average Ct values for each gene for each sample, to determine the normalized average Ct value (DCt). When expression was profiled across developmen- tal stages, the results are represented as expression relative to the endogenous control gene (22DCt), when expression was compared within a single developmental stage, the results were determined by subtracting the average DCt for D2 ovaries according to the 22DDCt method [66]. In pairwise comparisons of gene expression levels with a priori predictions about the direction of the effect, such as detecting lower expression of Wt1 and Sf1 or higher expression of Wnt4 and other ovarian genes in B6 samples, statistically significant differences were identified by one-tailed unpaired Student’s t-tests (a = 0.05) using Microsoft Excel. For statistical analysis of Sry expression, one-way ANOVA was used to test for a significant effect of genotype in multiple comparisons and two- tailed unpaired Student’s t-tests (a = 0.05) in pairwise comparisons within a developmental stage using JMP version 8 software (SAS Institute Inc.). Multi-gene quantitative real time RT-PCR was conducted using RNA extracted from E12 gonad-mesonephros complexes and E14 gonads as described in [44]. Only E14 XYAKR mutant gonads lacking any testicular cords (classified as ovaries) were used for this analysis. Genotyping assay for the presence of Sf1A172 versus Sf1S172 alleles The presence of the Sf1A172 and Sf1S172 alleles was determined using a previously published genotyping assay [41]. Briefly, a region of Sf1 exon 3 including codon 172 was amplified from genomic DNA using 59-TGCGTGCTGATCGAATGC and 59- CCAGTCGACAATGGAGATAAAGG as primers and the PCR product digested using NcoI. Genomic DNA containing the Sf1A172 allele contains a NcoI recognition site resulting in two fragments of 331 and 262 bp, while the Sf1S172 allele lacks the NcoI recognition site resulting in an undigested 593 bp fragment. Genomic DNA was obtained from mice in the Eicher Laboratory mouse colony or purchased from the JAX Mice DNA Resource [C58/J (000669), PLoS Genetics | www.plosgenetics.org 18 April 2012 | Volume 8 | Issue 4 | e1002569