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Foxl2 codes for a forkhead/HNF3 transcription factor essential for follicular maturation and maintenance of ovarian identity. FOXL2 mutations are associated with Blepharophimosis, Ptosis and Epicanthus inversus Syndrome (BPES) characterized by eyelid malformations (types I and II) and premature ovarian insufficiency (type I). We show that Foxl2 is not only expressed by the ovary, but also by other components of the mouse female reproductive tract, including the uterus, the cervix and the oviduct. In the uterus, Foxl2 expression is first observed in the neonatal mesenchyme and, during uterine maturation, persists in the stroma and in the deep inner myometrial layer (IML). In the adult, Foxl2 is expressed in the differentiated stromal layer, but no longer in the myometrium. Conditional deletion of Foxl2 in the postnatal (PN) uterus using Progesterone Receptor-cre (Pgr(cre/+)) mice results in infertility. During PN uterine maturation Pgr(cre/+); Foxl2(flox/flox) mice present a severely reduced thickness of the stroma layer and an hypertrophic, disorganized IML. In adult Pgr(cre/+); Foxl2(flox/flox) mice a supplementary muscular layer is present at the stroma/myometrium border and vascular smooth muscle cells fail to form a coherent layer around uterine arteries. Wnt signalling pathways play a central role in uterine maturation; in Pgr(cre/+); Foxl2(flox/flox) mice, Wnt genes are deregulated suggesting that Foxl2 acts through these signals. In humans, thickening of the IML (also called "junctional zone") is associated with reduced fertility, endometriosis and adenomyosis. Our data suggest that Foxl2 has a crucial role in PN uterine maturation and could help to understand sub-fertility predisposition in women., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Blepharophimosis, ptosis, epicanthus-inversus syndrome (BPES) is an autosomal dominant genetic disorder characterized by narrow palpebral fissures and eyelid levator muscle defects. BPES is often associated to premature ovarian insufficiency (BPES type I). FOXL2, a member of the forkhead transcription factor family, is the only gene known to be mutated in BPES. Foxl2 is essential for maintenance of ovarian identity, but the developmental origin of the facial malformations of BPES remains, so far, unexplained. In this study, we provide the first detailed account of the developmental processes leading to the craniofacial malformations associated to Foxl2. We show that, during development, Foxl2 is expressed both by Cranial Neural Crest Cells (CNCCs) and by Cranial Mesodermal Cells (CMCs), which give rise to skeletal (CNCCs and CMCs) and muscular (CMCs) components of the head. Using mice in which Foxl2 is selectively inactivated in either CNCCs or CMCs, we reveal that expression of Foxl2 in CNCCs is essential for the development of extraocular muscles. Indeed, inactivation of Foxl2 in CMCs has only minor effects on muscle development, whereas its inactivation in CNCCs provokes a severe hypoplasia of the levator palpabrae superioris and of the superior and inferior oblique muscles. We further show that Foxl2 deletion in either CNCCs or CMCs prevents eyelid closure and induces subtle skeletal developmental defects. Our results provide new insights in the complex developmental origin of human BPES and could help to understand the origin of other ocular anomalies associated to this syndrome., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

FOXL2 transcription factor is responsible for the Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES), a genetic disease involving craniofacial malformations often associated with ovarian failure. Recently, a somatic FOXL2 mutation (p.C134W) has been reported in >95% of adult-type granulosa cell tumors. Here, we have identified 10 novel FOXL2 partners by yeast-two-hybrid screening and co-immunoprecipitation. Most BPES-inducing mutated FOXL2 proteins display aggregation in cultured cells. Here, we show that two of the partners (NR2C1 and GMEB1) can be sequestered in such aggregates. This co-aggregation can contribute to the pathogenesis of FOXL2 mutations. We have also measured the effects of FOXL2 interactants on the transcriptional regulation of a series of target promoters. Some of the partners (CXXC4, CXXC5, BANF1) were able to repress FOXL2 activity indistinctively of the promoter. Interestingly, CREM-τ2α, which acted as a repressor on most promoters, increased wild-type (WT) FOXL2 activity on two promoters (PTGS2 and CYP19A1), but was unable to increase the activity of the oncogenic mutant p.C134W. Conversely, GMEB1, which also acted as a repressor on most promoters and increased WT FOXL2 activity on the Per2 promoter, increased to a greater extent the activity of the p.C134W variant. Interestingly, partners with intrinsic pro-apoptotic effect were able to increase apoptosis induction by WT FOXL2, but not by the p.C134W mutant, whereas partners with an anti-apoptotic effect decreased apoptosis induction by both FOXL2 versions. Altogether, these results suggest that the p.C134W mutated form fails to integrate signals through protein-protein interactions to regulate target promoter subsets and in particular to induce cell death.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, lead to the Blepharophimosis Syndrome. Here, we have systematically replaced the amino acids of the helices of the forkhead domain (FHD) of FOXL2 by glycine residues to assess the impact of such substitutions. A number of mutations lead to protein mislocalization, aggregation and to partial or complete loss of transactivation ability on a series of luciferase reporter systems. To rationalize the results of this glycine mutation scan, we have modeled the structure of the FHD by comparison with crystallographic data available for other FHDs. We failed to detect a clear-cut correlation between protein mislocalization or aggregation and the position of the mutation. However, we found that the localization of the side chain of each amino acid strongly correlated with the impact of its mutation on FOXL2 transactivation capacity. Indeed, when the side chains of the amino acids involved in the helices of the forkhead are supposed to point towards the hydrophobic core formed by the three main helices, a loss of function was observed. On the contrary, if the side chains point outward the hydrophobic core, protein function was preserved. The extension of this analysis to natural mutants shows that a similar correlation can be found for BPES mutations associated or not with ovarian dysfunction. Our findings reveal new insights into the molecular effects of FOXL2 mutations affecting the FHD, which represent two-thirds of intragenic mutations, and provide the first  predictive tool of their effects.

Primary ovarian insufficiency (POI) is characterized by the loss of ovarian function before the age of 40 in humans. Although most cases of POI are idiopathic, many are familial, underlying a genetic origin of the disease. Mutations in genes involved in the control of steroidogenesis, such as NR5A1 (SF-1, Steroidogenic Factor 1), CYP17, CYP19A1 (aromatase), StAR (Steroidogenic Acute Regulatory), and the forkhead transcription factor FOXL2 have been associated with different forms of POI. In males, the homeobox transcription factors Dlx5 and Dlx6 are involved in the control of steroidogenesis through the activation of GATA4-induced-StAR transcription. Here, we analyze the potential involvement of Dlx5 and Dlx6 in female reproduction. To this end, we make use of an existing mouse model in which Dlx5 and Dlx6 are simultaneously disrupted. We show that: (i) allelic reduction of Dlx5 and Dlx6 in the mouse results in a POI-like phenotype, characterized by reduced fertility and early follicular exhaustion; (ii) in granulosa cell lines, a reciprocal regulation exists between Dlx5 and Foxl2; (iii) in the mouse ovary, allelic reduction of Dlx5/6 results in the upregulation of Foxl2. We propose that the mutual regulation between Dlx5/6 and Foxl2 and their opposite effects on StAR expression might contribute to determine the homeostatic control of steroidogenesis within the ovary. Dysregulation of this homeostatic control would result in abnormal follicular maturation and reduced fertility. Our results bring new elements to our conceptual model of follicle maturation and maintenance and provide new potential genetic targets for cases of familial POI.

Mutations of FOXL2 are responsible for the Blepharophimosis-Ptotsis-Epicantus-inversus Syndrome (BPES), involving complex eyelid malformations often associated with premature ovarian failure (POF). Loss-of-function mutations are expected to lead to BPES associated with POF, whereas hypomorphic mutations would lead to BPES without ovarian dysfunction. However, multiple exceptions to the genotype-phenotype correlation have been described and missense mutations in the forkhead domain can lead to either type of BPES. This renders almost impossible the prediction of a POF condition from a given genotype. Moreover, no clear-cut correlation between nuclear and/or cytoplasmic aggregation or cytoplasmic retention of mutant FOXL2 forms and the BPES type has been established thus far. Here, we dissect the molecular and functional effects of 10 FOXL2 mutants, known to induce BPES associated with POF or not. We found a correlation between the transcriptional activity of FOXL2 variants on two different reporter promoters and the type of BPES. We used this functional classification framework to explore the behavior of 18 missense mutations leading to BPES of unknown type. The reporters used enabled us to assess the risk of POF associated with these mutations. Moreover, we document a previously overlooked correlation between subcellular mislocalization and aggregation of mutant FOXL2 and the type of BPES, known or predicted using our reporter assays. Thus, intranuclear aggregation and cytoplasmic mislocalization of mutant FOXL2 may be considered as loose predictors of ovarian dysfunction. The functional classification tool described here is a first step towards circumventing the lack of a clear-cut genotype-phenotype correlation in BPES.

FOXL2 is a forkhead transcription factor, essential for ovarian function, whose mutations are responsible for the blepharophimosis syndrome, characterized by craniofacial defects, often associated with premature ovarian failure. Here, we show that cell stress upregulates FOXL2 expression in an ovarian granulosa cell model. Increased FOXL2 transcription might be mediated at least partly by self-activation. Moreover, using 2D-western blot, we show that the response of FOXL2 to stress correlates with a dramatic remodeling of its post-translational modification profile. Upon oxidative stress, we observe an increased recruitment of FOXL2 to several stress-response promoters, notably that of the mitochondrial manganese superoxide dismutase (MnSOD). Using several reporter systems, we show that FOXL2 transactivation is enhanced in this context. Models predict that gene upregulation in response to a signal should eventually be counterbalanced to restore the initial steady state. In line with this, we find that FOXL2 activity is repressed by the SIRT1 deacetylase. Interestingly, we demonstrate that SIRT1 transcription is, in turn, directly upregulated by FOXL2, which closes a negative-feedback loop. The regulatory relationship between FOXL2 and SIRT1 prompted us the test action of nicotinamide, an inhibitor of sirtuins, on FoxL2 expression/activity. According to our expectations, nicotinamide treatment increases FoxL2 transcription. Finally, we show that 11 disease-causing mutations in the ORF of FOXL2 induce aberrant regulation of FOXL2 and/or regulation of the FOXL2 stress-response target gene MnSOD. Taken together, our results establish that FOXL2 is an actor of the stress response and provide new insights into the pathogenic consequences of FOXL2 mutations.

The Forkhead transcription factor FOXL2 plays a crucial role in ovarian development and maintenance. In humans, its mutations lead to craniofacial abnormalities, isolated or associated with ovarian dysfunction. Using a combinatorial approach, we identified and characterized a FoxL2 response element (FLRE) and showed that it is highly specific and that it diverges from that of other Forkhead transcription factors. This specificity should prevent aberrant regulation of FOXL2 targets by other members of the family and should prevent ectopic activation of the ovarian differentiation program in testes. We provide evidence that the FLRE is used in naturally occurring promoters. We show that polyAlanine expansions of FOXL2, which are the most frequent pathogenic mutations, induce a length-dependent loss of response on different artificial promoter reporters depending on the number and sequence of the FLREs that they contain. Thus, we provide clear mechanistic evidence explaining how the architecture of promoters influences their sensitivity to decreased transcription factor availability. Furthermore, we speculate that the generally absent ovarian phenotype of patients carrying the most frequent polyAlanine expansion should come from its ability to properly regulate high-affinity ovarian targets. The existence of critical high-affinity ovarian targets would be compatible with the role of FOXL2 in reproduction and ensure developmental and functional robustness. Taken together, our results give mechanistic insights on the molecular pathogenesis of FOXL2 polyAlanine expansions.

Polyalanine (polyAla) tract expansions have been associated with an increasing number of human diseases. Here, we have undertaken a functional study of the effects of polyAla expansions in the context of the transcription factor FOXL2, involved in cranio-facial and ovarian development. Using two cellular models, we show that FOXL2 polyAla expansions lead to protein mislocalization and aggregation in a length-dependent manner. The fraction of cells containing cytoplasmic staining displays a sigmoidal relationship with respect to the length of the polyAla tract, suggesting the existence of a threshold length above which protein mislocalization occurs. The existence of such a threshold might be rationalized if we consider that the longer the polyAla tract is, the higher its tendency to misfolding or to inducing spurious interactions with cytoplasmic components. To study the intranuclear dynamics of polyAla-expanded FOXL2, we performed fluorescence recovery after photobleaching experiments. The most unexpected result concerned the pathogenic protein containing 19 Ala residues in the run, which was virtually immobile, although this variant does not present a classical aggregation pattern. Luciferase assays and real time RT-PCR of many potential target genes showed that polyAla expansions induce different losses of activity according to the target promoters tested. We provide molecular explanations for these findings. Although our main focus is the mechanisms of pathogenesis of polyAla-expanded proteins, we discuss the potential relevance of polyAla length variation in micro- and macroevolution because polyAla-containing proteins tend to be transcription factors.

The discovery that the SRY gene induces male sex in humans and other mammals led to speculation about a possible equivalent for female sex. However, only partial effects have been reported for candidate genes experimentally tested so far. Here we demonstrate that inactivation of two ovarian somatic factors, Wnt4 and Foxl2, produces testis differentiation in XX mice, resulting in the formation of testis tubules and spermatogonia. These genes are thus required to initiate or maintain all major aspects of female sex determination in mammals. The two genes are independently expressed and show complementary roles in ovary morphogenesis. In addition, forced expression of Foxl2 impairs testis tubule differentiation in XY transgenic mice, and germ cell-depleted XX mice lacking Foxl2 and harboring a Kit mutation undergo partial female-to-male sex reversal. The results are all consistent with an anti-testis role for Foxl2. The data suggest that the relative autonomy of the action of Foxl2, Wnt4 and additional ovarian factor(s) in the mouse should facilitate the dissection of their respective contributions to female sex determination.

Mutations of FOXL2, a gene encoding a forkhead transcription factor, have been shown to cause the blepharophimosis-ptosis-epicanthus inversus syndrome. This genetic disorder is characterized by eyelid and craniofacial abnormalities associated or not with premature ovarian failure. We have previously shown that mutant FOXL2 with an expanded polyAlanine (polyAla) tract forms large aggregates both in the nucleus and in the cytoplasm of transfected cells, whereas the wild-type protein localizes in the nucleus in a rather diffuse manner. Premature stop codons in FOXL2 have been considered so far as null alleles. However, we demonstrate here that such nonsense mutations may lead to the production of N-terminally truncated proteins by re-initiation of translation downstream of the stop codon. Surprisingly, the truncated proteins strongly aggregate in the nucleus, partially localize in the cytoplasm and retain a fraction of the wild-type protein. We also show that a complete deletion of the polyAla tract of FOXL2 induces a significant intranuclear aggregation. Our results enlarge the spectrum of mutations inducing FOXL2 aggregation.

Genetic control of female sex differentiation from a bipotential gonad in mammals is poorly understood. We find that mouse XX gonads lacking the forkhead transcription factor Foxl2 form meiotic prophase oocytes, but then activate the genetic program for somatic testis determination. Pivotal Foxl2 action thus represses the male gene pathway at several stages of female gonadal differentiation. This suggests the possible continued involvement of sex-determining genes in maintaining ovarian function throughout female reproductive life.

FOXL2 mutations cause gonadal dysgenesis or premature ovarian failure (POF) in women, as well as eyelid/forehead dysmorphology in both sexes (the 'blepharophimosis-ptosis-epicanthus inversus syndrome', BPES). Here we report that mice lacking Foxl2 recapitulate relevant features of human BPES: males and females are small and show distinctive craniofacial morphology with upper eyelids absent. Furthermore, in mice as in humans, sterility is confined to females. Features of Foxl2 null animals point toward a new mechanism of POF, with all major somatic cell lineages failing to develop around growing oocytes from the time of primordial follicle formation. Foxl2 disruption thus provides a model for histogenesis and reproductive competence of the ovary.

Mutations in FOXL2, a forkhead transcription factor gene, have recently been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II, a rare genetic disorder. In BPES type I a complex eyelid malformation is associated with premature ovarian failure (POF), whereas in BPES type II the eyelid defect occurs as an isolated entity. In this study, we describe the identification of novel mutations in the FOXL2 gene in BPES types I and II families, in sporadic BPES patients, and in BPES families where the type could not be established. In 67% of the patients studied, we identified a mutation in the FOXL2 gene. In total, 21 mutations (17 of which are novel) and one microdeletion were identified. Thirteen of these FOXL2 mutations are unique. In this study, we demonstrate that there is a genotype--phenotype correlation for either types of BPES by the finding that mutations predicted to result in a truncated protein either lacking or containing the forkhead domain lead to BPES type I. In contrast, duplications within or downstream of the forkhead domain, and a frameshift downstream of them, all predicted to result in an extended protein, cause BPES type II. In addition, in 30 unrelated patients with isolated POF no causal mutations were identified in FOXL2. Our study provides further evidence that FOXL2 haploinsufficiency may cause BPES types I and II by the effect of a null allele and a hypomorphic allele, respectively. Furthermore, we propose that in a fraction of the BPES patients the genetic defect does not reside within the coding region of the FOXL2 gene and may be caused by a position effect.