Gerd Scherer

Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9

A human autosomal XY sex reversal locus, SRA1, associated with the skeletal malformation syndrome campomelic dysplasia (CMPD1), has been placed at distal 17q. The SOX9 gene, a positional candidate from the chromosomal location and expression pattern reported for mouse Sox9, was isolated and characterized. SOX9 encodes a putative transcription factor structurally related to the testis-determining factor SRY and is expressed in many adult tissues, and in fetal testis and skeletal tissue. Inactivating mutations on one SOX9 allele identified in nontranslocation CMPD1-SRA1 cases point to haploinsufficiency for SOX9 as the cause for both campomelic dysplasia and autosomal XY sex reversal. The 17q breakpoints in three CMPD1 translocation cases map 50 kb or more from SOX9.

Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene.

ABSTRACT For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

SOX9 is required for maintenance of the pancreatic progenitor cell pool

The factors necessary to maintain organ-specific progenitor cells are poorly understood and yet of extreme clinical importance. Here, we identify the transcription factor SOX9 as the first specific marker and maintenance factor of multipotential progenitors during pancreas organogenesis. In the developing pancreas, SOX9 expression is restricted to a mitotically active, Notch-responsive subset of PDX1+ pluripotent progenitors and is absent from committed endocrine precursors or differentiated cells. Similar to Notch mutations, organ-specific Sox9 inactivation in mice causes severe pancreatic hypoplasia resulting from depletion of the progenitor cell pool. We show that Sox9 maintains pancreatic progenitors by stimulating their proliferation, survival, and persistence in an undifferentiated state. Our finding that SOX9 regulates the Notch-effector HES1 suggests a Notch-dependent mechanism and establishes a possible genetic link between SOX factors and Notch. These findings will be of major significance for the development of in vitro protocols for cell replacement therapies.

Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium.

The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active β-catenin–Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis.

Homozygous Inactivation of Sox9 Causes Complete XY Sex Reversal in Mice

Abstract 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.

Two Independent Nuclear Localization Signals Are Present in the DNA-binding High-mobility Group Domains of SRY and SOX9

SRY and SOX9, members of the family of high-mobility group (HMG) domain transcription factors, are both essential for testis formation during human embryonic development. The HMG domain is a DNA-binding and DNA-bending motif comprising about 80 amino acid residues. It has been shown that SRY and SOX9 are nuclear proteins. Using normal or mutant SRY-β-galactosidase and SOX9-β-galactosidase fusion proteins in transfection studies involving COS-7 cells, we have identified two nuclear localization signals (NLSs) within the HMG domains of both proteins that can independently direct the fusion proteins into the nucleus. Only mutational inactivation of both NLS motifs resulted in complete exclusion of the fusion proteins from the nucleus. The NLS sequences are located at the N and C termini of the HMG domain and are a bipartite NLS motif and a basic cluster NLS motif, respectively. Both NLS motifs are conserved in the HMG domains of other transcription factors. The implications of the present results are discussed regarding (a) the apparent dual function of certain basic amino acid residues in the HMG domain of SRY in both DNA binding and in nuclear localization and (b) the possible control of SOX9 in early gonadal differentiation at the level of nuclear translocation.

Male specific expression suggests role of DMRT1 in human sex determination.

Sex determination in mammals is controlled by various transcription factors. Following the identification of SRY on the Y chromosome, several other factors have been identified. They can normally be identified as being involved in sex determination by the identification of sex reversal mutations or deletions, functional studies, and also by male-specific expression patterns in embryos. Here, it is shown that DMRT1, recently demonstrated to be deleted in 9p monosomies associated with sex reversal, is specifically expressed during sex determination in the genital ridge of human male, but not female, embryos, similar to SRY.

Testis cord differentiation after the sex determination stage is independent of Sox9 but fails in the combined absence of Sox9 and Sox8

Sox9 and Sox8 are transcription factors expressed in embryonic and postnatal Sertoli cells of the mouse testis. Sox9 inactivation prior to the sex determination stage leads to complete XY sex reversal. In contrast, there is normal embryonic testis development in Sox8 mutants which are initially fertile, but later develop progressive seminiferous tubule failure and infertility. To determine whether Sox9 is required for testis development after the initial steps of sex determination, we crossed Sox9(flox) mice with an AMH-Cre transgenic line thereby completely deleting Sox9 in Sertoli cells by E14.0. Conditional Sox9 null mutants show normal embryonic testis development and are initially fertile, but, like Sox8(-/-) mutants, become sterile from dysfunctional spermatogenesis at about 5 months. To see whether Sox8 may compensate for the absence of Sox9 during embryonic testis differentiation, we generated a Sox9 conditional knockout on a Sox8 mutant background. In the double mutants, differentiation of testis cords into seminiferous testis tubules ceases after P6 in the absence of one Sox8 allele, and after P0 in the absence of both Sox8 alleles, leading to complete primary infertility. Sox9,Sox8 double nullizygous testes show upregulation of early ovary-specific markers and downregulation of Sertoli intercellular junctions at E15.5. Their very low Amh levels still cause complete regression of the Müllerian duct but with reduced penetrance. This study shows that testis cord differentiation is independent of Sox9, and that concerted Sox9 and Sox8 function in post E14.0 Sertoli cells is essential for the maintenance of testicular function.

The PGD2 pathway, independently of FGF9, amplifies SOX9 activity in Sertoli cells during male sexual differentiation.

Activation by the Y-encoded testis determining factor SRY and maintenance of expression of the Sox9 gene encoding the central transcription factor of Sertoli cell differentiation are key events in the mammalian sexual differentiation program. In the mouse XY gonad, SOX9 upregulates Fgf9, which initiates a Sox9/Fgf9 feedforward loop, and Sox9 expression is stimulated by the prostaglandin D2 (PGD2) producing lipocalin prostaglandin D synthase (L-PGDS, or PTDGS) enzyme, which accelerates commitment to the male pathway. In an attempt to decipher the genetic relationships between Sox9 and the L-Pgds/PGD2 pathway during mouse testicular organogenesis, we found that ablation of Sox9 at the onset or during the time window of expression in embryonic Sertoli cells abolished L-Pgds transcription. By contrast, L-Pgds-/- XY embryonic gonads displayed a reduced level of Sox9 transcript and aberrant SOX9 protein subcellular localization. In this study, we demonstrated genetically that the L-Pgds/PGD2 pathway acts as a second amplification loop of Sox9 expression. Moreover, examination of Fgf9-/- and L-Pgds-/- XY embryonic gonads demonstrated that the two Sox9 gene activity amplifying pathways work independently. These data suggest that, once activated and maintained by SOX9, production of testicular L-PGDS leads to the accumulation of PGD2, which in turn activates Sox9 transcription and nuclear translocation of SOX9. This mechanism participates together with FGF9 as an amplification system of Sox9 gene expression and activity during mammalian testicular organogenesis.

A familial mutation in the testis-determining gene SRY shared by both sexes

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRYF109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.