(000661), BALB/cBY NZB/BlNJ (000684), C3H/HeSnJ (000650), SM/J (000687)]. Supporting Information Figure S1 Transmitted light micrographs illustrating the phenotypic range of Wt1+/2 B6 XYAKR E15.5 gonads. Only Wt1+/2 gonad/mesonephros complexes are shown because Wt1+/2 and Sf1+/2 B6 XYAKR gonads had similar morphology. All images at 106 magnification, cranial is to the left, and the gonad is above the dotted black line with the mesonephros below it. Heterozygous XYAKR ovaries (C) did not have testicular cords, lacked obvious structure including the coelomic vessel, and were more similar in size to age-matched +/+ XX ovaries (A) than +/+ XYAKR testes (B). Heterozygous XYAKR ovotestes (D) usually had central regions with testicular cords (outlined with dotted white lines) and regions at the cranial (anterior) and caudal (posterior) poles that lacked obvious structure and appeared to be ovarian. The regions with cords were associated with a coelomic vessel (arrowhead) whereas this vasculature was not present in the flanking regions. The ovary in (C) and the ovotestis in (D) were present in the same fetus. Some heterozygous XYAKR testes were slightly abnormal (E) in that they were smaller than +/+ B6 XYAKR testes and appeared to have delayed testis cord formation. However, these did not contain obvious ovarian tissue. In contrast, some +/2 B6 XYAKR testes had well-developed cords throughout (F) and were very similar to +/+ B6 XYAKR testes. The gonads in (A, B, E, and F) were dissected from fetuses from the same litter. (TIF) Figure S2 Expression of ovary-specific markers in B6 XYAKR E13.5 gonads analyzed by WIHC and WISH. A) WIHC analysis. FOXL2, which is normally expressed in ovarian somatic cells, often was expressed in the poles of B6 XYAKR gonads (left panel, 206 magnification). In some cases, FOXL2-expressing cells were References 1. Achermann JC, Ito M, Hindmarsh PC, Jameson JL (1999) A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans. Nat Genet 22: 125–126. 2. Correa RV, Domenice S, Bingham NC, Billerbeck AE, Rainey WE, et al. (2004) A microdeletion in the ligand binding domain of human steroidogenic factor 1 causes XY sex reversal without adrenal insufficiency. J Clin Endocrinol Metab 89: 1767–1772. 3. Mallet D, Bretones P, Michel-Calemard L, Dijoud F, David M, et al. (2004) Gonadal dysgenesis without adrenal insufficiency in a 46, XY patient heterozygous for the nonsense C16X mutation: a case of SF1 haploinsufficiency. J Clin Endocrinol Metab 89: 4829–4832. 4. Lourenco D, Brauner R, Lin L, De Perdigo A, Weryha G, et al. (2009) Mutations in NR5A1 associated with ovarian insufficiency. N Engl J Med 360: 1200–1210. 5. Philibert P, Zenaty D, Lin L, Soskin S, Audran F, et al. (2007) Mutational analysis of steroidogenic factor 1 (NR5a1) in 24 boys with bilateral anorchia: a French collaborative study. Hum Reprod 22: 3255–3261. 6. Lin L, Philibert P, Ferraz-de-Souza B, Kelberman D, Homfray T, et al. (2007) Heterozygous missense mutations in steroidogenic factor 1 (SF1/Ad4BP, NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function. J Clin Endocrinol Metab 92: 991–999. 7. Pelletier J, Bruening W, Kashtan CE, Mauer SM, Manivel JC, et al. (1991) Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell 67: 437–447. 8. Pelletier J, Bruening W, Li FP, Haber DA, Glaser T, et al. (1991) WT1 mutations contribute to abnormal genital system development and hereditary Wilms’ tumour. Nature 353: 431–434. 9. Patek CE, Little MH, Fleming S, Miles C, Charlieu JP, et al. (1999) A zinc finger truncation of murine WT1 results in the characteristic urogenital abnormalities of Denys-Drash syndrome. Proc Natl Acad Sci U S A 96: 2931–2936. 10. Jordan BK, Mohammed M, Ching ST, Delot E, Chen XN, et al. (2001) Up- regulation of WNT-4 signaling and dosage-sensitive sex reversal in humans. Am J Hum Genet 68: 1102–1109. 11. Jager RJ, Harley VR, Pfeiffer RA, Goodfellow PN, Scherer G (1992) A familial mutation in the testis-determining gene SRY shared by both sexes. Hum Genet 90: 350–355. Modeling Dosage-Sensitive Sex Reversal in Mice found in regions containing AMH-expressing Sertoli cells and incipient testicular cords (right panel, 406 magnification). B) WISH analysis. The ovary-specific somatic cell marker Irx3, and meiotic germ cell markers Stra8 and Rec8 were expressed in the cranial (anterior) and/or caudal (posterior) poles of B6 XYAKR gonads. When Stra8 and Rec8 were expressed at both poles in B6 XYAKR gonads at this developmental stage, expression was higher at the cranial (left) vs. the caudal pole (right). The gonad/ mesonephros complex in each panel is oriented with the gonad above the mesonephros and the anterior pole to the left. (TIF) Table S1 WIHC and WISH markers. Pertinent information on the antibodies and riboprobes used to analyze morphology and marker gene expression during fetal gonad differentiation. (DOCX) Acknowledgments Mice carrying alleles of Wt1, Sf1, and Wnt4 were kindly provided by Jordan Kreidberg, Keith Parker, and Andrew McMahon, respectively. For generously supplying reagents, we thank Marc Fellous (FOXL2 antibody), Francis Poulat (SOX9 antibody), Ken-ichirou Morohashi (SF1 antibody), David Page (Stra8 riboprobe plasmid), Joan Jorgensen (Irx3 riboprobe plasmid), and Andrew McMahon (Wnt4 and Ptch1 riboprobe plasmids). We thank Ron Myers, Andrew Recknagel, Lisa Somes, and Meredith Crane for mouse husbandry and technical support; Steve Munger for comments on the manuscript; and Dorothy Pazin and Caryn Navarro for helpful discussions during the course of this work and for critical comments on the manuscript. Author Contributions Conceived and designed the experiments: KHA EME. Performed the experiments: SMC LLW RSK MCM GJB EME KHA. Analyzed the data: SMC LLW RSK MCM GJB EME KHA. Wrote the paper: SMC EME KHA. 12. Vilain E, McElreavey K, Jaubert F, Raymond JP, Richaud F, et al. (1992) Familial case with sequence variant in the testis-determining region associated with two sex phenotypes. Am J Hum Genet 50: 1008–1011. 13. Jordan BK, Shen JH, Olaso R, Ingraham HA, Vilain E (2003) Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/beta-catenin synergy. Proc Natl Acad Sci U S A 100: 10866–10871. 14. Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, et al. (1993) WT-1 is required for early kidney development. Cell 74: 679–691. 15. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, et al. (1995) Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dyn 204: 22–29. 16. Luo X, Ikeda Y, Parker KL (1994) A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77: 481–490. 17. Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA, et al. (1995) Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci U S A 92: 10939–10943. 18. Veitia RA, Salas-Cortes L, Ottolenghi C, Pailhoux E, Cotinot C, et al. (2001) Testis determination in mammals: more questions than answers. Mol Cell Endocrinol 179: 3–16. 19. Eicher EM, Washburn LL, Whitney JB, III, Morrow KE (1982) Mus poschiavinus Y chromosome in the C57BL/6J murine genome causes sex reversal. Science 217: 535–537. 20. Washburn LL, Eicher EM (1983) Sex reversal in XY mice caused by dominant mutation on chromosome 17. Nature 303: 338–340. 21. Nagamine CM, Taketo T, Koo GC (1987) Studies on the genetics of tda-1 XY sex reversal in the mouse. Differentiation 33: 223–231. 22. Bouma GJ, Albrecht KH, Washburn LL, Recknagel AK, Churchill GA, et al. (2005) Gonadal sex reversal in mutant Dax1 XY mice: a failure to upregulate Sox9 in pre-Sertoli cells. Development 132: 3045–3054. 23. Bouma GJ, Washburn LL, Albrecht KH, Eicher EM (2007) Correct dosage of Fog2 and Gata4 transcription factors is critical for fetal testis development in mice. Proc Natl Acad Sci U S A 104: 14994–14999. PLoS Genetics | www.plosgenetics.org 19 April 2012 | Volume 8 | Issue 4 | e1002569