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During sex determination in the mouse, fibroblast growth factor 9 signals through the fibroblast growth factor receptor 2c isoform (FGFR2c) to trigger Sertoli cell and testis development from 11.5 days post coitum (dpc). In the XX gonad, the FOXL2 and WNT4/RSPO1 pathways drive granulosa cell and ovarian development. The function of FGFR2 in the developing ovary, and whether FGFR2 is required in the testis after sex determination, is not clear. In fetal mouse gonads from 12.5 dpc, FGFR2 shows sexually dimorphic expression. In XX gonads, FGFR2c is coexpressed with FOXL2 in pregranulosa cells, whereas XY gonads show FGFR2b expression in germ cells. Deletion of Fgfr2c in XX mice led to a marked decrease/absence of germ cells by 13.5 dpc in the ovary. This indicates that FGFR2c in the somatic pregranulosa cells is required for the maintenance of germ cells. Surprisingly, on the Fgfr2c-/- background, the germ cell phenotype could be rescued by ablation of Foxl2, suggesting a novel mechanism whereby FGFR2 and FOXL2 act antagonistically during germ cell development. Consistent with low/absent FGFR2 expression in the Sertoli cells of 12.5 and 13.5 dpc XY gonads, XY AMH:Cre; Fgfr2flox/flox mice showed normal testis morphology and structures during fetal development and in adulthood. Thus, FGFR2 is not essential for maintaining Sertoli cell fate after sex determination. Combined, these data show that FGFR2 is not necessary for Sertoli cell function after sex determination but does play an important role in the ovary.

46,XY gonadal dysgenesis (GD) is a Disorder/Difference of Sex Development (DSD) that can present with phenotypes ranging from ambiguous genitalia to complete male-to-female sex reversal. Around 50% of 46,XY DSD cases receive a molecular diagnosis. In mice, Fibroblast growth factor 9 (FGF9) is an important component of the male sex-determining pathway. Two FGF9 variants reported to date disrupt testis development in mice, but not in humans. Here, we describe a female patient with 46,XY GD harbouring the rare FGF9 variant (missense mutation), NM_002010.2:c.583G A;p.(Asp195Asn) (D195N). By biochemical and cell-based approaches, the D195N variant disrupts FGF9 protein homodimerisation and FGF9-heparin-binding, and reduces both Sertoli cell proliferation and Wnt4 repression. XY Fgf9

The transcription factor SOX9 is essential for the development of multiple organs including bone, testis, heart, lung, pancreas, intestine and nervous system. Mutations in the human SOX9 gene led to campomelic dysplasia, a haploinsufficiency disorder with several skeletal malformations frequently accompanied by 46, XY sex reversal. The mechanisms underlying the diverse SOX9 functions during organ development including its post-translational modifications, the availability of binding partners, and tissue-specific accessibility to target gene chromatin. Here we summarize the expression, activities, and downstream target genes of SOX9 in molecular genetic pathways essential for organ development, maintenance, and function. We also provide an insight into understanding the mechanisms that regulate the versatile roles of SOX9 in different organs.

In mice, male sex determination depends on FGF9 signalling via FGFR2c in the bipotential gonads to maintain the expression of the key testis gene SOX9. In humans, however, while FGFR2 mutations have been linked to 46,XY disorders of sex development (DSD), the role of FGF9 is unresolved. The only reported pathogenic mutations in human FGF9, FGF9S99N and FGF9R62G, are dominant and result in craniosynostosis (fusion of cranial sutures) or multiple synostoses (fusion of limb joints). Whether these synostosis-causing FGF9 mutations impact upon gonadal development and DSD etiology has not been explored. We therefore examined embryonic gonads in the well-characterized Fgf9 missense mouse mutants, Fgf9S99N and Fgf9N143T, which phenocopy the skeletal defects of FGF9S99N and FGF9R62G variants, respectively. XY Fgf9S99N/S99N and XY Fgf9N143T/N143T fetal mouse gonads showed severely disorganized testis cords and partial XY sex reversal at 12.5 days post coitum (dpc), suggesting loss of FGF9 function. By 15.5 dpc, testis development in both mutants had partly recovered. Mitotic analysis in vivo and in vitro suggested that the testicular phenotypes in these mutants arise in part through reduced proliferation of the gonadal supporting cells. These data raise the possibility that human FGF9 mutations causative for dominant skeletal conditions can also lead to loss of FGF9 function in the developing testis, at least in mice. Our data suggest that, in humans, testis development is largely tolerant of deleterious FGF9 mutations which lead to skeletal defects, thus offering an explanation as to why XY DSDs are rare in patients with pathogenic FGF9 variants.

Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. We show that Mmd2 is expressed during the sex-determining period in XY but not XX gonads, specifically in the Sertoli cell lineage which orchestrates early testis development. Analysis of knockout mice deficient in Sox9 and Sf1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, when we used CRISPR to ablate Mmd2 in the mouse, fetal testis development appeared to progress normally. To determine if other genes might have compensated for the loss of Mmd2, we identified the closely related PAQR family members Paqr8 and Mmd as also being expressed during testis development. We used CRISPR to generate mouse strains deficient in Paqr8 and Mmd, but both knockout lines appeared phenotypically normal and fertile. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. These results may reflect functional redundancy among these factors. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes, and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

Heterozygous mutations in the human SOX9 gene cause the skeletal malformation syndrome campomelic dysplasia which in 75% of 46, XY individuals is associated with male-to-female sex reversal. Although studies in homozygous Sox9 knockout mouse models confirmed that SOX9 is critical for testis development, mice heterozygous for the Sox9-null allele were reported to develop normal testes. This led to the belief that the SOX9 dosage requirement for testis differentiation is different between humans, which often require both alleles, and mice, in which one allele is sufficient. However, in prior studies, gonadal phenotypes in heterozygous Sox9 XY mice were assessed only by either gross morphology, histological staining or analyzed on a mixed genetic background. In this study, we conditionally inactivated Sox9 in somatic cells of developing gonads using the Nr5a1-Cre mouse line on a pure C57BL/6 genetic background. Section and whole-mount immunofluorescence for testicular and ovarian markers showed that XY Sox9 heterozygous gonads developed as ovotestes. Quantitative droplet digital PCR confirmed a 50% reduction of Sox9 mRNA as well as partial sex reversal shown by an upregulation of ovarian genes. Our data show that haploinsufficiency of Sox9 can perturb testis development in mice, suggesting that mice may provide a more accurate model of human disorders/differences of sex development than previously thought.

International audience; Disorders/differences of sex development (DSD) cause profound psychological and reproductive consequences for the affected individuals, however, most are still unexplained at the molecular level. Here, we present a novel gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), encoding a metabolic enzyme in the liver important for energy production from fatty acids, that shows an unusual expression pattern in developing fetal mouse gonads. Shortly after gonadal sex determination it is up-regulated in the developing testes following a very similar spatial and temporal pattern as the male-determining gene Sry in Sertoli cells before switching to ovarian enriched expression. To test if Hmgcs2 is important for gonad development in mammals, we pursued two lines of investigations. Firstly, we generated Hmgcs2-null mice using CRISPR/Cas9 and found that these mice had gonads that developed normally even on a sensitized background. Secondly, we screened 46,XY DSD patients with gonadal dysgenesis and identified two unrelated patients with a deletion and a deleterious missense variant in HMGCS2 respectively. However, both variants were heterozygous, suggesting that HMGCS2 might not be the causative gene. Analysis of a larger number of patients in the future might shed more light into the possible association of HMGCS2 with human gonadal development.

Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan., Author summary Fertility is a concern in women’s health, especially when the reported average age of mothers is rising (2016, CDC DB232). Of concern in aging mothers is ovarian follicle health, which requires active communication between its cell components that include a single egg and its surrounding granulosa support cells. The process used to establish cell-cell communication within follicles is unknown but begins during fetal development. We discovered that two related genes, Irx3 and Irx5, direct network construction. If Irx3 and Irx5 are eliminated in mice, oocyte-granulosa cell contact is lost, and all follicles die within short order. Analysis of additional Irx3 and Irx5 mutant mice highlighted that each factor is important for establishing follicle integrity. During fetal development, they work together in the same cell to establish the identity of future granulosa support cells. Then, as the follicles form, they continue to work cooperatively, but in different cells. While Irx5 expression remains in granulosa cells, Irx3 expression transitions to the oocyte and together, they promote cell patterns and interacting zones to synchronize the follicle as an interactive unit. Although the work of Irx3 and Irx5 is finished in the perinatal ovary, their impact lasts a lifetime as it establishes long-term follicle integrity and therefore, fertility.

Male sex determination in mammals relies on sex determining region Y-mediated upregulation of sex determining region-box 9 (SOX9) expression in XY gonads, whereas Wnt family member (WNT)/R-spondin 1 signaling and forkhead box L2 (FOXL2) drive female sex determination in XX gonads. Fibroblast growth factor (FGF) 9 signaling ensures sustained SOX9 expression through repression of one of the ovarian pathways (WNT signaling), whereas the significance of FGF-mediated repression of the FOXL2 pathway has not been studied. Previously, we demonstrated that FGFR2 is the receptor for FGF9 in the XY gonad. Whether a specific isoform (FGFR2b or FGFR2c) is required was puzzling. Here, we show that FGFR2c is required for male sex determination. Initially, in developing mouse embryos at 12.5 to 13.5 days postcoitum (dpc), XY Fgfr2c-/- gonads appear as ovotestes, with SOX9 and FOXL2 expression predominantly localized to the posterior and anterior gonadal poles, respectively. However, by 15.5 dpc, XY Fgfr2c-/- gonads show complete male-to-female sex reversal, evident by the lack of SOX9 and ectopic expression of FOXL2 throughout the gonads. Furthermore, ablation of the Foxl2 gene leads to partial or complete rescue of gonadal sex reversal in XY Fgfr2c-/- mice. Together with previous findings, our data suggest that testis determination involves FGFR2c-mediated repression of both the WNT4- and FOXL2-driven ovarian-determining pathways.

In most mammals, the Y chromosomal Sry gene initiates testis formation within the bipotential gonad, resulting in male development. SRY is a transcription factor and together with SF1 it directly up-regulates the expression of the pivotal sex-determining gene Sox9 via a 1.3-kb cis-regulatory element (TESCO) which contains an evolutionarily conserved region (ECR) of 180 bp. Remarkably, several rodent species appear to determine sex in the absence of Sry and a Y chromosome, including the mole voles Ellobius lutescens and Ellobius tancrei, whereas Ellobius fuscocapillus of the same genus retained Sry. The sex-determining mechanisms in the Sry-negative species remain elusive. We have cloned and sequenced 1.1 kb of E. lutescens TESCO which shares 75% sequence identity with mouse TESCO indicating that testicular Sox9 expression in E. lutescens might still be regulated via TESCO. We have also cloned and sequenced the ECRs of E. tancrei and E. fuscocapillus. While the three Ellobius ECRs are highly similar (94-97% sequence identity), they all display a 14-bp deletion (Δ14) removing a highly conserved SOX/TCF site. Introducing Δ14 into mouse TESCO increased both basal activity and SF1-mediated activation of TESCO in HEK293T cells. We propose a model whereby Δ14 may have triggered up-regulation of Sox9 in XX gonads leading to destabilization of the XY/XX sex-determining mechanism in Ellobius. E. lutescens/E. tancrei and E. fuscocapillus could have independently stabilized their sex determination mechanisms by Sry-independent and Sry-dependent approaches, respectively.

Sex determination in fetal germ cells depends on a balance between exposure to retinoic acid (RA) and the degradation of RA achieved by the testis-specific expression of the catabolic cytochrome P450 enzyme, CYP26B1. Therefore, identification of factors regulating the expression of the Cyp26b1 gene is an important goal in reproductive biology. We used in situ hybridization to demonstrate that Cyp26b1 and transcription factor genes steroidogenic factor-1 (Sf1) and Sry-related HMG box 9 (Sox9) are coexpressed in Sertoli cells, whereas Cyp26b1 and Sf1 are coexpressed in Leydig cells in mouse fetal testes. In the mouse gonadal somatic cell line TM3, transfection of constructs expressing SOX9 and SF1 activated Cyp26b1 expression, independently of the positive regulator RA. In embryonic gonads deficient in SOX9 or SF1, Cyp26b1 expression was decreased relative to wild-type (WT) controls, as measured by quantitative RT-PCR (qRT-PCR). Furthermore, qRT-PCR showed that Cyp26b1 up-regulation by SOX9/SF1 was attenuated by the ovarian transcription factor Forkhead box L2 (FOXL2) in TM3 cells, whereas in Foxl2-null mice, Cyp26b1 expression in XX gonads was increased ∼20-fold relative to WT controls. These data support the hypothesis that SOX9 and SF1 ensure the male fate of germ cells by up-regulating Cyp26b1 and that FOXL2 acts to antagonize Cyp26b1 expression in ovaries.—Kashimada, K., Svingen, T., Feng, C.-W., Pelosi, E., Bagheri-Fam, S., Harley, V. R., Schlessinger, D., Bowles, J., Koopman, P. Antagonistic regulation of Cyp26b1 by transcription factors SOX9/SF1 and FOXL2 during gonadal development in mice.

Sexual reproduction is essential for the propagation and the maintenance of fitness of our species, and is dependent on the correct development of the bipotential genital ridges into testes and ovaries. Although several transcription factors, secreted signaling molecules, and their receptors have been found to be important for testis determination and early gonad development, comparatively little research has been carried out into intracellular signal transduction pathways activated during these processes. Focal adhesion kinase (FAK) and protein tyrosine kinase 2 beta (PTK2B) form one group of cytosolic tyrosine kinases that are known to be important for processes such as cell proliferation, differentiation, and motility. Here, we describe the temporal and spatial expression patterns of Fak and Ptk2b mRNA and protein during sex determination and early gonadogenesis in mouse embryos. Ptk2b mRNA and PTK2B protein were expressed in testes from 11.5 days post coitum onward, predominantly in developing Sertoli cells, in a SOX9-dependent manner. Fak mRNA and FAK protein were expressed in gonads of both sexes at all stages examined. Our data suggest cell type- and stage-specific roles for PTK2B during early testis development. Developmental Dynamics 239:2735–2741, 2010. © 2010 Wiley-Liss, Inc.

Balanced production and degradation of retinoids is important in regulating development of several organ systems in the vertebrate embryo. Among these, it is known that retinoic acid (RA), and the retinoid-catabolyzing enzyme CYP26B1 together regulate the sex-specific behavior of germ cells in developing mouse gonads. We report here that the gene encoding a cytosolic class-1 aldehyde dehydrogenase, ALDH1A1, a weak catalyst of RA production, is strongly expressed in a male-specific manner in somatic cells of the developing mouse testis, beginning shortly after Sry expression is first detectable. This expression pattern is conserved in the developing male gonad of the chicken and is dependent on the testis-specific transcription factor SOX9. Our data suggest that low levels of RA may be required for early developmental events in the testis, or that Aldh1a1 expression in the fetus may prefigure a later requirement for ALDH1A1 in regulating spermatogenesis postnatally. Developmental Dynamics 238:2073-2080, 2009. (C) 2009 Wiley-Liss, Inc.

GlutathioneS-transferases (GSTs) are an important family of multifunctional enzymes that play a role in the protection of tissues by the detoxification of hazardous and carcinogenic compounds. We found previously thatGstm6is upregulated in the somatic cells of male mouse fetal gonads relative to female gonads. In this study, we describe the spatial and temporal expression pattern ofGstm6during mouse development. We show thatGstm6is predominantly expressed in the reproductive system, at significantly higher levels in XY gonads compared with XX gonads from 11.5 dpc onwards, and remains expressed in the testes in adult mice. Its expression is associated with the Sertoli cell lineage, and is dependent on the expression of the male sex-determining geneSox9. Our data suggest thatGstm6plays a male-specific role in gonad development or function, possibly by modulating the exposure of somatic tissue and/or germ cells to endogenous or exogenous toxicants.

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.

In the presence of the Y-chromosomal gene Sry, the bipotential mouse gonads develop as testes rather than as ovaries. The autosomal gene Sox9, a likely and possibly direct Sry target, can induce testis development in the absence of Sry. Sox9 is thus sufficient but not necessarily essential for testis induction. Mutational inactivation of one allele of SOX9/Sox9 causes sex reversal in humans but not in mice. Because Sox9 –/– embryos die around Embryonic Day 11.5 (E11.5) at the onset of testicular morphogenesis, differentiation of the mutant XY gonad can be analyzed only ex vivo in organ culture. We have therefore conditionally inactivated both Sox9 alleles in the gonadal anlagen using the CRE/loxP recombination system, whereby CRE recombinase is under control of the cytokeratin 19 promoter. Analysis of resulting Sox9 –/– XY gonads up to E15.5 reveals immediate, complete sex reversal, as shown by expression of the early ovary-specific markers Wnt4 and Foxl2 and by lack of testis cord and Leydig cell formation. Sry expression in mutant XY gonads indicates that downregulation of Wnt4 and Foxl2 is dependent on Sox9 rather than on Sry. Our results provide in vivo proof that, in contrast to the situation in humans, complete XY sex reversal in mice requires inactivation of both Sox9 alleles and that Sox9 is essential for testogenesis in mice. developmental biology, gene regulation, ovary, Sertoli cells, testis

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.

MicroRNAs are important regulators of developmental gene expression, but their contribution to fetal gonad development is not well understood. We have identified the evolutionarily conserved gonadal microRNAs miR-202-5p and miR-202-3p as having a potential role in regulating mouse embryonic gonad differentiation. These microRNAs are expressed in a sexually dimorphic pattern as the primordial XY gonad differentiates into a testis, with strong expression in Sertoli cells. In vivo, ectopic expression of pri-miR-202 in XX gonads did not result in molecular changes to the ovarian determination pathway. Expression of the primary transcript of miR-202-5p/3p remained low in XY gonads in a conditional Sox9-null mouse model, suggesting that pri-miR-202 transcription is downstream of SOX9, a transcription factor that is both necessary and sufficient for male sex determination. We identified the pri-miR-202 promoter that is sufficient to drive expression in XY but not XX fetal gonads ex vivo. Mutation of SOX9 and SF1 binding sites reduced ex vivo transactivation of the pri-miR-202 promoter, demonstrating that pri-miR-202 may be a direct transcriptional target of SOX9/SF1 during testis differentiation. Our findings indicate that expression of the conserved gonad microRNA, miR-202-5p/3p, is downstream of the testis-determining factor SOX9, suggesting an early role in testis development.

MicroRNAs are important regulators of developmental gene expression, but their contribution to fetal gonad development is not well understood. We have identified the evolutionarily conserved gonadal microRNAs miR-202-5p and miR-202-3p as having a potential role in regulating mouse embryonic gonad differentiation. These microRNAs are expressed in a sexually dimorphic pattern as the primordial XY gonad differentiates into a testis, with strong expression in Sertoli cells. In vivo, ectopic expression of pri-miR-202 in XX gonads did not result in molecular changes to the ovarian determination pathway. Expression of the primary transcript of miR-202-5p/3p remained low in XY gonads in a conditional Sox9-null mouse model, suggesting that pri-miR-202 transcription is downstream of SOX9, a transcription factor that is both necessary and sufficient for male sex determination. We identified the pri-miR-202 promoter that is sufficient to drive expression in XY but not XX fetal gonads ex vivo. Mutation of SOX9 and SF1 binding sites reduced ex vivo transactivation of the pri-miR-202 promoter, demonstrating that pri-miR-202 may be a direct transcriptional target of SOX9/SF1 during testis differentiation. Our findings indicate that expression of the conserved gonad microRNA, miR-202-5p/3p, is downstream of the testis-determining factor SOX9, suggesting an early role in testis development.

Background Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU). Methodology/Principal Findings ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1. Conclusions/Significance In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

Human DAX1 duplications cause dosage-sensitive sex reversal (DSS) whereby chromosomally XY individuals can develop as females due to gonadal dysgenesis. However, the mechanism of DSS-adrenal hypoplasia congenita on X, gene 1 (DAX1) action in the fetal testis is unknown. We show that in fetal testes from XY Dax1-overexpressing transgenic mice, the expression of the key testis-promoting gene sex-determining region on Y (SRY)-box-9 (Sox9) is reduced. Moreover, in XY Sox9 heterozygotes, in which testis development is usually normal, Dax1 overexpression results in ovotestes, suggesting a DAX1-SOX9 antagonism. The ovarian portion of the XY ovotestes was characterized by expression of the granulosa cell marker, Forkhead box-L2, with complete loss of the Sertoli cell markers, SOX9 and anti-Müllerian hormone, and the Leydig cell marker CYP17A1. However, the expression of SRY and steroidogenic factor-1 (SF1), two key transcriptional regulators of Sox9, was retained in the ovarian portion of the XY ovotestes. Using reporter mice, Dax1 overexpression reduced activation of TES, the testis enhancer of Sox9, indicating that DAX1 might repress Sox9 expression via TES. In cultured cells, increasing levels of DAX1 antagonized SF1-, SF1/SRY-, and SF1/SOX9-mediated activation of TES, due to reduced binding of SF1 to TES, providing a likely mechanism for DSS.

X-linked ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome in males is characterized by mental retardation, facial dysmorphism, alpha thalassemia and urogenital abnormalities, including small testes. It is unclear how mutations in the chromatin-remodeling protein ATRX cause these highly specific clinical features, since ATRX is widely expressed during organ development. To investigate the mechanisms underlying the testicular defects observed in ATR-X syndrome, we generated ScAtrxKO (Sertoli cell Atrx knockout) mice with Atrx specifically inactivated in the supporting cell lineage (Sertoli cells) of the mouse testis. ScAtrxKO mice developed small testes and discontinuous tubules, due to prolonged G2/M phase and apoptosis of proliferating Sertoli cells during fetal life. Apoptosis might be a consequence of the cell cycle defect. We also found that the onset of spermatogenesis was delayed in postnatal mice, with a range of spermatogenesis defects evident in adult ScAtrxKO mice. ATRX and the androgen receptor (AR) physically interact in the testis and in the Sertoli cell line TM4 and co-operatively activate the promoter of Rhox5, an important direct AR target. We also demonstrate that ATRX directly binds to the Rhox5 promoter in TM4 cells. Finally, gene expression of Rhox5 and of another AR-dependent gene, Spinlw1, was reduced in ScAtrxKO testes. These data suggest that ATRX can directly enhance the expression of androgen-dependent genes through physical interaction with AR. Recruitment of ATRX by DNA sequence-specific transcription factors could be a general mechanism by which ATRX achieves tissue-specific transcriptional regulation which could explain the highly specific clinical features of ATR-X syndrome when ATRX is mutated.

The transcription factor sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, because mutations in SRY cause disorders of sex development in XY individuals. During gonadal development, Sry in pre-Sertoli cells activates Sox9 gene transcription, committing the fate of the bipotential gonad to become a testis rather than an ovary. The high-mobility group domain of human SRY contains two independent nuclear localization signals, one bound by calmodulin (CaM) and the other by importin-β. Although XY females carry SRY mutations in these nuclear localization signals that affect SRY nuclear import in transfected cells, it is not known whether these transport mechanisms are essential for gonadal development and sex determination. Here, we show that mouse Sry protein binds CaM and that a CaM antagonist reduces CaM binding, nuclear accumulation, and transcriptional activity of Sry in transfected cells. CaM antagonist treatment of cultured, sexually indifferent XY mouse fetal gonads led to reduced expression of the Sry target gene Sox9, defects in testicular cord formation, and ectopic expression of the ovarian markers Rspondin1 and forkhead box L2. These results indicate the importance of CaM for SRY nuclear import, transcriptional activity, testis differentiation, and sex determination.

The HMG-domain-containing transcription factor Sox9 is an important regulator of chondrogenesis, testis formation and development of several other organs. Sox9 is expressed in the otic placodes, the primordia of the inner ear, and studies in Xenopus have provided evidence that Sox9 is required for otic specification. Here we report novel and different functions of Sox9 during mouse inner ear development. We show that in mice with a Foxg1 Cre -mediated conditional inactivation of Sox9 in the otic ectoderm, otic placodes form and express markers of otic specification. However, mutant placodes do not attach to the neural tube, fail to invaginate, and subsequently degenerate by apoptosis, resulting in a complete loss of otic structures. Transmission-electron microscopic analysis suggests that cell–cell contacts in the Sox9 mutant placodes are abnormal, although E-cadherin, N-cadherin, and beta-catenin protein expression are unchanged. In contrast, expression of Epha4 was downregulated in mutant placodes. In embryos with a Keratin-19 Cre -mediated mosaic inactivation of Sox9 , Sox9-negative and Sox9-positive cells in the otic ectoderm sort out from one another. In these embryos only Sox9-positive cells invaginate and form one or several micro-vesicles, whereas Sox9-negative cells stay behind and die. Our findings demonstrate that, in contrast to Xenopus , Sox9 is not required for the initial specification of the otic placode in the mouse, but instead controls adhesive properties and invagination of placodal cells in a cell-autonomous manner.

In the presence of the Y-chromosomal gene Sry, the bipotential mouse gonads develop as testes rather than as ovaries. The autosomal gene Sox9, a likely and possibly direct Sry target, can induce testis development in the absence of Sry. Sox9 is thus sufficient but not necessarily essential for testis induction. Mutational inactivation of one allele of SOX9/Sox9 causes sex reversal in humans but not in mice. Because Sox9(-/-) embryos die around Embryonic Day 11.5 (E11.5) at the onset of testicular morphogenesis, differentiation of the mutant XY gonad can be analyzed only ex vivo in organ culture. We have therefore conditionally inactivated both Sox9 alleles in the gonadal anlagen using the CRE/loxP recombination system, whereby CRE recombinase is under control of the cytokeratin 19 promoter. Analysis of resulting Sox9(-/-) XY gonads up to E15.5 reveals immediate, complete sex reversal, as shown by expression of the early ovary-specific markers Wnt4 and Foxl2 and by lack of testis cord and Leydig cell formation. Sry expression in mutant XY gonads indicates that downregulation of Wnt4 and Foxl2 is dependent on Sox9 rather than on Sry. Our results provide in vivo proof that, in contrast to the situation in humans, complete XY sex reversal in mice requires inactivation of both Sox9 alleles and that Sox9 is essential for testogenesis in mice.

BackgroundIn human embryogenesis, loss of SRY (sex determining region on Y), SOX9 (SRY-related HMG box 9) or SF1 (steroidogenic factor 1) function causes disorders of sex development (DSD). A defining event of vertebrate sex determination is male-specific upregulation and maintenance of SOX9 expression in gonadal pre-Sertoli cells, which is preceded by transient SRY expression in mammals. In mice, Sox9 regulation is under the transcriptional control of SRY, SF1 and SOX9 via a conserved testis-specific enhancer of Sox9 (TES). Regulation of SOX9 in human sex determination is however poorly understood.Methodology/principal findingsWe show that a human embryonal carcinoma cell line (NT2/D1) can model events in presumptive Sertoli cells that initiate human sex determination. SRY associates with transcriptionally active chromatin in NT2/D1 cells and over-expression increases endogenous SOX9 expression. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1. SOX9 also activates hTES, augmented by SF1, suggesting a mechanism for maintenance of SOX9 expression by auto-regulation. Analysis of mutant SRY, SF1 and SOX9 proteins encoded by thirteen separate 46,XY DSD gonadal dysgenesis individuals reveals a reduced ability to activate hTES.Conclusions/significanceWe demonstrate how three human sex-determining factors are likely to function during gonadal development around SOX9 as a hub gene, with different genetic causes of 46,XY DSD due a common failure to upregulate SOX9 transcription.

SOX9 is an evolutionary conserved transcription factor that is expressed in a variety of tissues, with essential functions in cartilage, testis, heart, glial cell, inner ear and neural crest development. By comparing human and pufferfish genomic sequences, we previously identified eight highly conserved sequence elements between 290 kb 5′ and 450 kb 3′ to human SOX9. In this study, we assayed the regulatory potential of elements E1 to E7 in transgenic mice using a lacZ reporter gene driven by a 529 bp minimal mouse Sox9 promoter. We found that three of these elements and the Sox9 promoter control distinct subsets of the tissue-specific expression pattern of Sox9. E3, located 251 kb 5′ to SOX9, directs lacZ expression to cranial neural crest cells and to the inner ear. E1 is located 28 kb 5′ to SOX9 and controls expression in the node, notochord, gut, bronchial epithelium and pancreas. Transgene expression in the neuroectoderm is mediated by E7, located 95 kb 3′ to SOX9, which regulates expression in the telencephalon and midbrain, and by the Sox9 minimal promoter which controls expression in the ventral spinal cord and hindbrain. We show that E3-directed reporter gene expression in neural crest cells of the first but not of the second and third pharyngeal arch is dependent on beta-catenin, revealing a complex regulation of Sox9 in cranial neural crest cells. Moreover, we identify and discuss highly conserved transcription factor binding sites within enhancer E3 that are in good agreement with current models for neural crest and inner ear development. Finally, we identify enhancer E1 as a cis-regulatory element conserved between vertebrates and invertebrates, indicating that some cis-regulatory sequences that control developmental genes in vertebrates might be phylogenetically ancient.