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First published online 31 March 2004
doi: 10.1242/dev.01087

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Functional analysis of Sox8 and Sox9 during sex determination in the mouse

¹ INSERM U470, Centre de Biochimie, Parc Valrose, 06108 Nice Cedex 2, France
² Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
³ Department of Molecular Genetics, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA
⁴ Departments of Endocrinology and of Cell Biology, Utrecht University, 3584-CH Utrecht, The Netherlands
⁵ Institute for Biochemistry, University of Erlangen, D-91054 Erlangen, Germany

View larger version (64K): [in a new window]   Fig. 9. Defects in testis formation in Sox9 heterozygous knock-out mice on a Sox8/Sox8 knock-out background. (A-C) Urogenital ridges isolated at E13.5 were analyzed for the presence of sex cords. Cords in Sox8/Sox8; Sox9/+ gonads were poorly outlined and the formation of the coelomic vessel was disturbed. (D-F) Immunofluorescent analysis of gonads at E15.5 (laminin, red; Mis, green). Sox8/Sox8; Sox9/+ gonads show a reduced number of sex cords, reduced expression of Mis and areas with poor differentiation (white arrowhead).

View larger version (69K): [in a new window]   Fig. 1. Analysis of tissue specific deletion of Sox9 in Sf1:Cre; Sox9flox/Sox9flox animals. Transgenic lines carrying the Cre recombinase under a 674 bp Sf1 promoter fragment were crossed onto the homozygous Sox9flox/Sox9flox background. (A) The floxed and deleted Sox9 locus. Orange arrows indicate the position of primers used for the detection of the Sox9flox and the Sox9 allele. (B) Detection of Sox9 deletion in urogenital ridges (E13.5) on the genomic level. Only mice carrying the Sf1:Cre transgene (lanes 3 and 4) show the deleted band Sox9. Incomplete deletion of the floxed allele may be due to inefficient expression of Sf1:Cre or may reflect the contamination of cells from the mesonephros. (C-E) In situ hybridization analysis for Sox9 at E13.5. Dotted lines indicate the outline of the developing gonads. Embryos homozygous for the Sox9flox allele and carrying the Sf1:Cre transgene (Cre) show reduced (D) or absent (E) expression of Sox9 in the developing gonad. Note the persistent signal of Sox9 in other tissues (e.g. arrow in panel E indicates expression in the neural tube).

View larger version (103K): [in a new window]   Fig. 2. Sox9 or Sox8/Sox9 knock-out mice show defects in sex cord formation. E13.5 gonads from Sox9 tissue-specific knock-out animals show a variable degree of sex cord formation ranging from normal (not shown) to severely abnormal (arrow in C). Note the presence of abnormal vascularization (arrowhead in C). Sox8/Sox8 knock-out gonads develop normal testis cords (D). By contrast, Sox9 tissue-specific knock-out animals (Cre/+ Sox9flox/Sox9flox) either heterozygous (E) or homozygous (F) for the Sox8 knock-out allele often show complete absence of sex cord formation. (For frequencies of abnormal gonadal development see Table 1.)

View larger version (166K): [in a new window]   Fig. 3. Histological analysis of tissue-specific knock-out gonads at E18.5. (A-C) Control females. In contrast to control males (D-F), Sox9 knock-out animals show somewhat irregular and poorly differentiated sex cords (G, and arrow in H). Next to quiescent gonocytes (arrows in I), several gonocytes in meiotic prophase could be detected (asterisks), suggesting differentiation of these primordial germ cells along the female pathway (compare with C). (J-L) Mice homozygous for the Sox8/Sox8 knock-out allele, but without the Sox9 tissue-specific knock-out showed normal sex cord formation and no sign of meiosis. By contrast, embryos heterozygous for the Sox8 knock-out and homozygous for the Sox9 knock-out (M-O) often showed complete sex reversal (compare with female development in A-C). See Table 1 for the frequencies of the sex reversal phenotype.

View larger version (20K): [in a new window]   Fig. 4. Real-time RT-PCR analysis of markers involved in the sex determination process. Sox9 expression (shown on the X axis) in individual E13.5 knock-out gonads was variable with levels in some gonads being comparable to those in female mice. (A) Mis expression was directly dependent on Sox9 levels indicating direct activation of its promoter (correlation coefficient r=0.9905; P<0.0001). Note that Mis expression in mice with low levels of Sox9 was still higher than that in control females. (B) Expression of Sox8 also showed a clear dependence on Sox9 levels (r=0.8444; P<0.0001). (C) No such direct relationship for the steroidogenic factor Sf1 was observed (r=0.3173; P=0.2689). (D) Gonads with very low levels of Sox9 showed persistent expression of the Sry gene.

View larger version (27K): [in a new window]   Fig. 5. Conditional knock-out of Sox9 in male and female germ cells. (A) Schematic diagram of breeding strategy. Although Sox9 was deleted in almost all oocytes of Sox9flox/flox; Zp3:Cre females, Sox9 was deleted in only about half of the sperm of in Sox9flox/flox; Prm:Cre males. Therefore, when Prm:Cre males and Zp3:Cre females were mated, Sox9/ and Sox9flox/ embryos were obtained at a 1:1 ratio (n=251). Only one Sox9flox/flox embryo was obtained (0.4%). (B) PCR genotyping of Sox9 mutants obtained from the germ cell knock-out. No mosaicism was observed in homozygous Sox9 mutants. Sox9flox, conditional (active) Sox9 allele; Sox9, recombined (inactive) Sox9 allele.

View larger version (75K): [in a new window]   Fig. 6. Organ culture of urogenital tissues from Sox9 mutants. (A-D) Lateral view of embryos at E11.5. (E-H) Laminin immunostaining of sectioned cultured urogenital systems. (I-L) High magnification of the gonad of cultured urogenital systems. No cord formation was observed in gonads from XY Sox9/ embryos. g, gonad; tc, testis cords; arrowhead, Wolffian duct; arrow, Müllerian duct.

View larger version (56K): [in a new window]   Fig. 7. Whole-mount in situ hybridization of cultured urogenital systems. Mis (A-D), P450-Scc (E-H), Bmp2 (I-L) and follistatin (M-P). Arrows indicate the gonad. (E-H) Arrowheads indicate Scc expression in the adrenal gland. The cultured XY Sox9/ gonad is sex reversed to female.

View larger version (106K): [in a new window]   Fig. 8. Immunofluorescent analysis of Sox8, Sox9 double knock-out mice. Double staining of E15.5 gonads for Mis (red) and Sox9 (green). (A,B) Control ovary and testis. Sf1:Cre; Sox9/Sox9 knock-out mice (C,D) showed a varying degree of sex cord formation (D shows the most severe phenotype observed during this analysis). (E,F) Control ovary and testis. By contrast, Sox8/Sox9 double knock-out mice showed a much more severe phenotype, often resulting in complete sex reversal (G,H). The formation of sex cords and the expression of Mis was restricted to cells expressing Sox9. Note, embryos shown in A-D were derived from a different litter to those shown in E-H.