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FOXP1 encodes a transcription factor involved in tissue regulation and cell-type-specific functions. Haploinsufficiency of FOXP1 is associated with a neurodevelopmental disorder: autosomal dominant mental retardation with language impairment with or without autistic features. More recently, heterozygous FOXP1 variants have also been shown to cause a variety of structural birth defects including central nervous system (CNS) anomalies, congenital heart defects, congenital anomalies of the kidney and urinary tract, cryptorchidism, and hypospadias. In this report, we present a previously unpublished case of an individual with congenital diaphragmatic hernia (CDH) who carries an approximately 3.8 Mb deletion. Based on this deletion, and deletions previously reported in two other individuals with CDH, we define a CDH critical region on chromosome 3p13 that includes FOXP1 and four other protein-coding genes. We also provide detailed clinical descriptions of two previously reported individuals with CDH who carry de novo, pathogenic variants in FOXP1 that are predicted to trigger nonsense-mediated mRNA decay. A subset of individuals with putatively deleterious FOXP4 variants has also been shown to develop CDH. Since FOXP proteins function as homo- or heterodimers and the homologs of FOXP1 and FOXP4 are expressed at the same time points in the embryonic mouse diaphragm, they may function together as a dimer, or in parallel as homodimers, to regulate gene expression during diaphragm development. Not all individuals with heterozygous, loss-of-function changes in FOXP1 develop CDH. Hence, we conclude that FOXP1 acts as a susceptibility factor that contributes to the development of CDH in conjunction with other genetic, epigenetic, environmental, and/or stochastic factors.

RationaleCongenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood.ObjectivesTo establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing.MethodsWe developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from CDH patients and preterm and term, non-CDH controls were derived and analyzed by bulk RNA-sequencing, ATAC-sequencing, and air-liquidinterface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH.Measurements and Main ResultsTranscriptomic and epigenetic profiling of CDH and non-CDH basal stem cells reveals a disease-specific, proinflammatory signature independent of severity or hernia size. In addition, CDH basal stem cells exhibit defective epithelial differentiationin vitrothat recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, steroid treatment normalizes epithelial differentiation phenotypes of human CDH basal stem cellsin vitroand in nitrofen rat tracheasin vivo.ConclusionsOur findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

We identified the anti-Mullerian hormone (also known as Müllerian inhibiting substance or MIS) as an inhibitory hormone that induces long-term contraception in mammals. The type II receptor to this hormone, AMHR2 (also known as MISR2), represents a promising druggable target for the modulation of female reproduction with a mechanism of action distinct from steroidal contraceptives. We designed an in vitro platform to screen and validate small molecules that can activate MISR2 signaling and suppress ovarian folliculogenesis. Using a bone morphogenesis protein (BMP)–response element luciferase reporter cell–based assay, we screened 5,440 compounds from a repurposed drug library. Positive hits in this screen were tested for specificity and potency in luciferase dose–response assays, and biological activity was tested in ex vivo Mullerian duct regression bioassays. Selected candidates were further evaluated in ex vivo follicle/ovary culture assays and in vivo in mice and rats. Here, we report that SP600125, CYC-116, gandotinib, and ruxolitinib can specifically inhibit primordial follicle activation and repress folliculogenesis by stimulating the MISR2 pathway.

PURPOSE: To enhance the understanding of the clinical significance of anti-Müllerian hormone (AMH) in follicular fluid, we aimed to determine the variability of AMH concentrations in follicular fluid within and across IVF cycles and whether high follicular fluid AMH concentrations are associated with improved clinical IVF outcomes. METHODS: This was a retrospective cohort study of companion follicular fluid and serum samples from 162 women enrolled in the Environment and Reproductive Health (EARTH) Study between 2010 and 2016. AMH concentrations were quantified using a sandwich enzyme-linked immunosorbent assay. Spearman correlation and intra-class correlation (ICC) were calculated to assess variability of follicular fluid AMH, and generalized linear mixed models were used to evaluate the associations of FF AMH with IVF outcomes. RESULTS: The median (interquartile range, IQR) age of the 162 women was 34.0 years (32.0, 37.0). Follicular fluid AMH concentrations were highly correlated between follicles within each IVF cycle (Spearman r = 0.78 to 0.86) and across cycles for each woman (ICC 0.87 (95% CI 0.81 to 0.92)). Compared with women in the highest tertile of FF AMH (mean AMH = 2.3 ng/ml), women in the lowest tertile (mean AMH = 0.2 ng/ml) had lower serum AMH (T1 = 0.1 ng/ml vs. T3 = 0.6 ng/ml, p

Background A test that helps predict the time to the final menstrual period (FMP) has been sought for many years. Objective To assess the ability of antimullerian hormone (AMH) measurements to predictions the time to FMP. Design Prospective longitudinal cohort study. Setting The Study of Women’s Health Across the Nation. Participants and Measurements AMH and FSH were measured in 1537 pre- or early perimenopausal women, mean age 47.5 ± 2.6 years at baseline, then serially until 12 months of amenorrhea occurred. AMH was measured using a 2-site ELISA with a detection limit of 1.85 pg/mL. Main Outcome Measure Areas under the receiver operating curves (AUC) for AMH-based and FSH-based predictions of time to FMP, stratified by age. Probabilities that women would undergo their FMP in the next 12, 24, or 36 months across a range of AMH values were assessed. Results AUCs for predicting that the FMP will occur within the next 24 months were significantly greater for AMH-based than FSH-based models. The probability that a woman with an AMH 100 pg/mL would not undergo her FMP within the next 12 months ranged from 97% in women Conclusions AMH measurement helps estimate when a woman will undergo her FMP, and, in general, does so better than FSH.

The estrous cycle is regulated by rhythmic endocrine interactions of the nervous and reproductive systems, which coordinate the hormonal and ovulatory functions of the ovary. Folliculogenesis and follicle progression require the orchestrated response of a variety of cell types to allow the maturation of the follicle and its sequela, ovulation, corpus luteum formation, and ovulatory wound repair. Little is known about the cell state dynamics of the ovary during the estrous cycle and the paracrine factors that help coordinate this process. Herein, we used single-cell RNA sequencing to evaluate the transcriptome of >34,000 cells of the adult mouse ovary and describe the transcriptional changes that occur across the normal estrous cycle and other reproductive states to build a comprehensive dynamic atlas of murine ovarian cell types and states.

Proper lung development and function requires two independent but interrelated processes: branching morphogenesis to form the airway tree, and alveolar cell differentiation for peripheral gas exchange. The disruption of either branching or differentiation results in severe respiratory deficiencies and often in neonatal death. The molecular mechanisms that control branching patterns and the transition to alveolar differentiation are not completely understood. Here we report on the in vitro and in vivo characterization of the lungs of mouse embryos lacking a functional Svep1 gene. Our data demonstrate that the SVEP1 extracellular matrix protein is critical for airway patterning and for the process of transitioning from branching to alveolar maturation. Svep1-/- embryos on a C57BL/6J genetic background are characterized by hypoplastic lungs and a disorganized increase in distal airway tips which disrupts airway architecture and lobe shape. The lungs of Svep1 knockout embryos also have defects in alveolar differentiation. In vitro lung explant experiments demonstrated that SVEP1 normally inhibits branching morphogenesis and that treatment with a SVEP1 peptide can rescue the branching defects observed in Svep1 knockouts. Our findings reveal for the first time that Svep1 is essential for constructing the basic airway architecture and for the transition from lung branching to alveolar differentiation. Our results suggest therapeutic strategies to enhance lung development in patients with life-threatening respiratory disorders such as the lung hypoplasia and prematurity observed in neonates with congenital diaphragmatic hernia (CDH).

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with a novel X-linked condition characterized by diaphragm defects, variable anterior body wall anomalies, and/or facial dysmorphism. Using linkage analysis and whole exome or whole genome sequencing, we identified novel missense variants in the actin binding domains of plastin 3 (PLS3), a gene encoding an actin bundling protein, that co-segregate with disease in all families. Loss-of-function variants in PLS3 have been described previously in association with X-linked osteoporosis. To address these seemingly disparate clinical phenotypes, we performed in silico protein modeling and cellular overexpression experiments, which suggest that the affected residues in individuals with CDH are important for actin binding and result in disorganization of the actin cytoskeleton and a reduction in normal actin stress fiber formation. A mouse knock-in model of a variant identified in one of the families, p.W499C, shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal wall defects. Both the mouse model and one surviving adult patient with a PLS3 variant were observed to have increased, rather than decreased, bone mineral density. Together, these clinical and functional data in human and mouse reveal that specific missense variants affecting the actin binding domains of PLS3 may have a gain-of-function effect and cause a new Mendelian disorder.

Significance Anti-Müllerian hormone (AMH) plays a crucial role in male sex differentiation and female reproductive development. As such, AMH is widely used as a biomarker for measuring a woman’s fertility, estimating onset of menopause, and has been implicated in reproductive syndromes such as polycystic ovarian syndrome and premature ovarian failure. Despite its biological relevance, how AMH functions on the molecular level is not well understood. In this study, we show that AMH engages its receptor, AMHR2, using an extensive interface distinct from other type II receptors. Furthermore, we identify several regions in both AMH and AMHR2 that are responsible for specificity and required for AMH signaling., Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-β) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-β family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (Lon Peptidase 1, Mitochondrial) and ALYREF (Aly/REF Export Factor) as novel candidate CDH genes based on de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 cases and 11,220 ancestry-matched population controls and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in familial cases. Approximately 3% of our CDH cohort was heterozygous with ultra-rare predicted damaging variants in LONP1 who have a range of clinical phenotypes including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium specific deletion of Lonp1 die immediately after birth and have reduced lung growth and branching that may at least partially explain the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies.

Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is thought to be a negative regulator of primordial follicle activation. We have previously reported that treatment with exogenous MIS can induce complete ovarian suppression within 5 weeks of treatment in mice. To investigate the kinetics of the return of folliculogenesis following the reversal of suppression, we treated animals with recombinant human MIS (rhMIS) protein for 40 days in adult female Nu/Nu mice and monitored the recovery of each follicle type over time. Following cessation of MIS therapy, secondary, and antral follicles returned within 30 days, along with the normalization of reproductive hormones, including LH, FSH, MIS, and Inhibin B. Furthermore, 30 days following MIS pretreatment, the number of antral follicles were significantly higher than controls, and superovulation with timed pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation at this time point resulted in an approximately threefold increased yield of eggs. Use of the combined rhMIS-gonadotropin superovulation regimen in a diminished ovarian reserve (DOR) mouse model, created by 4-vinylcyclohexene dioxide treatment, also resulted in a twofold improvement in the yield of eggs. In conclusion, treatment with rhMIS can induce a reversible ovarian suppression, following which a rapid and synchronized large initial wave of growing follicles can be harnessed to enhance the response to superovulation. Therapies modulating MIS signaling may therefore augment the response to current ovarian stimulation protocols and could be particularly useful to women with DOR or poor responders to controlled ovarian hyperstimulation during in vitro fertilization.

PURPOSE OF REVIEW The nascent field of oncofertility is quickly gaining traction as novel experimental treatments are being developed, driving a renewed interest in Mullerian inhibiting substance (MIS) as an ovarian fertoprotectant. RECENT FINDINGS MIS is unique in its mechanisms of ovarian protection by virtue of acting directly on granulosa cells of primordial follicles and for being a benign reproductive hormone, with few side effects. We will explore in this review how it may be utilized to protect the ovary from chemotherapy, or to enhance ovarian tissue cryopreservation therapy. We will also examine potential mechanisms of action of MIS across multiple cell types, as well as current limitations in our understanding of the pharmacology of recombinant MIS. SUMMARY The usefulness of MIS as a fertoprotectant may be dependent on the mechanisms of gonadotoxicity of each chemotherapeutic. Further investigation is needed to determine how to best deliver and combine MIS treatment to existing fertility management strategies.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies.

Significance This study improves our understanding of the role of Müllerian inhibiting substance (MIS/AMH) in antagonizing primordial follicle activation. By applying scRNA-seq methods to neonatal mouse ovaries, we revealed transcriptional signatures associated with MIS treatment in ovarian cell types present during the first wave of folliculogenesis and follicle development. We showed that MIS inhibits pregranulosa cell differentiation and the proliferation of ovarian surface epithelium and stromal cells. MIS also uncoupled granulosa and germ cell maturation, leading to abnormal development of activated follicles. These findings identify markers and pathways related to primordial follicle quiescence that could be targeted in contraception, preservation of ovarian reserve during aging or chemotherapy, or synchronization of preantral follicle growth for IVF., Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

Anti-Müllerian hormone (AMH) or Müllerian inhibiting substance is a unique member of the TGF-β family responsible for development and differentiation of the reproductive system. AMH signals through its own dedicated type II receptor, anti-Müllerian hormone receptor type II (AMHR2), providing an exclusive ligand-receptor pair within the broader TGF-β family. In this study, we used previous structural information to derive a model of AMH bound to AMHR2 to guide mutagenesis studies to identify receptor residues important for AMH signaling. Nonconserved mutations were introduced in AMHR2 and characterized in an AMH-responsive cell-based luciferase assay and native PAGE. Collectively, our results identified several residues important for AMH signaling within the putative ligand binding interface of AMHR2. Our results show that AMH engages AMHR2 at a similar interface to how activin and BMP class ligands bind the type II receptor, ACVR2B; however, there are significant molecular differences at the ligand interface of these 2 receptors, where ACVR2B is mostly hydrophobic and AMHR2 is predominately charged. Overall, this study shows that although the location of ligand binding on the receptor is similar to ACVR2A, ACVR2B, and BMPR2; AMHR2 uses unique ligand-receptor interactions to impart specificity for AMH.

Research question Is formulated and lyophilized, recombinant human Mullerian inhibiting substance, also known as anti-Mullerian hormone (AMH), suitable for the preparation of a WHO international standard to calibrate AMH immunoassays? Design The AMH content of a trial preparation, coded SS-581, was determined by five laboratories using seven immunoassay methods. Participants were requested to report the content of the preparation in terms of their method calibrators through the measurement of a minimum of five concentrations in the linear part of the dose-response curve. Participants were also asked to measure, concomitantly, a panel of six serum samples containing AMH at concentrations of 0.1–13.0 ng/ml. Results Across all assays, including two automated assays in development, the geometric mean content was 361.76 ng/ampoule with a geometric coefficient of variation (GCV%) of 39.95%. When measured by immunoassays that were commercially available at the time of the study, the mean content was 423.08 ng/ampoule, with a GCV% of 26.67%. The inter-method geometric means of five serum samples with an AMH concentration >0.3 ng/ml and measured concomitantly with dilutions of SS-581 varied with a range of GCV% of 14.90–22.35%, which may reflect the use of serum sample value transfer to calibrate current immunoassays, some of which use non-human AMH calibrators. The AMH in trial preparation SS-581 was shown to be biologically active in the Mullerian duct regression assay. Conclusions A reference material prepared using human recombinant AMH is a promising candidate for the preparation of an international standard for AMH for immunoassays calibrated to recombinant human AMH.

Context There is increasing evidence for Mullerian-inhibiting substance (MIS)/anti-Mullerian hormone (AMH) physiologic activity in the human uterus, so it is relevant to study how MIS/AMH levels impact pregnancy. Objective To investigate the association of MIS/AMH levels with the risk of adverse obstetric outcomes. Design Retrospective cohort study. Setting Academic fertility center. Patients Women who became pregnant through in vitro fertilization between January 2012 and October 2016. Exclusion criteria were: oocyte donation, gestational carrier, multiple gestations, miscarriage before 20 weeks, or medically indicated preterm deliveries. Interventions None. Main Outcome Measures There were two primary outcomes, preterm birth and cesarean delivery for arrest of labor. Because MIS/AMH level is highly skewed by certain infertility diagnoses, the preterm birth analysis was stratified by polycystic ovary syndrome (PCOS) diagnosis, and the cesarean delivery for arrest of labor analysis was stratified by diminished ovarian reserve diagnosis. χ2, Mann-Whitney, and t tests were used as appropriate. A P value of

Significance This study describes the results of a large-scale case control analysis of copy number variants (CNVs) in a cohort of patients with congenital diaphragmatic hernia (CDH) and a large number of healthy population-matched controls. Using a customized array comparative genomic hybridization system, we have identified six CNVs that are associated with CDH with statistical significance (P < 0.05). These regions validate several hypothesized CDH candidate genes and identify additional genes and pathways that contribute to the pathogenesis of CDH. The estimated frequency of pathogenic CNVs in this cohort is 13%, which underscores the critical contribution of CNVs in CDH. This study also provides a model approach that is broadly applicable to other structural birth defects and identifies candidates for future functional studies., Congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and hypoplasia of the lungs, is one of the most common and severe birth defects, and is associated with high morbidity and mortality rates. There is growing evidence demonstrating that genetic factors contribute to CDH, although the pathogenesis remains largely elusive. Single-nucleotide polymorphisms have been studied in recent whole-exome sequencing efforts, but larger copy number variants (CNVs) have not yet been studied on a large scale in a case control study. To capture CNVs within CDH candidate regions, we developed and tested a targeted array comparative genomic hybridization platform to identify CNVs within 140 regions in 196 patients and 987 healthy controls, and identified six significant CNVs that were either unique to patients or enriched in patients compared with controls. These CDH-associated CNVs reveal high-priority candidate genes including HLX, LHX1, and HNF1B. We also discuss CNVs that are present in only one patient in the cohort but have additional evidence of pathogenicity, including extremely rare large and/or de novo CNVs. The candidate genes within these predicted disease-causing CNVs form functional networks with other known CDH genes and play putative roles in DNA binding/transcription regulation and embryonic development. These data substantiate the importance of CNVs in the etiology of CDH, identify CDH candidate genes and pathways, and highlight the importance of ongoing analysis of CNVs in the study of CDH and other structural birth defects.

Müllerian inhibiting substance/anti-Müllerian hormone (MIS/AMH) has been suggested as a biotherapeutic agent in gynecological cancers that highly express the MIS/AMH type II receptors (MISRII/AMHRII) but the anticancer mechanisms by which MIS/AMH acts are not fully understood. Our experiments show that MIS/AMH inhibits ovarian cancer by deregulating the Wnt signal pathway via the β-catenin interacting protein (ICAT). MIS/AMH inhibition of ICAT by small interfering RNAs (siRNA) decreased ICAT driven ovarian cancer cell viability as measured by the methylthiazoltetrazolium assay, reversed cell cycle arrest and annexin V expression and diminished migration by scratch wound assay. Changes in expression of regulatory proteins were shown by western blotting. We determined that MIS/AMH upregulated ICAT in ovarian cancer cell line which caused decreased cell viability, cell cycle arrest and apoptosis. This effect, however, was blocked when ICAT was downregulated by siRNA. The present study demonstrates a role for ICAT in MIS/AMH mediated inhibition of the Wnt signaling pathway in ovarian cancer.

The Mullerian ducts are the anlagen of the female reproductive tract, which regress in the male fetus in response to MIS. This process is driven by subluminal mesenchymal cells expressing Misr2, which trigger the regression of the adjacent Mullerian ductal epithelium. In females, these Misr2+ cells are retained, yet their contribution to the development of the uterus remains unknown. Here, we report that subluminal Misr2+ cells persist postnatally in the uterus of rodents, but recede by week 37 of gestation in humans. Using single-cell RNA sequencing, we demonstrate that ectopic postnatal MIS administration inhibits these cells and prevents the formation of endometrial stroma in rodents, suggesting a progenitor function. Exposure to MIS during the first six days of life, by inhibiting specification of the stroma, dysregulates paracrine signals necessary for uterine development, eventually resulting in apoptosis of the Misr2+ cells, uterine hypoplasia, and complete infertility in the adult female., eLife digest In the womb, mammals possess all of the preliminary sexual structures necessary to become either male or female. This includes the Mullerian duct, which develops into the Fallopian tubes, uterus, cervix, and vagina in female fetuses. In male fetuses, the testis secretes a hormone called Mullerian inhibiting substance (MIS). This triggers the activity of a small group of cells, known as Misr2+ cells, that cause the Mullerian duct to degenerate, preventing males from developing female sexual organs. It was not clear what happens to Misr2+ cells in female fetuses or if they affect how the uterus develops. Saatcioglu et al. now show that in newborn female mice and rats, a type of Misr2+ cell that sits within a thin inner layer of the developing uterus still responds to MIS. At this time, the uterus is in a critical early period of development. Treating the mice and rats with MIS protein during their first six days of life eventually caused the Misr2+ cells to die. The treatment also prevented a layer of connective tissue, known as the endometrial stroma, from forming in the uterus. As a result, the mice and rats were infertile and had severely underdeveloped uteri. While the Misr2+ cells are present in newborn rats and mice, Saatcioglu et al. found that they disappeared before birth in humans. However, the overall results suggest that Misr2+ cells act as progenitor cells that develop into the cells of the endometrial stroma. Future work could investigate the roles these cells play in causing uterine developmental disorders and infertility disorders. Furthermore, the finding that MIS inhibits the Misr2+ cells could help researchers to develop treatments for uterine cancer and other conditions where the cells of the uterus grow and divide too much.

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10-8), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH., Author summary Congenital diaphragmatic hernia (CDH) is a life-threatening condition affecting about 1 every 3000 newborns. Although the role of genetics in the pathogenesis of CDH has been well established, only a handful of disease genes have been identified so far. We and other have previously shown that de novo variants, those carried by the cases but not inherited from parents, are enriched in sporadic CDH cases consistent with their negative effects on reproductive fitness. To further investigate the genetics of CDH, we analyzed de novo variants in 362 proband-father-mother trios from whole exome or genome sequencing data and identified four patients carrying damaging variants in MYRF, a membrane associated transcription factor that is highly expressed in developing diaphragm and heart. We then ascertained a total of 12 patients with MYRF de novo variants, and found they shared common phenotype characteristics including congenital abnormalities in diaphragm, heart and reproductive organs. The high rate of recurrence and similar phenotypic manifestations suggest that de novo variants of MYRF have pleiotropic effects and cause a novel syndrome. The identified new gene is reminiscent of previously identified CDH genes (e.g., GATA4, GATA6, NR2F2, ZFPM2, and WT1) that are also associated with other developmental disorders. Indeed, we found in our cohort more than 20 damaging de novo variants in genes implicated in other developmental disorders but not previously linked to CDH. The overlap was unlikely to occur by chance and can be best explained by their pleiotropic effects. We also showed that, despite the shared genetic basis with other disorders, damaging de novo variants in CDH as a whole were enriched in specific functional pathways that recapitulated our current knowledge about diaphragm development. So additional candidate genes can be prioritized based on the genetic pleiotropy and functional specificity. The findings have general implications in design and analysis in genetic studies of rare birth defects.

Congenital diaphragmatic hernia (CDH) is one of the most common and lethal congenital anomalies, and significant evidence is available in support of a genetic contribution to its etiology, including single-gene knockout mice associated with diaphragmatic defects, rare monogenetic disorders in humans, familial aggregation, and association of CDH with chromosomal abnormalities. Structural lung defects in the form of lung hypoplasia are almost invariably seen in patients with CDH and frequently in animal models of this condition. Better understanding of the mechanisms of pulmonary defects in CDH has the potential for creating targeted therapies, particularly in postnatal stages, when therapeutics can have maximum clinical impact on the surviving cohorts. Successful treatment of CDH is dependent on the integration of human genomic and genetic data with developmental expression profiling, mouse knockouts, and gene network and pathway modeling, which have generated a large number of candidate genes and pathways for follow-up studies. In particular, defective alveolarization appears to be a common and potentially actionable phenotype in both patients and animal models.

COUP-TFII (NR2F2) is mapped to the 15q26 deletion hotspot associated with the common and highly morbid congenital diaphragmatic hernia (CDH). Conditional homozygous deletions of COUP-TFII in mice result in diaphragmatic defects analogous to the human Bochdalek-type hernia phenotype. Despite evidence from animal models however, mutations in the coding sequence of COUP-TFII have not been reported in patients, prompting the speculation that additional coding or non-coding sequences in the 15q26 locus are necessary for diaphragmatic hernias to develop. In this report, we describe a case of a patient with a heterozygous de novo COUP-TFII frameshift mutation, presenting with CDH and an atrial septal defect. The p.Pro33AlafsTer77 mutation specifically disrupts protein isoform 1 which contains the DNA binding domain. In addition, we review other COUP-TFII sequence variations and deletions that have been described in cases of CDH. We conclude that COUP-TFII mutations can cause diaphragmatic hernias, and should be included in the differential diagnosis of CDH patients, particularly those with comorbid congenital heart defects. © 2016 Wiley Periodicals, Inc.

Background: Epithelial ovarian cancers are the fifth leading cause of cancer-related deaths in American women, with a five-year overall survival rate for patients diagnosed at advanced stages of less than 30%. High-grade serous ovarian cancers (HGSOCs), the most common histologic subtype, often initially responds to platinum-based chemotherapy. However, over 80% of patients experience chemoresistant recurrence in advanced-stage disease. Methods: We have established over 40 patient-derived HGSOC cell lines from ascites of patients with recurrences and hypothesized that these refractory HGSOC cell lines may each possess genetic liabilities and be dependent on unique pathways for survival that can be identified using an unbiased screening approach for the development of personalized therapies. In one such example, we identified candidate genes that are uniquely essential to a single HGSOC patient (ptD), as proof of concept, using a genome-wide lentiviral sgRNA library, containing 187,536 sgRNAs targeting 18,543 protein-coding human genes and 1,504 nontargeting control sgRNAs in a Cas9-containing vector. Results: The unbiased CRISPR screen identified BRAF (p=2E-6) as a specific susceptibility of ptD, confirming the known oncogene addiction of this primary cell line carrying a V600E mutation, as well as a significant sensitivity to JUNB deletion (p=6E-6). Knockout of the JUNB gene, using lentiviruses expressing guide RNAs and Cas9 protein targeted to JUNB, repressed cell proliferation in vitro in ptD cells but not other primary cell lines. Moreover, in a xenograft mouse model, JUNB KO ptD cells displayed slower tumor growth than control JUNB WT cells. Conclusion: We have confirmed in a single patient cell line our ability to identify and validate unique genetic liabilities, suggesting the approach can be scaled to rapidly screen primary HGSOC samples for personalized therapy. Citation Format: Nobuhiro Takahashi, Heather Keys, Raghav Mohan, Lihua Zhang, Yi Li, Hatice D. Saatcioglu, Nicholas Nagykery, Patricia K. Donahoe, David Sabatini, David Pépin. Whole-genome CRISPR/CAS9 screen using patient samples reveals JunB as a unique genetic liability [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A63.

Measurement of serum concentrations of Mullerian inhibiting substance (MIS), also known as anti-Mullerian Hormone (AMH) by immunoassay is gaining clinical acceptance and widespread use for the diagnosis of ovarian conditions and for prediction of the response to ovarian stimulation protocols as part of assisted reproductive therapies. Provision of an International Standard to harmonize immunoassay methods is required. It is desirable for the content of a future International Standard to be assigned in mass units for consistency with the units reported by current methods. Isotope dilution mass spectrometry (IDMS), a physicochemical method with traceability to the SI (Systeme International d'Unites) unit of mass, is a candidate approach to provide orthogonal data to support this mass assignment. Here, we report on the development of an IDMS method for quantitation of AMH using three peptides from different regions of the AMH monomer as surrogates for the measurement of AMH. We show the sensitivity and linearity of the standard peptides and demonstrate the reproducibility and consistency of the measurement amongst the three peptides for determining the AMH content in buffered preparations and in trial preparations of recombinant AMH, lyophilised in the presence of an excess of bovine casein.

Müllerian inhibiting substance (MIS) has been shown to inhibit growth of a number of tumors in vitro and/or in vivo, but the downstream pathways which it regulates are not fully understood. In the present study we show that MIS type II receptor was highly expressed in AN3CA cells, a cell line derived from human endometrial cancer cell in which MIS-treatment caused a reduction of cell viability, and induced cellular apoptosis and genes involved cell cycle arrest. To understand the genome-wide effects of MIS on gene regulation, we performed serial gene expression analyses from 0 to 96 h at 24 h intervals after treating AN3CA cells with MIS. Transcriptomic analysis of molecular changes induced by MIS identified 2,688 differentially expressed genes that were significantly up- or down-regulated during the 96 h study period. When the 2,688 differentially expressed genes were mapped to known biological processes, Wnt-, cancer-, proteolysis-, cytoskeleton-, cell cycle-, apoptosis-, and MAPK-signaling pathways emerged as the functions most significantly changed by MIS in AN3CA cells. Furthermore, western blot analysis validated that protein expression of cell cycle inhibitory genes, apoptotic protease activating factor-1 (APAF-1), β-catenin-interacting protein (ICAT), Rb related protein 130 (p130), and inhibitor of disheveled Dvl and Axin complex (IDAX), were gradually increased over the time of the study, whereas downstream cell cycle activating genes, cyclin-dependent kinase 2 (CDK2) and phospho-c-Jun were downregulated in MIS-treated AN3CA cells. These transcriptome analyses support previous observations that MIS functions as a tumor suppressor, potentially by regulating signaling pathways that could contribute to endometrial carcinogenesis, and indicating that MIS should be considered as a potential treatment for endometrial cancer.

Congenital diaphragmatic hernia (CDH) is one of the most common and lethal birth defects. Previous studies using exome sequencing support a significant contribution of codingde novovariants in complex CDH cases with additional anomalies and likely gene-disrupting (LGD) variants in isolated CDH cases. To further investigate the genetic architecture of CDH, we performed exome or genome sequencing in 283 proband-parent trios. Combined with data from previous studies, we analyzed a total of 357 trios, including 148 complex and 209 isolated cases. Complex and isolated cases both have a significant burden of deleteriousde novocoding variants (1.7~fold, p= 1.2×10−5for complex, 1.5~fold, p= 9.0×10−5for isolated). Strikingly, in isolated CDH, almost all of the burden is carried by female cases (2.1~fold, p=0.004 for likely gene disrupting and 1.8~fold, p= 0.0008 for damaging missense variants); whereas in complex CDH, the burden is similar in females and males. Additionally,de novoLGD variants in complex cases are mostly enriched in genes highly expressed in developing diaphragm, but distributed in genes with a broad range of expression levels in isolated cases. Finally, we identified a new candidate risk geneMYRF(4de novovariants, p-value=2×10−10), a transcription factor intolerant of mutations. Patients withMYRFmutations have additional anomalies including congenital heart disease and genitourinary defects, likely representing a novel syndrome.

This chapter reviews the pathogenesis and treatment of Disorders of Sex Development (DSD). “Disorders of Sex Development” is a more comprehensive term than the older terms “Ambiguous Genitalia” or “Intersex” and covers a broad clinical spectrum of hormonal, metabolic, and chromosomal abnormalities resulting in abnormal genital development. Patients with DSD are best evaluated and treated by a multidisciplinary team of medical and surgical specialists. In this era of shared decision-making, the diagnosis and treatment plan must be thoroughly discussed with parents, with the goal of giving the child the most satisfactory quality of life possible. It is essential that surgeons taking care of these complex patients feel comfortable with all the available techniques, so they can achieve satisfactory anatomical and functional repairs that support either the male of female gender.

Congenital Diaphragmatic Hernia (CDH) is a common and often lethal birth defect characterized by diaphragmatic structural defects and pulmonary hypoplasia. CDH is isolated in 60% of newborns, but may also be part of a complex phenotype with additional anomalies. We performed whole exome sequencing (WES) on 87 individuals with isolated or complex CDH and on their unaffected parents, to assess the contribution of de novo mutations in the etiology of diaphragmatic and pulmonary defects and to identify new candidate genes. A combined analysis with 39 additional trios with complex CDH, previously published, revealed a significant genome-wide burden of de novo variants compared to background mutation rate and 900 control trios. We identified an increased burden of likely gene-disrupting (LGD, i.e. nonsense, frameshift, and canonical splice site) and predicted deleterious missense (D-mis) variants in complex and isolated CDH patients. Overall, an excess of predicted damaging de novo LGD and D-mis variants relative to the expected frequency contributed to 21% of complex cases and 12% of isolated CDH cases. The burden of de novo variants was higher in genes expressed in the developing mouse diaphragm and heart. Some overlap with genes responsible for congenital heart defects and neurodevelopmental disorders was observed in CDH patients within our cohorts. We propose that de novo variants contribute significantly to the development of CDH.

The ovarian reserve represents the stock of quiescent primordial follicles in the ovary which is gradually depleted during a woman’s reproductive lifespan, resulting in menopause. Mullerian inhibiting substance (MIS) (or anti-Mullerian hormone/AMH), which is produced by granulosa cells of growing follicles, has been proposed as a negative regulator of primordial follicle activation. Here we show that long-term parenteral administration of superphysiological doses of MIS, using either an adeno-associated virus serotype 9 (AAV9) gene therapy vector or recombinant protein, resulted in a complete arrest of folliculogenesis in mice. The ovaries of MIS-treated mice were smaller than those in controls and did not contain growing follicles but retained a normal ovarian reserve. When mice treated with AAV9/MIS were paired with male breeders, they exhibited complete and permanent contraception for their entire reproductive lifespan, disrupted vaginal cycling, and hypergonadotropic hypogonadism. However, when ovaries from AAV9-MIS–treated mice were transplanted orthotopically into normal recipient mice, or when treatment with the protein was discontinued, folliculogenesis resumed, suggesting reversibility. One of the important causes of primary ovarian insufficiency is chemotherapy-induced primordial follicle depletion, which has been proposed to be mediated in part by increased activation. To test the hypothesis that MIS could prevent chemotherapy-induced overactivation, mice were given carboplatin, doxorubicin, or cyclophosphamide and were cotreated with AAV9-MIS, recombinant MIS protein, or vehicle controls. We found significantly more primordial follicles in MIS-treated animals than in controls. Thus treatment with MIS may provide a method of contraception with the unique characteristic of blocking primordial follicle activation that could be exploited to prevent the primary ovarian insufficiency often associated with chemotherapy.

Congenital diaphragmatic hernia (CDH) is a common and severe birth defect. Despite its clinical significance, the genetic and developmental pathways underlying this disorder are incompletely understood. In this study, we report a catalog of variants detected by a whole exome sequencing study on 275 individuals with CDH. Predicted pathogenic variants in genes previously identified in either humans or mice with diaphragm defects are enriched in our CDH cohort compared with 120 size-matched random gene sets. This enrichment was absent in control populations. Variants in these critical genes can be found in up to 30.9% of individuals with CDH. In addition, we filtered variants by using genes derived from regions of recurrent copy number variations in CDH, expression profiles of the developing diaphragm, protein interaction networks expanded from the known CDH-causing genes, and prioritized genes with ultrarare and highly disruptive variants, in 11.3% of CDH patients. These strategies have identified several high priority genes and developmental pathways that likely contribute to the CDH phenotype. These data are valuable for comparison of candidate genes generated from whole exome sequencing of other CDH cohorts or multiplex kindreds and provide ideal candidates for further functional studies. Furthermore, we propose that these genes and pathways will enhance our understanding of the heterogeneous molecular etiology of CDH.

Zinc finger protein, FOG2 family member 2 (ZFPM2) (previously named FOG2) gene defects result in the highly morbid congenital diaphragmatic hernia (CDH) in humans and animal models. In a cohort of 275 CDH patient exomes, we estimated the prevalence of damaging ZFPM2 mutations to be almost 5%. Genetic analysis of a multigenerational family identified a heritable intragenic ZFPM2 deletion with an estimated penetrance of 37.5%, which has important implications for genetic counseling. Similarly, a low penetrance ZFPM2 frameshift mutation was observed in a second multiplex family. Isolated CDH was the predominant phenotype observed in our ZFPM2 mutation patients. Findings from the patients described herein indicate that ZFPM2 point mutations or deletions are a recurring cause of CDH.

A key event during mammalian sexual development is regression of the Müllerian ducts (MDs) in the bipotential urogenital ridges (UGRs) of fetal males, which is caused by the expression of Müllerian inhibiting substance (MIS) in the Sertoli cells of the differentiating testes. The paracrine signaling mechanisms involved in MD regression are not completely understood, particularly since the receptor for MIS, MISR2, is expressed in the mesenchyme surrounding the MD, but regression occurs in both the epithelium and mesenchyme. Microarray analysis comparing MIS signaling competent and Misr2 knockout embryonic UGRs was performed to identify secreted factors that might be important for MIS-mediated regression of the MD. A seven-fold increase in the expression of Wif1, an inhibitor of WNT/β-catenin signaling, was observed in the Misr2-expressing UGRs. Whole mount in situ hybridization of Wif1 revealed a spatial and temporal pattern of expression consistent with Misr2 during the window of MD regression in the mesenchyme surrounding the MD epithelium that was absent in both female UGRs and UGRs knocked out for Misr2. Knockdown of Wif1 expression in male UGRs by Wif1-specific siRNAs beginning on embryonic day 13.5 resulted in MD retention in an organ culture assay, and exposure of female UGRs to added recombinant human MIS induced Wif1 expression in the MD mesenchyme. Knockdown of Wif1 led to increased expression of β-catenin and its downstream targets TCF1/LEF1 in the MD mesenchyme and to decreased apoptosis, resulting in partial to complete retention of the MD. These results strongly suggest that WIF1 secretion by the MD mesenchyme plays a role in MD regression in fetal males.

Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells both in vivo and in vitro. We have previously shown that its receptor, MISRII, is expressed in a majority of ovarian serous adenocarcinomas. However the determinants of response and the pathways elicited by MIS treatment have been difficult to elucidate due the heterogeneity of tumors both across the patient population and within each sample. To address this complexity we have developed an assay in which patient cancer cells and immune cells are isolated from ascites of recurrent chemoresistant ovarian cancer and incubated in cleared supernatant from autologous ascites for 24h in presence of drug (MIS 10ug/ml) or vehicle control (N=3). This protocol allows for short-term culture in conditions nearly identical to the peritoneal environment. A parallel experiment was conducted using matching pure primary cancer cell lines (derived from the ascites samples) treated in media (MIS 10ug/ml or vehicle for 24h), but lacking immune cells and soluble factors otherwise present in ascites. The effect of treatment on cell sates, and the interaction between immune cells and cancer cells was interrogated by clustering analysis of single cell RNA sequencing (inDROP). We uncovered a high degree of heterogeneity of expression of known and novel markers related to epithelial-mesenchymal states and stemness markers, both across patients and within patient samples, suggesting dynamically coexisting cancer cell states and hierarchies that are patient-specific. These markers were validated in vitro across a larger cohort of primary patient cells lines (N=12) grown as tumor spheroids (with and without MIS 10ug/ml for 24h). Additionally, we have identified a varying abundance of immune cell types (Macrophages, dendritic cells, NK cells, T cells, B cells), and an unexpected effect of MIS on gene expression in both macrophage and cancer cells. The knowledge gained on the response rate in patients, and the biomarkers identified in this study will be fundamental for the successful translation of MIS to the clinic, and further our understanding of the role of non-cell autonomous inhibition of cancer cells by immune components and patient heterogeneity. Citation Format: Raghav Mohan, Motohiro Kano, Hatice Saatcioglu, Nobuhiro Takahashi, LiHua Zhang, Nicholas Nagykery, Joy Poulo, Patricia K Donahoe, David Pepin. A SINGLE CELL RNA-SEQUENCING APPROACH TO UNCOVERING HUMAN OVARIAN TUMOR AND IMMUNE CELL HETEROGENEITY, AND THEIR RESPONSE TO MULLERIAN INHIBITING SUBSTANCE USING PATIENT ASCITES SAMPLES [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP29.

Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. Furthermore, recent evidence suggests that MIS may effectively target a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin- cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here we show that, compared to wild type, the substitution of the MIS leader sequence to that of human serum albumin, combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased C-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing these alterations retains its capacity to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays.

Type IV collagen is the main component of the basement membrane that gives strength to the blood-gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown.We have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal-epithelial septal-like structures in co-culture.Our study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum.

Background Type IV collagen is the main component of the basement membrane that gives strength to the blood–gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown. Results We have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal–epithelial septal-like structures in co-culture. Conclusions Our study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0281-2) contains supplementary material, which is available to authorized users.

COUP-TFII (NR2F2) is mapped to the 15q26 deletion hotspot associated with the common and highly morbid congenital diaphragmatic hernia (CDH). Conditional homozygous deletions of COUP-TFII in mice result in diaphragmatic defects analogous to the human Bochdalek-type hernia phenotype. Despite evidence from animal models however, mutations in the coding sequence of COUP-TFII have not been reported in patients, prompting the speculation that additional coding or non-coding sequences in the 15q26 locus are necessary for diaphragmatic hernias to develop. In this report, we describe a case of a patient with a heterozygous de novo COUP-TFII frameshift mutation, presenting with CDH and an atrial septal defect. The p.Pro33AlafsTer77 mutation specifically disrupts protein isoform 1 which contains the DNA binding domain. In addition, we review other COUP-TFII sequence variations and deletions that have been described in cases of CDH. We conclude that COUP-TFII mutations can cause diaphragmatic hernias, and should be included in the differential diagnosis of CDH patients, particularly those with comorbid congenital heart defects. © 2016 Wiley Periodicals, Inc.