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This dataset represents genes that are dysregulated in the postnatal day 12 (P12) mouse testis when ATRX is specifically inactivated in Sertoli cells (

In humans, dysregulation of the sex determining gene SRY-box 9 (SOX9) leads to disorders of sex development (DSD). In mice, knock-out of Sox9 prior to sex determination leads to XY sex reversal, while Sox9 inactivation after sex determination leads to spermatogenesis defects. SOX9 specifies the differentiation and function of Sertoli cells from somatic cell precursors, which then orchestrate the development and maintenance of other testicular cell types, largely through unknown mechanisms. Here, we describe a novel testicular target gene of SOX9, Ets variant factor 5 (ETV5), a transcription factor responsible for maintaining the spermatogonial stem cell niche. Etv5 was highly expressed in wild-type XY but not XX mouse fetal gonads, with ETV5 protein localized in the Sertoli cells, interstitial cells and germ cells of the testis. In XY Sox9 knock-out gonads, Etv5 expression was strongly down-regulated. Similarly, knock-down of SOX9 in the human Sertoli-like cell line NT2/D1 caused a decrease in ETV5 gene expression. Transcriptomic analysis of NT2/D1 cells over-expressing SOX9 showed that ETV5 expression was increased in response to SOX9. Moreover, chromatin immunoprecipitation of these cells, as well as of embryonic mouse gonads, showed direct binding of SOX9 to ETV5 regulatory regions. We demonstrate that SOX9 was able to activate ETV5 expression via a conserved SOX site in the 5' regulatory region, mutation of which led to loss of activation. In conclusion, we present a novel target gene of SOX9 in the testis, and suggest that SOX9 regulation of ETV5 contributes to the control of male fertility.

While the primary sex determining switch varies between vertebrate species, a key downstream event in testicular development, namely the male-specific up-regulation of Sox9, is conserved. To date, only two sex determining switch genes have been identified, Sry in mammals and the Dmrt1-related gene Dmy (Dmrt1bY) in the medaka fish Oryzias latipes. In mice, Sox9 expression is evidently up-regulated by SRY and maintained by SOX9 both of which directly activate the core 1.3 kb testis-specific enhancer of Sox9 (TESCO). How Sox9 expression is up-regulated and maintained in species without Sry (i.e. non-mammalian species) is not understood. In this study, we have undertaken an in-depth comparative genomics approach and show that TESCO contains an evolutionarily conserved region (ECR) of 180 bp which is present in marsupials, monotremes, birds, reptiles and amphibians. The ECR contains highly conserved modules that predict regulatory roles for SOX, TCF/LEF, Forkhead, DMRT, and GATA proteins in vertebrate sex determination/differentiation. Our data suggest that tetrapods share common aspects of Sox9 regulation in the testis, despite having different sex determining switch mechanisms. They also suggest that Sox9 autoregulation is an ancient mechanism shared by all tetrapods, raising the possibility that in mammals, SRY evolved by mimicking this regulation. The validation of ECR regulatory sequences conserved from human to frogs will provide new insights into vertebrate sex determination.

In mammals, sex is determined in the bipotential embryonic gonad by a balanced network of gene actions which when altered causes disorders of sexual development (DSD, formerly known as intersex). In the XY gonad, presumptive Sertoli cells begin to differentiate when SRY up-regulates SOX9, which in turn activates FGF9 and PGDS to maintain its own expression. This study identifies a new and essential component of FGF signaling in sex determination. Fgfr2 mutant XY mice on a mixed 129/C57BL6 genetic background had either normal testes, or developed ovotestes, with predominantly testicular tissue. However, backcrossing to C57BL6 mice resulted in a wide range of gonadal phenotypes, from hypoplastic testes to ovotestes with predominantly ovarian tissue, similar to Fgf9 knockout mice. Since typical male-specific FGF9-binding to the coelomic epithelium was abolished in Fgfr2 mutant XY gonads, these results suggest that FGFR2 acts as the receptor for FGF9. Pgds and SOX9 remained expressed within the testicular portions of Fgfr2 mutant ovotestes, suggesting that the Prostaglandin pathway acts independently of FGFR2 to maintain SOX9 expression. We could further demonstrate that double-heterozygous Fgfr2/Sox9 knockout mice developed ovotestes, demonstrating that both Fgfr2 and Sox9 can act as modifier intersex genes in the heterozygous state. In summary, we provide evidence that FGFR2 is important for male sex determination in mice, thereby rendering human FGFR2 a candidate gene for unsolved DSD cases such as 10q26 deletions.

Campomelic dysplasia (CD), a human skeletal malformation syndrome with XY sex reversal, is caused by heterozygous mutations in and around the gene SOX9. SOX9 has an extended 5' control region, as indicated by CD translocation breakpoints scattered over 1 Mb proximal to SOX9 and by expression data from mice transgenic for human SOX9-spanning yeast artificial chromosomes. To identify long-range regulatory elements within the SOX9 5' control region, we compared approximately 3.7 Mb and 195 kb of sequence around human and Fugu rubripes SOX9, respectively. We identified only seven and five protein-coding genes in the human and F. rubripes sequences, respectively. Four of the F. rubripes genes have been mapped in humans; all reside on chromosome 17 but show extensive intrachromosomal gene shuffling compared with the gene order in F. rubripes. In both species, very large intergenic distances separate SOX9 from its directly flanking genes: 2 Mb and 500 kb on either side of SOX9 in humans, and 68 and 97 kb on either side of SOX9 in F. rubripes. Comparative sequence analysis of the intergenic regions revealed five conserved elements, E1-E5, up to 290 kb 5' to human SOX9 and up to 18 kb 5' to F. rubripes SOX9, and three such elements, E6-E8, 3' to SOX9. Where available, mouse sequences confirm conservation of the elements. From the yeast artificial chromosome transgenic data, elements E3-E5 are candidate enhancers for SOX9 expression in limb and vertebral column, and 8 of 10 CD translocation breakpoints separate these elements from SOX9.