Transcription Factor FOXL2 Research Articles

FOXL2 interaction with different binding partners regulates the dynamics of ovarian development.

The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing of Foxl2 deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes, suggesting different roles across development. Here, we comprehensively investigated FOXL2's role through a multi-omics approach to characterize gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in somatic cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle formation and steroidogenesis. Deletion of one interactor, ubiquitin-specific protease 7 (Usp7), results in impairment of somatic cell differentiation, germ cell nest breakdown, and ovarian development, leading to sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.

ERK1/2-SOX9/FOXL2 axis regulates ovarian steroidogenesis and favors the follicular-luteal transition.

Estradiol and progesterone are the primary sex steroids produced by the ovary. Upon luteinizing hormone surge, estradiol-producing granulosa cells convert into progesterone-producing cells and eventually become large luteal cells of the corpus luteum. Signaling pathways and transcription factors involved in the cessation of estradiol and simultaneous stimulation of progesterone production in granulosa cells are not clearly understood. Here, we decipher that phosphorylated ERK1/2 regulates granulosa cell steroidogenesis by inhibiting estradiol and inducing progesterone production. Down-regulation of transcription factor FOXL2 and up-regulation of SOX9 by ERK underpin its differential steroidogenic function. Interestingly, the incidence of SOX9 is largely uncovered in ovarian cells and is found to regulate FOXL2 along with CYP19A1 and STAR genes, encoding rate-limiting enzymes of steroidogenesis, in cultured granulosa cells. We propose that the novel ERK1/2-SOX9/FOXL2 axis in granulosa cells is a critical regulator of ovarian steroidogenesis and may be considered when addressing pathophysiologies associated with inappropriate steroid production and infertility in humans and animals.

Abstract 453: Targeting FOXL2C402Gvulnerabilities in adult type ovarian granulosa cell tumors

Abstract Background: Nearly all adult-type granulosa cell tumors of the ovary (aGCTs) carry the same oncogenic mutation affecting the second winged helix domain of the Forkhead family transcription factor FOXL2 (c.C402G; p.C134W). There are no currently available therapies that exploit the high prevalence of FOXL2 mutations in this disease. Objectives: Exploit FOXL2-C402G oncogenic addiction in order to identify FDA-approved compounds with differential potency in KGN-FOXL2WT/C402G cells and isogenic KGN-FOXL2—/— cells. Methods: CRISPR/Cas9 was used to generate isogenic derivatives of KGN, a cell line derived from a recurrent aGCT. Parental cells (KGN-FOXL2WT/C402G) or isogenic cells lacking both FOXL2 alleles (KGN-FOXL2—/—) were functionally characterized and loss of Foxl2 protein was confirmed by immunoblot. Two drug libraries comprising a total of 2,416 compounds (majority FDA approved) were screened for effectiveness in KGN-FOXL2WT/C402G cells and KGN-FOXL2—/— cells. Our assay included staining with DAPI, followed by automated image analysis of each well to enumerate nuclei/cell numbers. Based on Fraction Affected (FA) statistics, compounds were ranked according to effectiveness in KGN-FOXL2WT/C402G cells. FA-area under the curve (FA-AUC) cutoff >0.1 yielded 152 drug candidates, categorized into 13 drug classes. 40 drug candidates, including the top 1-4 hits in each drug class were chosen for further validation. Validation rate in the confirmatory screen was at 95%. A novel primary tumor organoid culture system for aGCT was developed and validated using whole exome sequencing and immunophenotype. Selected candidate compounds were further investigated in aGCT organoid cultures from two relapsed tumors. Results: We identified classes of drugs such as the inhibitors of Bromodomain family proteins and Hydroxy-Camptothecin analogs, with enhanced activity in KGN-FOXL2WT/C402G cells relative to KGN-FOXL2—/— cells. Unexpectedly glucocorticoids as a class were found to stimulate proliferation of KGN-FOXL2WT/C402G cells whereas they lacked this activity in KGN-FOXL2—/— cells. For 36 glucocorticoids present in the screen, FA-AUC ranged from -0.3191 to -0.1342 (highest proliferation index among all test compounds) in KGN-FOXL2WT/C402G cells compared to -0.0353 to +0.1667 in KGN-FOXL2—/— cells. In recurrent aGCT organoids, the high-potency glucocorticoid dexamethasone induced cell proliferation and co-treatment with glucocorticoids increased the IC50 of paclitaxel in KGN-FOXL2WT/C402G cells from 1.43 nM to 4.04 nM. Conclusion: Mutant FOXL2 mediates a proliferative response to glucocorticoids in cultured cells and primary aGCT organoids. Paclitaxel is a standard of care chemotherapy in the treatment of aGCTs and is often administered with glucocorticoid pre-medication. The adverse effect of glucocorticoids on paclitaxel efficiency shown here should thus be taken into consideration in the clinic. Citation Format: Thomas Welte, Veena Vuttaradhi, Eleonora Khlebus, Barrett Lawson, Robert T. Hillman, Nghi Nguyen, Mary Sobieski, Reid T. Powell, Clifford Stephan. Targeting FOXL2C402Gvulnerabilities in adult type ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 453.

Integrated chromatin accessibility and DNA methylation analysis to reveal the critical epigenetic modification and regulatory mechanism in gonadal differentiation of the sequentially hermaphroditic fish, Monopterus albus

BackgroundMonopterus albus is a hermaphroditic and economically farmed fish that undergoes sex reversal from ovary to testis via ovotestis during gonadal development. The epigenetic changes that are associated with gonadal development in this species remain unclear.MethodsWe produced DNA methylome, transcriptome, and chromatin accessibility maps of the key stages of gonad development: ovary, ovotestis, and testis. The expression of the key candidate genes was detected using qRT-PCR and in situ hybridization and the methylation levels were analysed using bisulphite sequencing PCR. Promoter activity and regulation were assessed using dual-luciferase reporter assays.ResultsGonadal development exhibits highly dynamic transcriptomic, DNA methylation, and chromatin accessibility changes. We found that DNA methylation status, especially of the transcription start site, was significantly negatively correlated with gene expression while chromatin accessibility exhibited no correlation with gene expression during gonadal development. The epigenetic signatures revealed many novel regulatory elements and genes involved in sex reversal, which were validated. DNA methylation detection and site mutation of plastin-2 promoter, as a candidate gene, revealed that DNA methylation could impact the binding of transcription factor dmrt1 and foxl2 through methylation and demethylation to regulate plastin-2 expression during gonadal development.ConclusionsThese data provide novel insights into epigenetic modification and help elucidate the potential molecular mechanism by which dynamic modification of DNA methylation plays a crucial role in gonadal development.

The Oncogenic FOXL2 C134W Mutation Is a Key Driver of Granulosa Cell Tumors.

A newly generated mouse model carrying a FOXL2 mutation characteristic of adult-type granulosa cell tumors shows that FOXL2 C134W shifts the transcriptome towards a signature of granulosa cell cancer and drives tumorigenesis.

The forkhead factor Foxl2 participates in the ovarian differentiation of Chinese soft-shelled turtle Pelodiscus sinensis

The forkhead transcription factor Foxl2 plays a major role in ovarian development and function in mice and fish, and acts as a female sex-determining gene in goat. Its functional role in the sex determination and gonadal differentiation has not yet been investigated in reptiles. Here, we characterized Foxl2 gene in Chinese soft-shelled turtle Pelodiscus sinensis, exhibiting ZZ/ZW sex chromosomes. Foxl2 exhibited a female-specific embryonic expression pattern throughout the critical sex determination periods in P. sinensis. The expression of Foxl2 was induced at early stage in ZZ embryonic gonads that were feminized by estrogen treatment. Most importantly, Foxl2 knockdown in ZW embryos by RNA interference resulted in female-to-male sex reversal, characterized by obvious masculinization of gonads, significant up-regulation of testicular markers Dmrt1 and Sox9, and remarkable down-regulation of ovarian regulator Cyp19a1. Conversely, gain-of-function study showed that overexpression of Foxl2 in ZZ embryos led to largely feminized genetic males, production of Cyp19a1, and a decline in Dmrt1 and Sox9. These findings demonstrate that Foxl2 is both necessary and sufficient to initiate ovarian differentiation in P. sinensis, thereby acting as a key upstream regulator of the female pathway in a reptilian species.

Foxl2 is required for the initiation of the female pathway in a temperature-dependent sex determination system in Trachemys scripta

ABSTRACT KDM6B-mediated epigenetic modification of the testicular regulator Dmrt1 has previously been identified as the primary switch of the male pathway in a temperature-dependent sex-determination (TSD) system; however, the molecular network of the female pathway has not yet been established. Here, we have functionally characterized for the first time an upstream regulator of the female pathway, the forkhead transcription factor FOXL2, in Trachemys scripta, a turtle species with a TSD system. FOXL2 exhibited temperature-dependent female-specific expression patterns before the onset of gonadal differentiation and was preferentially localized in ovarian somatic cells. Foxl2 responded rapidly to temperature shifts and estrogen. Importantly, forced expression of Foxl2 at the male-producing temperature led to male-to-female sex reversal, as evidenced by the formation of an ovary-like structure, and upregulation of the ovarian regulators Cyp19a1 and R-spondin1. Additionally, knockdown of Foxl2 caused masculinization at the female-producing temperature, which was confirmed by loss of the female phenotype, development of seminiferous tubules, and elevated expression of Dmrt1 and Sox9. Collectively, we demonstrate that Foxl2 expression is necessary and sufficient to drive ovarian determination in T. scripta, suggesting a crucial role of Foxl2 in female sex determination in the TSD system.

MAP3K1 Variant Causes Hyperactivation of Wnt4/β-Catenin/FOXL2 Signaling Contributing to 46,XY Disorders/Differences of Sex Development.

Background: 46,XY disorders/differences of sex development (46,XY DSD) are congenital conditions that result from abnormal gonadal development (gonadal dysgenesis) or abnormalities in androgen synthesis or action. During early embryonic development, several genes are involved in regulating the initiation and maintenance of testicular or ovarian-specific pathways. Recent reports have shown that MAP3K1 genes mediate the development of the 46,XY DSD, which present as complete or partial gonadal dysgenesis. Previous functional studies have demonstrated that some MAP3K1 variants result in the gain of protein function. However, data on possible mechanisms of MAP3K1 genes in modulating protein functions remain scant. Methods: This study identified a Han Chinese family with the 46,XY DSD. To assess the history and clinical manifestations for the 46,XY DSD patients, the physical, operational, ultra-sonographical, pathological, and other examinations were performed for family members. Variant analysis was conducted using both trio whole-exome sequencing (trio WES) and Sanger sequencing. On the other hand, we generated transiently transfected testicular teratoma cells (NT2/D1) and ovary-derived granular cells (KGN), with mutant or wild-type MAP3K1 gene. We then performed functional assays such as determination of steady-state levels of gender related factors, protein interaction and luciferase assay system. Results: Two affected siblings were diagnosed with 46,XY DSD. Our analysis showed a missense c.556A > G/p.R186G variant in the MAP3K1 gene. Functional assays demonstrated that the MAP3K1R186G variant was associated with significantly decreased affinity to ubiquitin (Ub; 43–49%) and increased affinity to RhoA, which was 3.19 ± 0.18 fold, compared to MAP3K1. The MAP3K1R186G led to hyperphosphorylation of p38 and GSK3β, and promoted hyperactivation of the Wnt4/β-catenin signaling. In addition, there was increased recruitment of β-catenin into the nucleus, which enhanced the expression of pro-ovarian transcription factor FOXL2 gene, thus contributing to the 46,XY DSD. Conclusion: Our study identified a missense MAP3K1 variant associated with 46,XY DSD. We demonstrated that MAP3K1R186G variant enhances binding to the RhoA and improves its own stability, resulting in the activation of the Wnt4/β-catenin/FOXL2 pathway. Taken together, these findings provide novel insights into the molecular mechanisms of 46,XY DSD and promotes better clinical evaluation.

Identification and functional analysis of a sex-biased transcriptional factor Foxl2 in the bay scallop Argopecten irradians irradians.

Transcription factor Foxl2 is an evolutionarily conserved gene playing pivotal roles in regulation of early ovarian differentiation and maintenance in animals. However, the Foxl2 gene has not been thoroughly studied in hermaphroditic scallops. In this study, we cloned and characterized a Foxl2 (designated as AiFoxl2) from the bay scallop Argopecten irradians irradians. The open reading frame of AiFoxl2 was 1122bp encoding 373 amino acids residues and contained a conserved forkhead box domain. Quantitative real-time PCR showed that AiFoxl2 was mainly expressed in the ovary. Moreover, the highest expression of AiFoxl2 in the ovary was detected at proliferative stage and growing stage, while the lowest level was found at resting stage. During the embryonic and larval development, expression of AiFoxl2 was found first in fertilized eggs, increased significantly at the blastula stage, and reached peak value at the D-larvae stage. When AiFoxl2 was knocked down, testis development-related genes (Dmrt1, Sox7 and Sox9) were up-regulated significantly while the ovary development-related genes (Vg, HSD14, and GATA-1) were down-regulated manifestly. These findings suggested that AiFoxl2 was a female-related gene in A. i. irradians and may be involved in regulation of ovarian development and differentiation.

Conventional and unconventional interactions of the transcription factor FOXL2 uncovered by a proteome-wide analysis.

Beyond the study of its transcriptional target genes, the identification of the various interactors of a transcription factor (TF) is crucial to understand its diverse cellular roles. We focused on FOXL2, a winged-helix forkhead TF important for ovarian development and maintenance. FOXL2 has been implicated in diverse cellular processes, including apoptosis, the control of cell cycle or the regulation of steroid hormone synthesis. To reliably identify partners of endogenous FOXL2, we performed a proteome-wide analysis using co-immunoprecipitation in the murine granulosa cell-derived AT29c and the pituitary-derived alpha-T3 cell lines, using three antibodies targeting different parts of the protein. Following a stringent selection of mass spectrometry data on the basis of identification reliability and protein enrichment, we identified a core set of 255 partners common to both cell lines. Their analysis showed that we could co-precipitate several complexes involved in mRNA processing, chromatin remodeling and DNA replication and repair. We further validated (direct and/or indirect) interactions with selected partners, suggesting an unexpected role for FOXL2 in those processes. Overall, this comprehensive analysis of the endogenous FOXL2 interactome sheds light on its numerous and diverse interactors and unconventional cellular roles.

Mutational Landscape of Ovarian Adult Granulosa Cell Tumors from Whole Exome and Targeted TERT Promoter Sequencing.

Adult granulosa cell tumor (aGCT), the most common malignant ovarian sex cord-stromal tumor, is characterized by the forkhead transcription factor FOXL2 p.C134W somatic mutation. Late recurrences are relatively common but the molecular mechanisms of relapse or aggressive behavior are not known. The mutational landscape of FOXL2 p.C134W mutation-positive tumors (n = 22) was determined using whole-exome sequencing (WES). An average of 64 coding and essential splice-site variants were identified per tumor. As the TERT promoter region is poorly covered by the WES, targeted sequencing identified the TERT -124C>T promoter mutation as the only recurrent mutation (∼40% of cases). Pathway analysis suggested an association with DNA replication/repair and the EGFR family canonical pathways. Copy number analysis confirmed that gains of chromosomes 12 and 14 occur in approximately 30% of aGCT and loss of chromosome 22 occurs in approximately 40% of cases. In summary, exome-wide analysis of the mutational landscape of aGCT revealed that, except for the TERT promoter mutation, recurrence and/or aggressive behavior is not defined by activation or loss of specific genes. IMPLICATIONS: This study found that although aGCTs are defined by the presence of a common FOXL2 gene mutation, recurrence and/or aggressive behavior cannot be attributed to subsequent mutation of specific gene(s) or pathways; however, there is a high frequency of the TERT -124C>T promoter mutation, which is associated with more aggressive disease.

Genome-wide identification of FOXL2 binding and characterization of FOXL2 feminizing action in the fetal gonads.

The identity of the gonads is determined by which fate, ovarian granulosa cell or testicular Sertoli cell, the bipotential somatic cell precursors choose to follow. In most vertebrates, the conserved transcription factor FOXL2 contributes to the fate of granulosa cells. To understand FOXL2 functions during gonad differentiation, we performed genome-wide analysis of FOXL2 chromatin occupancy in fetal ovaries and established a genetic mouse model that forces Foxl2 expression in the fetal testis. When FOXL2 was ectopically expressed in the somatic cell precursors in the fetal testis, FOXL2 was sufficient to repress Sertoli cell differentiation, ultimately resulting in partial testis-to-ovary sex-reversal. Combining genome-wide analysis of FOXL2 binding in the fetal ovary with transcriptomic analyses of our Foxl2 gain-of-function and previously published Foxl2 loss-of-function models, we identified potential pathways responsible for the feminizing action of FOXL2. Finally, comparison of FOXL2 genome-wide occupancy in the fetal ovary with testis-determining factor SOX9 genome-wide occupancy in the fetal testis revealed extensive overlaps, implying that antagonistic signals between FOXL2 and SOX9 occur at the chromatin level.

Effects of exposure to BPF on development and sexual differentiation during early life stages of zebrafish (Danio rerio)

Bisphenol F (BPF) has become a predominant bisphenol contaminant in recent years. It has significant estrogenic properties in both in vivo and in vitro studies. We have previously studied the disrupting mechanisms of BPF on the hypothalamic–pituitary–gonadal axis of adult zebrafish. However, the effects of BPF exposure on development and sexual differentiation of zebrafish embryos/larvae remain unclear. To determine the effects of BPF on the critical stage of sex differentiation in zebrafish, zebrafish embryos/larvae were exposed to 1, 10, 100, and 1000 μg/L BPF from fertilization to 60 days post-fertilization (dpf). Developmental malformations were induced by exposure to BPF from 2 h post-fertilization (hpf), with a LC50 of 10,030 μg/L at 96 hpf and 9391 μg/L at 120 hpf. Long-term exposure during sex differentiation tended to result in a female sex ratio bias. Histological analyses at 60 dpf indicated that the development of ovo-testes and immature ovaries was induced by 100 and 1000 μg/L BPF. Homogenate testosterone levels decreased and 17β-estradiol levels increased in zebrafish in a concentration-dependent manner. BPF exposure suppressed gene expression of double sex, Mab3-related transcription factor 1(dmrt1), fushi tarazu factor 1d (ff1d), sry-box containing gene 9a (sox9a) and anti-Mullerian hormone (amh); induced expression of the forkhead box L2 transcription factor (foxl2), leading to increased expression of aromatase (cyp19a1a), which promoted production of estrogens, and further caused phenotypic feminization of zebrafish. These results suggest that developmental exposure to BPF has adverse effects on sexual differentiation, and the results were useful for a BPF risk assessment.

Abstract QS43: Blepharophimosis Ptosis Epicanthus Inversus Syndrome Caused by a ZC3H13 Gene Mutation

PURPOSE: Blepharophimosis Ptosis Epicanthus Inversus Syndrome (BPES) is a rare craniofacial autosomal dominant disorder characterized by severe bilateral ptosis, telecanthus, epicanthus inverses, belpharophimosis and orbital dysmorphology. BPES type 1 is accompanied by ovarian failure and attendant infertility, while type 2 manifests only eyelid and orbital involvement. While BPES is often associated with a dominantly inherited mutation in the forkhead transcription factor FOXL2 (3q23), we sought to capitalize on next generation whole exome sequencing to decipher the cause of BPES in a pedigree lacking a FOXL2 mutation. METHODS: A male patient with BPES was identified at the Yale-New Haven Hospital. Following informed consent, clinical data and blood samples from both affected and unaffected family members were obtained. Genomic DNA was extracted from whole blood samples and subjected to exome capture followed by next generation sequencing. Sequence reads were mapped to the human reference genome. Variants with minor allele frequencies in reference databases were selected and annotated for impact on the encoded protein and for conservation of the reference base and amino acid residue among orthologs across phylogeny. Sanger sequencing was employed to verify candidate mutations. In addition, all FOXL2 exons were subjected to Sanger sequencing to verify sequence status. RESULTS: A four-generation BPES pedigree was identified demonstrating autosomal dominant inheritance. In affected individuals, next generation whole exome sequencing identified a common non-sense mutation in ZC3H13 (13q14.13), which was confirmed by Sanger sequencing. A cytosine to thymine (C>T) alteration resulted in the conversion of arginine 1513 to a premature stop codon (R1513X). The variant 13:46538114 C / T was not found in dbSNP with an ExAC allele frequency of 1.651e-05. The amino acid residue arginine 1513 is evolutionarily conserved from Neurospora crassa (red bread mold) to Homo sapiens. All FOXL2 exons lacked disease-causing alterations. Conclusions: ZC3H13 is a putative tumor suppressor gene encoding a zinc finger CCCH-type containing protein. It localizes to nuclear speckles and is predicted to play a role in mRNA splicing and cell cycle progression. Our data suggest that ZC3H13 mutations represent an additional dominant subtype of BPES and point to a novel role for ZC3H13 in craniofacial development and disease pathogenesis. D.M. Balkin: None. C. Nelson-Williams: None. B.J. Sumpio: None. D.A. Scott: None. P. Le Pabic: None. T.F. Schilling: None. R.P. Lifton: None. D. Narayan: None.

The genetic make-up of ovarian development and function: the focus on the transcription factor FOXL2.

In a 46 XY individual, the presence of the Y chromosome harboring the testis-determining factor (SRY) triggers testis determination and differentiation. In a 46 XX individual, the absence of SRY and the activation of genes associated with the female pathway lead to ovarian development. The latter process has long been considered as a default pathway. However, recent studies have cast doubts on this dogma. Here, after a brief overview of the main steps of ovarian development, we focus on three genes WNT4, RSPO1 and FOXL2 that are essential for ovarian determination, differentiation and/or maintenance. Special attention is paid to FOXL2 whose mutations are responsible for the blepharophimosis syndrome, often associated with female infertility, and for cancer. We highlight the cooperation of WNT4, RSPO1 and FOXL2 within a regulatory network and the need for further research to better understand their role in defining and maintaining ovarian identity.

64 EPITHELIAL MESENCHYMAL TRANSITION AND DIFFERENTIATION OF STEROIDOGENIC FACTOR 1 MOUSE EMBRYONIC STEM CELLS INTO THE STEROIDOGENIC CELLS

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we transfected mouse embryonic stem cells (mESCs) with the mouse SF1 gene (SF1-mESCs) by using the nucleofector (Lonza), and selected SF1-mESCs by G418 250 μg mL–1. The selected cells were differentiated into granulosa-like cells through hanging-drops for 3 days, suspension culture for 1 day, then attachment onto 6-well plates. Expression of steroidogenesis-related genes and gonadal lineage-markers was analysed by real-time PCR. To test the phenotype for granulosa-like cells, transcripts of specific forkhead transcription factor (Foxl2) and follicle stimulating hormone receptor (Fshr) were measured. Also, expression of EMT-related genes, such as E-Cadherin (Cdh1), N-Cadherin (Cdh2), Snai1, Snai2 (Slug), Twist, and Vimentin, was monitored. SF1-mESCs were differentiated into the primitive streak‐mesendoderm and the steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (Hsd3b1), cytochrome P450-containing enzyme (Cyp)-11a1, and Cyp19a1 were time-dependently changed. Next, the mRNA levels of Foxl2 and Fshr representing granulosa-like cell were increased during differentiation of SF1-mESCs. Especially, the level of oestradiol and Cdh2 was increased at a specific differentiation time. We induced differentiation of mESCs into the functional granulosa-like cells through transfection of the mouse SF1 gene. These cells will be useful for further study and potential application of these cells in steroidogenesis. This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.

Combined comparative genomic hybridization and transcriptomic analyses of ovarian granulosa cell tumors point to novel candidate driver genes.

BackgroundOvarian granulosa cell tumors (GCTs) are the most frequent sex cord-stromal tumors. Several studies have shown that a somatic mutation leading to a C134W substitution in the transcription factor FOXL2 appears in more than 95% of adult-type GCTs. Its pervasive presence suggests that FOXL2 is the main cancer driver gene. However, other mutations and genomic changes might also contribute to tumor formation and/or progression.MethodsWe have performed a combined comparative genomic hybridization and transcriptomic analyses of 10 adult-type GCTs to obtain a picture of the genomic landscape of this cancer type and to identify new candidate co-driver genes.ResultsOur results, along with a review of previous molecular studies, show the existence of highly recurrent chromosomal imbalances (especially, trisomy 14 and monosomy 22) and preferential co-occurrences (i.e. trisomy 14/monosomy 22 and trisomy 7/monosomy 16q). In-depth analyses showed the presence of recurrently broken, amplified/duplicated or deleted genes. Many of these genes, such as AKT1, RUNX1 and LIMA1, are known to be involved in cancer and related processes. Further genomic explorations suggest that they are functionally related.ConclusionsOur combined analysis identifies potential candidate genes, whose alterations might contribute to adult-type GCT formation/progression together with the recurrent FOXL2 somatic mutation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1283-0) contains supplementary material, which is available to authorized users.

336 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS USING SF1 INTO THE STEROID-PRODUCING CELLS

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we obtained SF1 cDNA from mRNA of murine pituitary glands and constructed the SF1-expressing vector controlled by the CMV promoter. The SF1 transgenic mouse embryonic stem cells (SF1-mES cells) were established through transfection using the nucleofector (Lonza) and selection process using G418 at 250 μg mL–1. The SF1-mES cells were aggregated in hanging drops for 2 days and were transferred to suspension culture for an additional 1 day in mouse basal differentiation medium. Three-day-old SF1-mESC-derived EB were attached onto 6 well culture plates and differentiated into granulosa-like cells. Differentiated SF1-mES cells were analysed by expression of steroidogenesis-related genes and gonadal lineage-markers to the level of mRNA via real-time PCR method. To test the phenotype for granulosa-like cells, we confirmed transcripts of specific forkhead transcription factor FOXL2 and the follicle-stimulating hormone receptor (FSHR). On the other hand, we monitored some specific genes related to differentiation into testicular tissue. We observed the progress to primitive streak-mesendodermby gene expression analyses. In addition, we observed that differentiated SF1-mES cells express steroidogenic enzymes, such as 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, and CYP19A1. Using the advanced approach, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic and gonadal lineages. We also induced granulosa-like cells. We established the effective protocol to generate ovarian cells. The derivation of these cells explores new avenues for the further study and potential application of these cells in steroidogenesis.

A piggyBac insertion disrupts Foxl2 expression that mimics BPES syndrome in mice

Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) is an autosomal dominant genetic disorder characterized by small palpebral fissures and other craniofacial malformations, often with (type I) but could also without (type II) premature ovarian failure. While mutations of the forkhead transcription factor FOXL2 are associated with and likely be responsible for many BPES cases, how FOXL2 affects craniofacial development remain to be understood. Through a large-scale piggyBac (PB) insertion mutagenesis, we have identified a mouse mutant carrying a PB insertion ∼160 kb upstream of the transcription start site (TSS) of Foxl2. The insertion reduces, but not eliminates, the expression of Foxl2. This mutant, but not its revertant, displays BPES-like conditions such as midface hypoplasia, eyelid abnormalities and female subfertility. Further analysis indicates that the mutation does not affect mandible, but causes premature fusion of the premaxilla-maxilla suture, smaller premaxilla and malformed maxilla during midface development. We further identified an evolutionarily conserved fragment near the insertion site and observed enhancer activity of this element in tissue culture cells. Analyses using DNase I hypersensitivity assay and chromosome conformation capture assay in developing maxillary and periocular tissues suggest that the DNA region near the insertion site likely interacts with Foxl2 TSS. Therefore, this mutant presents an excellent animal model for mechanistic study of BPES and regulation of Foxl2.

Antagonists to the Wnt Cascade Exhibit Sex-Specific Expression in Gonads of Sexually Mature Shovelnose Sturgeon

Little is known regarding molecular mechanisms involved in sex determination and differentiation in sturgeon species. We addressed this knowledge gap by using next generation pyrosequencing technology to provide transcript libraries and species-specific sequences for mature gonads of shovelnose sturgeon, Scaphirhynchus platorynchus. We then mined these libraries to identify gender-specific transcripts and quantified relative transcript abundance using quantitative real-time polymerase chain reaction (qPCR). We detected a limited number of genes known to play a role in sex differentiation in other species. The sequence for dmrt1 was found only in the testes library. The abundance of dmrt1 differed slightly between the sexes, but the melt curve suggests that there may be different isoforms of dmrt1 in ovaries and testes of shovelnose sturgeon. The transcription factor foxl2 was 5.3-fold greater in ovaries than in testes. Two antagonists to the Wnt cascade, dickkopf-1 (dkk1) and dapper-1 (dact1), were found only in the ovary library. Results from qPCR indicated that dkk1 and dact1 were upregulated 1,819.1- and 207.5-fold, respectively, in ovaries compared with testes. These results suggest that antagonists to the Wnt cascade may play significant roles in sex differentiation and gonadal development in sturgeon and could serve as sex markers in this group of ancient fish.