Partial male-to-female reprogramming of mouse fetal testis by Sertoli cell ablation.

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions: near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3ßHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

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

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.

Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3/foxl2 in tilapia.

Germline sexual fate has long been believed to be determined by the somatic environment, but this idea is challenged by recent studies of foxl3 mutants in medaka. Here, we demonstrate that the sexual fate of tilapia germline is determined by the antagonistic interaction of dmrt1 and foxl3, which are transcriptionally repressed in male and female germ cells, respectively. Loss of dmrt1 rescued the germ cell sex reversal in foxl3Δ7/Δ7 XX fish, and loss of foxl3 partially rescued germ cell sex reversal but not somatic cell fate in dmrt1Δ5/Δ5 XY fish. Interestingly, germ cells lost sexual plasticity in dmrt1Δ5/Δ5 XY and foxl3Δ7/Δ7 XX single mutants, as aromatase inhibitor (AI) and estrogen treatment failed to rescue the respective phenotypes. However, recovery of germ cell sexual plasticity was observed in dmrt1/foxl3 double mutants. Importantly, mutation of somatic cell-specific foxl2 resulted in testicular development in foxl3Δ7/Δ7 or dmrt1Δ5/Δ5 mutants. Our findings demonstrate that sexual plasticity of germ cells relies on the presence of both dmrt1 and foxl3. The existence of dmrt1 and foxl3 allows environmental factors to influence the sex fate decision in vertebrates.

FOXL2 regulates RhoA expression to change actin cytoskeleton rearrangement in granulosa cells of chicken pre-ovulatory follicles†.

Forkhead box L2 (FOXL2) is an indispensable key regulator of female follicular development, and it plays important roles in the morphogenesis, proliferation, and differentiation of follicle granulosa cells, such as establishing normal estradiol signaling and regulating steroid hormone synthesis. Nevertheless, the effects of FOXL2 on granulosa cell morphology and the underlying mechanism remain unknown. Using FOXL2 ChIP-seq analysis, we found that FOXL2 target genes were significantly enriched in the actin cytoskeleton-related pathways. We confirmed that FOXL2 inhibited the expression of RhoA, a key gene for actin cytoskeleton rearrangement, by binding to TCATCCATCTCT in RhoA promoter region. In addition, FOXL2 overexpression in granulosa cells induced the depolymerization of F-actin and disordered the actin filaments, resulting in a slowdown in the expansion of granulosa cells, while FOXL2 silencing inhibited F-actin depolymerization and stabilized the actin filaments, thereby accelerating granulosa cell expansion. RhoA/ROCK pathway inhibitor Y-27632 exhibited similar effects to FOXL2 overexpression, even reversed the actin polymerization in FOXL2 silencing granulosa cells. This study revealed for the first time that FOXL2 regulated granulosa cell actin cytoskeleton by RhoA/ROCK pathway, thus affecting granulosa cell expansion. Our findings provide new insights for constructing the regulatory network of FOXL2 and propose a potential mechanism for facilitating rapid follicle expansion, thereby laying a foundation for further understanding follicular development.

Shifting the landscape: Dominant C-terminal rare missense FOXL2 variants in non-syndromic primary ovarian failure etiology.

Pathogenic germline variants in the FOXL2 gene are associated with Blepharophimosis, Ptosis, and Epicanthus Inversus syndrome (BPES) in humans, an autosomal dominant condition. Two forms of BPES have emerged: (i) type I (BPES-I), characterized by ocular signs and primary ovarian failure (POI), and (ii) type II (BPES-II) with no systemic associations. This study aimed to compare the distribution of FOXL2 variants in idiopathic POI/DOR (diminished ovarian reserve) and both types of BPES, and to determine the involvement of FOXL2 in non-syndromic forms of POI/DOR. We studied the whole coding region of the FOXL2 gene using next-generation sequencing in 1282 patients with non-syndromic POI/DOR. Each identified FOXL2 variant was compared to its frequency in the general population, considering ethnicity. Screening of the entire coding region of the FOXL2 gene allowed us to identify 10 different variants, including nine missense variants. Of the patients with POI/DOR, 14 (1%) carried a FOXL2 variant. Significantly, six out of nine missense variants (67%) were overrepresented in our POI/DOR cohort compared to the general or specific ethnic subgroups. Our findings strongly suggest that five rare missense variants, mainly located in the C-terminal region of FOXL2 are high-risk factors for non-syndromic POI/DOR, though FOXL2 gene implication accounts for approximately 0.54% of non-syndromic POI/DOR cases. These results support the implementation of routine genetic screening for patients with POI/DOR in clinical settings.

Transcriptional regulation of IAG by dsx and foxl-2 in mud crab (Scylla paramamosain).

Scylla paramamosain is an important cultured crab species on the southeast coast of China. However, the molecular regulation mechanism of its gonadal development still has not been thoroughly studied. Dsx (doublesex) and foxl-2 (forkhead transcription factor gene 2) are important transcription factors involved in gonadal development. So far, studies on the functions of dsx and foxl-2 in crustaceans are very limited. Insulin-like androgenic gland hormone (IAG) is an effector molecule that regulates the differentiation, development and sex maintenance of testes in crustaceans. In this study, the promoter region of Sp-IAG was predicted, and several potential binding sites of dsx and foxl-2 were found. Site-directed mutagenesis was performed on the predicted potential binding sites, and their promoter activity was analyzed. The results showed that there was a dsx and a foxl-2 binding site, respectively, that could regulate the expression of Sp-IAG. In order to verify the regulatory effect of these two transcription factors on Sp-IAG, we constructed the expression plasmids of dsx and foxl-2 and co-transfected them into HEK293T cell lines with the promoter of Sp-IAG, respectively. The results showed that dsx could significantly promote the expression of Sp-IAG, while foxl-2 could inhibit its expression substantially. Then we carried out in vivo RNA interference experiment on mud crabs. The expression of dsx and foxl-2 in crabs was interfered respectively. The results of qRT-PCR showed that the expression of Sp-IAG was significantly inhibited after interfering with dsx, while significantly increased after interfering with foxl-2, which was consistent with the cell experiment. In conclusion, dsx and foxl-2 transcription factors play opposite roles in regulating the expression of Sp-IAG.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

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.

Detection of FOXL2 C134W Mutation Status by a Novel BaseScope In Situ Hybridization Assay is Highly Sensitive and Specific for Adult Granulosa Cell Tumors.

Adult granulosa cell tumors (AGCTs) are a molecularly distinct group of malignant ovarian sex cord-stromal tumors (SCSTs) characterized by a nearly ubiquitous c.402C>G/p.C134W mutation in FOXL2 (hereafter referred to as "C134W"). In some cases, AGCT exhibits marked morphologic overlap with other SCSTs and has an identical immunophenotype, and molecular testing may be necessary to help confirm the diagnosis. However, molecular testing is time consuming, relatively expensive, and unavailable in many pathology laboratories. We describe the development and validation of an in situ hybridization (ISH) custom BaseScope assay for the detection of the FOXL2 C134W mutation. We evaluated 106 ovarian SCSTs, including 78 AGCTs, 9 juvenile granulosa cell tumors, 18 fibromas (cellular and conventional), and 1 SCST, not otherwise specified, as well as 53 epithelial ovarian tumors (42 endometrioid carcinomas and 11 carcinosarcomas) and 1 STK11 adnexal tumor for the presence or absence of FOXL2 wild-type and FOXL2 C134W RNA expression via BaseScope-ISH. Fifty-one tumors had previously undergone DNA sequencing of the FOXL2 gene. Across the entire cohort, the FOXL2 C134W probe staining was positive in 77 of 78 (98.7%) AGCTs. Two of 81 (2.5%) non-AGCTs also showed positive staining, both of which were epithelial ovarian tumors. The assay worked in tissue from blocks >20 years old. There was 100% concordance between the FOXL2 sequencing and BaseScope-ISH results. Overall, assessment of FOXL2 mutation status by custom BaseScope-ISH demonstrated 98.7% sensitivity and 97.5% specificity for the diagnosis of AGCT. BaseScope-ISH for FOXL2 C134W represents a reasonable alternative to sequencing, is quicker and less expensive, and is more easily incorporated than molecular testing into many pathology laboratories. It also has the advantage of requiring less tissue, and the neoplastic cells can be directly visualized on stained sections.

Conservation and contrast in cell states of echinoderm ovaries.

Echinoderms produce functional gametes throughout their lifespan, in some cases exceeding 200 years. The histology and ultrastructure of echinoderm ovaries has been described but how these ovaries function and maintain the production of high-quality gametes remains a mystery. Here, we present the first single cell RNA sequencing data sets of mature ovaries from two sea urchin species (Strongylocentrotus purpuratus [Sp] and Lytechinus variegatus [Lv]), and one sea star species (Patiria miniata [Pm]). We find 14 cell states in the Sp ovary, 16 cell states in the Lv ovary and 13 cell states in the ovary of the sea star. This resource is essential to understand the structure and functional biology of the ovary in echinoderms, and better informs decisions in the utilization of in situ RNA hybridization probes selective for various cell types. We link key genes with cell clusters in validation of this approach. This resource also aids in the identification of the stem cells for prolonged and continuous gamete production, is a foundation for testing changes in the annual reproductive cycle, and is essential for understanding the evolution of reproduction of this important phylum.

Estradiol promotes cell survival and induces Greb1 expression in granulosa cell tumors of the ovary through an ERα-dependent mechanism.

Granulosa cell tumor (GCT) is a form of ovarian tumor characterized by its tendency to recur years after surgical ablation. Little is known about the mechanisms involved in GCT development and progression. GCTs can produce estradiol (E2), but whether this hormone could play a role in this cancer through its nuclear receptors, i.e. ERα and ERß, remains unknown. Here, we addressed this issue by cell-based and molecular studies on human GCTs and GCT cell lines. Importantly, we observed that E2 significantly increased the growth of GCT cells by promoting cell survival. The use of selective agonists of each type of receptor, together with Esr1 (ERα) or Esr2 (ERß)-deleted GCT cells, revealed that E2 mediated its effects through ERα-dependent genomic mechanisms and ERß/ERα-dependent extra-nuclear mechanisms. Notably, the expression of Greb1, a prototypical ER target gene, was dose-dependently upregulated by E2 specifically through ERα in GCT cells. Accordingly, using GCTs from patients, we found that GREB1 mRNA abundance was positively correlated to intra-tumoral E2 concentrations. Tissue microarray analyses showed that there were various combinations of ER expression in primary and recurrent GCTs, and that ERα expression persisted only in combination with ERß in ~40% of recurrent tumors. Altogether, this study demonstrates that E2 can promote the progression of GCTs, with a clear dependence on ERα. In addition to demonstrating that GCTs can be classified as a hormone-related cancer, our results also highlight that the nature of ER forms present in recurrent GCTs could underlie the variable efficiency of endocrine therapies. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

FOXL2C134W : much ado about something!†.

Recent studies have suggested that the unique FOXL2C134W mutation, which is pathognomonic for adult granulosa cell tumours of the ovary, is a tumour suppressor gene. In a recent issue of The Journal of Pathology, a detailed study by Pilsworth et al seeks to rebut the proposition that the FOXL2C134W mutation, which uniquely characterises adult granulosa cell tumours of the ovary, leads to reduced transcript levels with the implication that FOXL2 is a tumour suppressor gene. The study provides compelling evidence that both wild-type and mutant FOXL2 transcripts and protein are expressed at equivalent levels. In the context of other recent studies, one is drawn to the conclusion that FOXL2C134W is a gain-of-function mutation whose impact is mediated through enhanced interactions with the SMAD transcription factor complex. © 2021 The Pathological Society of Great Britain and Ireland.

Molecular cloning and expression patterns of a sex-biased transcriptional factor Foxl2 in the giant freshwater prawn (Macrobrachium rosenbergii).

BACKGROUD: Macrobrachium rosenbergii is an economically important species that is widely cultivated in some Asian nations. Foxl2 is a transcriptional regulator of ovarian differentiation and development. The aim of this study was to study the bioinformatics features and expression patterns of M. rosenbergii Foxl2 (MrFoxl2). METHODS: In this study, all experimental animals were mature M. rosenbergii (9-12 cm) individuals. The foxl2 gene was identified and characterized in the genome of M. rosenbergii using molecular cloning, bioinformatic analysis, in situ hybridization, and quantitative analysis. RESULTS: The identified cDNA encoded a putative 489-amino-acid MrFoxl2 protein. Bioinformatics analysis revealed a low identity of MrFoxl2 to other crustacean orthologues. The closest phylogenetic relationship was to Foxl2 of Eriocheir sinensis. The result of in situ hybridization demonstrated that transcripts of MrFoxl2 in M. rosenbergii were identified in spermatocytes, oocytes, and secretory epithelial cells of the vas deferens. The result of q-PCR suggested that a high expression of MrFoxl2 was identified in the testis, vas deferens, and ovaries. During ovarian development, MrFoxl2 expression was the highest in stage I. CONCLUSION: Our findings suggest that MrFoxl2 may play a role in gonadal development in both female and male M. rosenbergii.

Expanded phenotypic spectrum of FOXL2 Variant c.672_701dup revealed by whole-exome sequencing in a rare blepharophimosis, ptosis, and epicanthus inversus syndrome family.

INTRODUCTION: Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is a rare genetic disease with diverse ocular malformations. This study aimed to investigate the disease-causing gene in members of a BPES pedigree presenting with the rare features of anisometropia, unilateral pathologic myopia (PM), and congenital cataracts. METHODS: The related BPES patients underwent a comprehensive ocular examination. Next, whole-exome sequencing (WES) was performed to screen for the disease-causing genetic variants. A step-wise variant filtering was performed to select candidate variants combined with the annotation of the variant's pathogenicity, which was assessed using several bioinformatic approaches. Co-segregation analysis and Sanger sequencing were then conducted to validate the candidate variant. RESULTS: The variant c.672_701dup in FOXL2 was identified to be the disease-causing variant in this rare BPES family. Combined with clinical manifestations, the two affected individuals were diagnosed with type II BPES. CONCLUSION: This study uncovered the variant c.672_701dup in FOXL2 as a disease causal variant in a rare-presenting BPES family with anisometropia, unilateral pathogenic myopia, and/or congenital cataracts, thus expanding the phenotypic spectrum of FOXL2.

A new insight into potential roles of Spfoxl-2 in the testicular development of Scylla paramamosain by RNAi and transcriptome analysis.

In our previous study, we found that the Spfoxl-2 transcript was highly expressed in gonads and explored its potential target genes in the ovary of Scylla paramamosain. In the current study, we primally analyzed its potential target genes in the testis through RNAi and RNA-Seq technology and compared with that in the ovary. The results showed that a total of 7892 unigenes were differentially expressed after Spfoxl-2 silencing in the testis, including plenty of conserved genes involved in testicular development, such as Dmrt family genes, Sox family genes, Caspase family genes, Cdk family genes, Kinesin family genes, Fox family genes and other genes. Further analysis revealed that these differentially expressed genes (DEGs) were enriched in crucial pathways involved in spermatogenesis, such as DNA replication, Cell cycle, Spliceosome, Homologous recombination, Meiosis and Apoptosis. The comparison results of potential target genes in the ovary and testis reveal 135 common potential target genes, including some genes involved in the immune response. According to our knowledge, the present work was the first to disclose the functions of foxl-2 in the testis of crustacean species using transcriptome analysis. It not only identifies key genes and pathways involved in the testicular development of S. paramamosain, but also reveals a new molecular-level understanding of the function of foxl-2 in testicular development.

Circular RNA expression profiles and CircSnd1-miR-135b/c-foxl2 axis analysis in gonadal differentiation of protogynous hermaphroditic ricefield eel Monopterus albus.

BACKGROUND: The expression and biological functions of circular RNAs (circRNAs) in reproductive organs have been extensively reported. However, it is still unclear whether circRNAs are involved in sex change. To this end, RNA sequencing (RNA-seq) was performed in gonads at 5 sexual stages (ovary, early intersexual stage gonad, middle intersexual stage gonad, late intersexual stage gonad, and testis) of ricefield eel, and the expression profiles and potential functions of circRNAs were studied. RESULTS: Seven hundred twenty-one circRNAs were identified, and the expression levels of 10 circRNAs were verified by quantitative real-time PCR (qRT-PCR) and found to be in accordance with the RNA-seq data, suggesting that the RNA-seq data were reliable. Then, the sequence length, category, sequence composition and the relationship between the parent genes of the circRNAs were explored. A total of 147 circRNAs were differentially expressed in the sex change process, and GO and KEGG analyses revealed that some differentially expressed (such as novel_circ_0000659, novel_circ_0004005 and novel_circ_0005865) circRNAs were closely involved in sex change. Furthermore, expression pattern analysis demonstrated that both circSnd1 and foxl2 were downregulated in the process of sex change, which was contrary to mal-miR-135b. Finally, dual-luciferase reporter assay and RNA immunoprecipitation showed that circSnd1 and foxl2 can combine with mal-miR-135b and mal-miR-135c. These data revealed that circSnd1 regulates foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. CONCLUSION: Our results are the first to demonstrate that circRNAs have potential effects on sex change in ricefield eel; and circSnd1 could regulate foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. These data will be useful for enhancing our understanding of sequential hermaphroditism and sex change in ricefield eel or other teleosts.

Functional Divergence of Multiple Duplicated Foxl2 Homeologs and Alleles in a Recurrent Polyploid Fish.

Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic-pituitary-gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.

FOXL2 regulates the expression of the Col4a1 collagen gene in chicken granulosa cells.

Forkhead box L2 (FOXL2), one member in the superfamily of forkhead transcription factors, is a core transcription factor specifically expressed in ovarian granulosa cells and is essential for the development of follicles. FOXL2 has been shown to regulate the transcription of genes encoding enzymes that synthesize steroid hormones and estrogen receptors and regulate the expression of collagen genes in granulosa cells. This study explored the effect of FOXL2 on collagen gene expression in granulosa cells by overexpressing Foxl2 in pregranulosa cells, prehierarchical follicles and preovulation follicle granulosa cells. The results showed that FOXL2 regulated the expression of several genes encoding collagens in chicken granulosa cells and that overexpression of Foxl2 significantly reduced the messenger RNA and protein levels of Col4a1 in different granulosa cells. Moreover, luciferase reporter and chromatin immunoprecipitation assays were performed to study how FOXL2 regulates the expression of collagen genes, and the results showed that FOXL2 directly regulated the expression of Col4a1 by binding to the motif of CAGCAGCACCAGCAG between -640 and -625 bp upstream of the coding region. The results indicated that FOXL2 could regulate the components of the extracellular matrix; however, the biological significance of this regulation needs further clarification.

Whole-exome sequencing identifies FOXL2, FOXA2 and FOXA3 as candidate genes for monogenic congenital anomalies of the kidneys and urinary tract.

BACKGROUND: Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the most common cause of chronic kidney disease in the first three decades of life. Variants in four Forkhead box (FOX) transcription factors have been associated with CAKUT. We hypothesized that other FOX genes, if highly expressed in developing kidneys, may also represent monogenic causes of CAKUT. METHODS: We here performed whole-exome sequencing (WES) in 541 families with CAKUT and generated four lists of CAKUT candidate genes: (A) 36 FOX genes showing high expression during renal development, (B) 4 FOX genes known to cause CAKUT to validate list A, (C) 80 genes that we identified as unique potential novel CAKUT candidate genes when performing WES in 541 CAKUT families and (D) 175 genes identified from WES as multiple potential novel CAKUT candidate genes. RESULTS: To prioritize potential novel CAKUT candidates in the FOX gene family, we overlapped 36 FOX genes (list A) with lists C and D of WES-derived CAKUT candidates. Intersection with list C identified a de novo FOXL2 in-frame deletion in a patient with eyelid abnormalities and ureteropelvic junction obstruction, and a homozygous FOXA2 missense variant in a patient with horseshoe kidney. Intersection with list D identified a heterozygous FOXA3 missense variant in a CAKUT family with multiple affected individuals. CONCLUSIONS: We hereby identified FOXL2, FOXA2 and FOXA3 as novel monogenic candidate genes of CAKUT, supporting the utility of a paralog-based approach to discover mutated genes associated with human disease.

FOXL2 in adult-type granulosa cell tumour of the ovary: oncogene or tumour suppressor gene?

A recurrent mutation in FOXL2 (c.402C>G; p.C134W) is present in over 95% of adult-type granulosa cell tumours (AGCTs). In contrast, various loss-of-function mutations in FOXL2 lead to the development of blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). BPES is characterised by an eyelid malformation often accompanied with primary ovarian insufficiency. Two recent studies suggest that FOXL2 C402G is a gain- or change-of-function mutation with altered DNA-binding specificity. Another study proposes that FOXL2 C402G is selectively targeted for degradation, inducing somatic haploinsufficiency, suggesting its role as a tumour suppressor. The latter study relies on data indicative of an FOXL2 allelic imbalance in AGCTs. Here we present RNA-seq data as genetic evidence that no real allelic imbalance is observed at the transcriptomic level in AGCTs. Additionally, there is no loss of protein expression in tumours harbouring the mutated allele. These data and other features of this mutation compared to other oncogenes and tumour suppressor genes argue strongly against FOXL2 being a tumour suppressor in this context. Given the likelihood that FOXL2 C402G is oncogenic, targeting the variant protein or its downstream consequences is the most viable path forward to identifying an effective treatment for this cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.