Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Mitochondrial dysfunction and oxidative stress contribute to cognitive and motor impairment in FOXP1 syndrome.

FOXP1 syndrome caused by haploinsufficiency of the forkhead box protein P1 (FOXP1) gene is a neurodevelopmental disorder that manifests motor dysfunction, intellectual disability, autism, and language impairment. In this study, we used a Foxp1+/- mouse model to address whether cognitive and motor deficits in FOXP1 syndrome are associated with mitochondrial dysfunction and oxidative stress. Here, we show that genes with a role in mitochondrial biogenesis and dynamics (e.g., Foxo1, Pgc-1α, Tfam, Opa1, and Drp1) were dysregulated in the striatum of Foxp1+/- mice at different postnatal stages. Furthermore, these animals exhibit a reduced mitochondrial membrane potential and complex I activity, as well as decreased expression of the antioxidants superoxide dismutase 2 (Sod2) and glutathione (GSH), resulting in increased oxidative stress and lipid peroxidation. These features can explain the reduced neurite branching, learning and memory, endurance, and motor coordination that we observed in these animals. Taken together, we provide strong evidence of mitochondrial dysfunction in Foxp1+/- mice, suggesting that insufficient energy supply and excessive oxidative stress underlie the cognitive and motor impairment in FOXP1 deficiency.

Experience with cultured thymus tissue in 105 children.

BACKGROUND: Currently, there are no approved therapies to treat congenital athymia, a condition of immune deficiency resulting in high early mortality due to infection and immune dysregulation. Multiple syndromic conditions, such as complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE (coloboma, heart defects, choanal atresia, growth or mental retardation, genital hypoplasia, and ear anomalies and/or deafness) syndrome, diabetic embryopathy, other genetic variants, and FOXN1 deficiency, are associated with congenital athymia. OBJECTIVE: Our aims were to study 105 patients treated with cultured thymus tissue (CTT), and in this report, to focus on the outcomes of 95 patients with treatment-naive congenital athymia. METHODS: A total of 10 prospective, single-arm open-label studies with patient enrollment from 1993 to 2020 form the basis of this data set. Patients were tested after administration of CTT for T-cell development; all adverse events and infections were recorded. RESULTS: A total of 105 patients were enrolled and received CTT (the full analysis set). Of those patients, 10 had diagnoses other than congenital athymia and/or received prior treatments. Of those 105 patients, 95 patients with treatment-naive congenital athymia were included in the efficacy analysis set (EAS). The Kaplan-Meier estimated survival rates at year 1 and year 2 after administration of CTT in the EAS were 77% (95% CI = 0.670-0.844) and 76% (95% CI = 0.657-0.834), respectively. In all, 21 patients died in the first year before developing naive T cells and 1 died in the second year after receipt of CTT; 3 subsequent deaths were not related to immunodeficiency. A few patients developed alopecia, autoimmune hepatitis, psoriasis, and psoriatic arthritis after year 1. The rates of infections, autologous graft-versus-host-disease manifestations, and autoimmune cytopenias all decreased approximately 1 year after administration of CTT. CONCLUSION: Treatment with CTT led to development of naive T cells with a 1-year survival rate of 77% and a median follow-up time of 7.6 years. Immune reconstitution sufficient to prevent infections and support survival typically develops 6 to12 months after administration of CTT.

Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.

Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.

Insulin and IGF-1 receptors regulate complex I-dependent mitochondrial bioenergetics and supercomplexes via FoxOs in muscle.

Decreased skeletal muscle strength and mitochondrial dysfunction are characteristic of diabetes. The actions of insulin and IGF-1 through the insulin receptor (IR) and IGF-1 receptor (IGF1R) maintain muscle mass via suppression of forkhead box O (FoxO) transcription factors, but whether FoxO activation coordinates atrophy in concert with mitochondrial dysfunction is unknown. We show that mitochondrial respiration and complex I activity were decreased in streptozotocin (STZ) diabetic muscle, but these defects were reversed in muscle-specific FoxO1, -3, and -4 triple-KO (M-FoxO TKO) mice rendered diabetic with STZ. In the absence of systemic glucose or lipid abnormalities, muscle-specific IR KO (M-IR-/-) or combined IR/IGF1R KO (MIGIRKO) impaired mitochondrial respiration, decreased ATP production, and increased ROS. These mitochondrial abnormalities were not present in muscle-specific IR, IGF1R, and FoxO1, -3, and -4 quintuple-KO mice (M-QKO). Acute tamoxifen-inducible deletion of IR and IGF1R also decreased muscle pyruvate respiration, complex I activity, and supercomplex assembly. Although autophagy was increased when IR and IGF1R were deleted in muscle, mitophagy was not increased. Mechanistically, RNA-Seq revealed that complex I core subunits were decreased in STZ-diabetic and MIGIRKO muscle, and these changes were not present with FoxO KO in STZ-FoxO TKO and M-QKO mice. Thus, insulin-deficient diabetes or loss of insulin/IGF-1 action in muscle decreases complex I-driven mitochondrial respiration and supercomplex assembly in part by FoxO-mediated repression of complex I subunit expression.

Foxo1 deletion promotes the growth of new lymphatic valves.

Patients with congenital lymphedema suffer from tissue swelling in part due to mutations in genes regulating lymphatic valve development. Lymphatic valve leaflets grow and are maintained throughout life in response to oscillatory shear stress (OSS), which regulates gene transcription in lymphatic endothelial cells (LECs). Here, we identified the first transcription factor, Foxo1, that repressed lymphatic valve formation by inhibiting the expression of valve-forming genes. We showed that both embryonic and postnatal ablation of Foxo1 in LECs induced additional valve formation in postnatal and adult mice in multiple tissues. Our quantitative analyses revealed that after deletion, the total number of valves in the mesentery was significantly (P < 0.01) increased in the Foxo1LEC-KO mice compared with Foxo1fl/fl controls. In addition, our quantitative real-time PCR (RT-PCR) data from cultured LECs showed that many valve-forming genes were significantly (P < 0.01) upregulated upon knockdown of FOXO1. To confirm our findings in vivo, rescue experiments showed that Foxc2+/- mice, a model of lymphedema-distichiasis, had 50% fewer lymphatic valves and that the remaining valves exhibited backleak. Both valve number and function were completely restored to control levels upon Foxo1 deletion. These findings established FOXO1 as a clinically relevant target to stimulate de novo lymphatic valve formation and rescue defective valves in congenital lymphedema.

Nanoparticle Delivery of STAT3 Alleviates Pulmonary Hypertension in a Mouse Model of Alveolar Capillary Dysplasia.

BACKGROUND: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. METHODS: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1WT/S52F mice and determine its effects on PH and RV hypertrophy. RESULTS: Foxf1WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. CONCLUSIONS: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

Identification of testicular Foxq1 as a critical modulator of lactate metabolism in mouse Sertoli cells.

Postmeiotic germ cells require the lactate produced by the adjacent Sertoli cells (SCs) as their sole energy fuels. Lactate production in SCs is elaborately regulated by monitoring the transcription of the lactate dehydrogenase A (Ldha) gene. However, the transcription factors that are responsible for the control of Ldha transcription in SCs remain ill defined. Herein, the expression of forkhead box Q1 (FOXQ1), a central modulator of glucose metabolism in liver, was demonstrated in mouse testis throughout postnatal development, with maximum levels in adult specimens. At this age, FOXQ1 was immunolocalized in the nuclei of the functionally mature SCs. Testicular levels of FOXQ1 were overtly modulated by germ cells (GCs)-derived IL-1α, in a dose- and time-dependent manner. To further clarify the biological functions of FOXQ1, we disrupted the mouse Foxq1 gene using a Cas9/RNA-mediated gene targeting strategy. Foxq1-/- males were subfertile and showed oligoasthenozoospermia due to lactate deficiency. Moreover, we provided the molecular evidence that FOXQ1 may regulate lactate production by directly targeting the transactivation of the Ldha gene in SCs. From a functional standpoint, overexpression of the exogenous Ldha ameliorated Foxq1 deficiency-impaired lactate synthesis in the SCsFoxq1-/- cells. Thus, these findings collectively underscore a reproductive facet of this recently characterized transcription factor, which may operate as a novel transcriptional integrator linking energy homeostasis and nursery function in SCs.

Loss of FOXO transcription factors in the liver mitigates stress-induced hyperglycemia.

OBJECTIVE: Stress-induced hyperglycemia is associated with poor outcomes in nearly all critical illnesses. This acute elevation in glucose after injury or illness is associated with increased morbidity and mortality, including multiple organ failure. Stress-induced hyperglycemia is often attributed to insulin resistance as controlling glucose levels via exogenous insulin improves outcomes, but the mechanisms are unclear. Forkhead box O (FOXO) transcription factors are direct targets of insulin signaling in the liver that regulate glucose homeostasis via direct and indirect pathways. Loss of hepatic FOXO transcription factors reduces hyperglycemia in chronic insulin resistance; however, the role of FOXOs in stress-induced hyperglycemia is unknown. METHODS: We subjected mice lacking FOXO transcription factors in the liver to a model of injury known to cause stress-induced hyperglycemia. Glucose, insulin, glycerol, fatty acids, cytokines, and adipokines were assessed before and after injury. Liver and adipose tissue were analyzed for changes in glycogen, FOXO target gene expression, and insulin signaling. RESULTS: Stress-induced hyperglycemia was associated with reduced hepatic insulin signaling and increased hepatic FOXO target gene expression while loss of FOXO1, 3, and 4 in the liver attenuated hyperglycemia and prevented hyperinsulinemia. Mechanistically, the loss of FOXO transcription factors mitigated the stress-induced hyperglycemia response by directly altering gene expression and glycogenolysis in the liver and indirectly suppressing lipolysis in adipose tissue. Reductions were associated with decreased IL-6, TNF-α, and follistatin and increased FGF21, suggesting that cytokines and FOXO-regulated hepatokines contribute to the stress-induced hyperglycemia response. CONCLUSIONS: This study implicates FOXO transcription factors as a predominant driver of stress-induced hyperglycemia through means that include cross-talk between the liver and adipose, highlighting a novel mechanism underlying acute hyperglycemia and insulin resistance in stress.

Single-cell analysis of FOXP3 deficiencies in humans and mice unmasks intrinsic and extrinsic CD4+ T cell perturbations.

FOXP3 deficiency in mice and in patients with immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome results in fatal autoimmunity by altering regulatory T (Treg) cells. CD4+ T cells in patients with IPEX syndrome and Foxp3-deficient mice were analyzed by single-cell cytometry and RNA-sequencing, revealing heterogeneous Treg-like cells, some very similar to normal Treg cells, others more distant. Conventional T cells showed no widespread activation or helper T cell bias, but a monomorphic disease signature affected all CD4+ T cells. This signature proved to be cell extrinsic since it was extinguished in mixed bone marrow chimeric mice and heterozygous mothers of patients with IPEX syndrome. Normal Treg cells exerted dominant suppression, quenching the disease signature and revealing in mutant Treg-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core FOXP3-dependent genes destabilizes Treg cells, de-repressing systemic mediators that imprint the disease signature on all T cells, furthering Treg cell dysfunction. Accordingly, interleukin-2 treatment improved the Treg-like compartment and survival.

Impairment of the Hif-1α regulatory pathway in Foxn1-deficient (Foxn1-/- ) mice affects the skin wound healing process.

Hypoxia and hypoxia-regulated factors (eg, hypoxia-inducible factor-1α [Hif-1α], factor inhibiting Hif-1α [Fih-1], thioredoxin-1 [Trx-1], aryl hydrocarbon receptor nuclear translocator 2 [Arnt-2]) have essential roles in skin wound healing. Using Foxn1-/- mice that can heal skin injuries in a unique scarless manner, we investigated the interaction between Foxn1 and hypoxia-regulated factors. The Foxn1-/- mice displayed impairments in the regulation of Hif-1α, Trx-1, and Fih-1 but not Arnt-2 during the healing process. An analysis of wounded skin showed that the skin of the Foxn1-/- mice healed in a scarless manner, displaying rapid re-epithelialization and an increase in transforming growth factor ß (Tgfß-3) and collagen III expression. An in vitro analysis revealed that Foxn1 overexpression in keratinocytes isolated from the skin of the Foxn1-/- mice led to reduced Hif-1α expression in normoxic but not hypoxic cultures and inhibited Fih-1 expression exclusively under hypoxic conditions. These data indicate that in the skin, Foxn1 affects hypoxia-regulated factors that control the wound healing process and suggest that under normoxic conditions, Foxn1 is a limiting factor for Hif-1α.

Loss of FOXC1 contributes to the corneal epithelial fate switch and pathogenesis.

Forkhead box C1 (FOXC1) is required for neural crest and ocular development, and mutations in FOXC1 lead to inherited Axenfeld-Rieger syndrome. Here, we find that FOXC1 and paired box 6 (PAX6) are co-expressed in the human limbus and central corneal epithelium. Deficiency of FOXC1 and alternation in epithelial features occur in patients with corneal ulcers. FOXC1 governs the fate of the corneal epithelium by directly binding to lineage-specific open promoters or enhancers marked by H3K4me2. FOXC1 depletion not only activates the keratinization pathway and reprograms corneal epithelial cells into skin-like epithelial cells, but also disrupts the collagen metabolic process and interferon signaling pathways. Loss of interferon regulatory factor 1 and PAX6 induced by FOXC1 dysfunction is linked to the corneal ulcer. Collectively, our results reveal a FOXC1-mediated regulatory network responsible for corneal epithelial homeostasis and provide a potential therapeutic target for corneal ulcer.

Dermal White Adipose Tissue (dWAT) Is Regulated by Foxn1 and Hif-1α during the Early Phase of Skin Wound Healing.

Dermal white adipose tissue (dWAT) is involved in the maintenance of skin homeostasis. However, the studies concerning its molecular regulation are limited. In the present paper, we ask whether the introduction of two transcription factors, Foxn1 and Hif-1α, into the post-wounded skin of Foxn1-/- mice regulates dWAT during wound healing (days 3 and 6). We have chosen lentivirus vectors (LVs) as a tool to deliver Foxn1 and Hif-1α into the post-wounded skin. We documented that combinations of both transgenes reduces the number, size and diameter of dermal adipocytes at the wound bed area. The qRT-PCR analysis of pro-adipogenic genes, revealed that LV-Hif-1α alone, or combined with LV-Foxn1, increases the mRNA expression of Pparγ, Glut 4 and Fasn at post-wounding day 6. However, the most spectacular stimulatory effect of Foxn1 and/or Hif-1α was observed for Igf2, the growth factor participating in adipogenic signal transduction. Our data also shows that Foxn1/Hif-1α, at post-wounding day 3, reduces levels of CD68 and MIP-1γ mRNA expression and the percentage of CD68 positive cells in the wound site. In conclusion, the present data are the first to document that Foxn1 and Hif-1α cooperatively (1) regulate dWAT during the proliferative phase of skin wound healing through the Igf2 signaling pathway, and (2) reduce the macrophages content in the wound site.

The Effect of FOXC2-AS1 on White Adipocyte Browning and the Possible Regulatory Mechanism.

Obesity has become a worldwide epidemic, and obesity-related problems are becoming more severe in public health. Increasing brown adipose tissue (BAT) mass or/and activity in mice and humans has been demonstrated to help lose weight and improve whole-body metabolism. Studies on the conversion of white adipose tissue (WAT) to BAT under certain conditions have provided new possibilities for treating obesity and the related disorders. It has been established that long non-coding RNAs (lncRNAs) play an important role in the regulation of mouse adipocyte differentiation and thermogenic programs; however, the function and potential mechanism of lncRNA in the process of human white adipocyte browning remains unclear. In the present study, we identified a lncRNA called Forkhead Box C2 antisense RNA 1 (FOXC2-AS1), which was first identified in osteosarcoma, and it was highly expressed in human adipocytes but decreased during the white adipocyte differentiation program. FOXC2-AS1 expression was also induced by the thermogenic agent forskolin. Lentivirus-mediated overexpression of FOXC2-AS1 in human white adipocytes did not affect lipid drop accumulation, but significantly promoted the browning phenotype, as revealed by the increased respiratory capacity and the enhanced protein expression levels of brown adipocyte-specific markers. In contrast, inhibiting FOXC2-AS1 with small interfering RNA led to attenuated thermogenic capacity in human white adipocytes. RNA-sequencing analysis and western blot were used to identify a possible regulatory role of the autophagy signaling pathway in FOXC2-AS1 to mediate white-to-brown adipocyte conversion. The autophagy inhibitor 3-methyladenine restored the reduced UCP1 protein level and thermogenic capacity caused by inhibiting FOXC2-AS1. Overall, the present study characterized the potential role of FOXC2-AS1 and further identified a lncRNA-mediated mechanism for inducing browning of human white adipocytes and maintaining thermogenesis, further providing a potential strategy for treating obesity and related disorder.

Knockdown of Foxg1 in Sox9+ supporting cells increases the trans-differentiation of supporting cells into hair cells in the neonatal mouse utricle.

Foxg1 plays important roles in regeneration of hair cell (HC) in the cochlea of neonatal mouse. Here, we used Sox9-CreER to knock down Foxg1 in supporting cells (SCs) in the utricle in order to investigate the role of Foxg1 in HC regeneration in the utricle. We found Sox9 an ideal marker of utricle SCs and bred Sox9CreER/+Foxg1loxp/loxp mice to conditionally knock down Foxg1 in utricular SCs. Conditional knockdown (cKD) of Foxg1 in SCs at postnatal day one (P01) led to increased number of HCs at P08. These regenerated HCs had normal characteristics, and could survive to at least P30. Lineage tracing showed that a significant portion of newly regenerated HCs originated from SCs in Foxg1 cKD mice compared to the mice subjected to the same treatment, which suggested SCs trans-differentiate into HCs in the Foxg1 cKD mouse utricle. After neomycin treatment in vitro, more HCs were observed in Foxg1 cKD mice utricle compared to the control group. Together, these results suggest that Foxg1 cKD in utricular SCs may promote HC regeneration by inducing trans-differentiation of SCs. This research therefore provides theoretical basis for the effects of Foxg1 in trans-differentiation of SCs and regeneration of HCs in the mouse utricle.

VPS-22/SNF8 regulates longevity via modulating the activity of DAF-16 in C. elegans.

Aging is regulated by complex signaling networks, the details of which remain poorly understood. Here, we demonstrate that VPS-22/SNF8, a component of endosomal sorting complex required for transport-II (ESCRT-II), regulates the lifespan of C. elegans. In this study we show that worms with vps-22/snf8 gene knockdown had a shorter lifespan than wild-type worms. The expression pattern of VPS-22/SNF8 in C. elegans was highly similar to that of DAF-16. Knockout of daf-16 in C. elegans shortened the worms' lifespan; however, reducing the expression of vps-22/snf8 in daf-16 null worms did not further shorten their lifespan, indicating that vps-22/snf8 and daf-16 may act in the same signaling pathway to regulate longevity. Over-expression of daf-16 rescued the short-lived phenotype of vps-22/snf8 knockdown worms. Moreover, down-regulation of vps-22/snf8 decreased the nuclear localization of DAF-16 and modulated the expression of daf-16 downstream genes that regulate longevity in C. elegans. In summary, our results indicate that vps-22/snf8 can regulate the longevity of C. elegans by partially modulating the activity of daf-16. These findings may help us to better understand the mechanisms of aging.

NOTCH1 signaling establishes the medullary thymic epithelial cell progenitor pool during mouse fetal development.

The cortical and medullary thymic epithelial cell (cTEC and mTEC) lineages are essential for inducing T cell lineage commitment, T cell positive selection and the establishment of self-tolerance, but the mechanisms controlling their fetal specification and differentiation are poorly understood. Here, we show that notch signaling is required to specify and expand the mTEC lineage. Notch1 is expressed by and active in TEC progenitors. Deletion of Notch1 in TECs resulted in depletion of mTEC progenitors and dramatic reductions in mTECs during fetal stages, consistent with defects in mTEC specification and progenitor expansion. Conversely, forced notch signaling in all TECs resulted in widespread expression of mTEC progenitor markers and profound defects in TEC differentiation. In addition, lineage-tracing analysis indicated that all mTECs have a history of receiving a notch signal, consistent with notch signaling occurring in mTEC progenitors. These data provide strong evidence for a requirement for notch signaling in specification of the mTEC lineage.

Canonical Notch signaling controls the early thymic epithelial progenitor cell state and emergence of the medullary epithelial lineage in fetal thymus development.

Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). However, the mechanisms controlling cTEC and mTEC production from the common TEPC are not understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPCs at the expense of cTEC differentiation. Finally, we uncover a cross-regulatory relationship between NOTCH and FOXN1, a master regulator of TEC differentiation. These data establish NOTCH as a potent regulator of TEPC and mTEC fate during fetal thymus development, and are thus of high relevance to strategies aimed at generating/regenerating functional thymic tissue in vitro and in vivo.

Single-Cell Analysis of Foxp1-Driven Mechanisms Essential for Striatal Development.

The striatum is a critical forebrain structure integrating cognitive, sensory, and motor information from diverse brain regions into meaningful behavioral output. However, the transcriptional mechanisms underlying striatal development at single-cell resolution remain unknown. Using single-cell RNA sequencing (RNA-seq), we examine the cellular diversity of the early postnatal striatum and show that Foxp1, a transcription factor strongly linked to autism and intellectual disability, regulates the cellular composition, neurochemical architecture, and connectivity of the striatum in a cell-type-dependent fashion. We also identify Foxp1-regulated target genes within distinct cell types and connect these molecular changes to functional and behavioral deficits relevant to phenotypes described in patients with FOXP1 loss-of-function mutations. Using this approach, we could also examine the non-cell-autonomous effects produced by disrupting one cell type and the molecular compensation that occurs in other populations. These data reveal the cell-type-specific transcriptional mechanisms regulated by Foxp1 that underlie distinct features of striatal circuitry.

Foxf1 knockdown promotes BMSC osteogenesis in part by activating the Wnt/ß-catenin signalling pathway and prevents ovariectomy-induced bone loss.

BACKGROUND: Forkhead box protein f1 (Foxf1) is associated with cell differentiation, and may be a key player in bone homoeostasis. However, the effect of Foxf1 on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) and ovariectomy-induced bone loss, as well as its clinical implications, is unknown. METHODS: By quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, we assayed Foxf1 expression in bone tissue, BMSCs, and bone marrow-derived macrophages (BMMs), derived from ovariectomised (OVX) mice, and during osteogenic differentiation and osteoclast differentiation. Using a loss-of-function approach (small interfering RNA [siRNA]-mediated knockdown) in vitro, we examined whether Foxf1 regulates osteoblast differentiation of BMSCs via the Wnt/ß-catenin signalling pathway. Furthermore, we assessed the anabolic effect of Foxf1 knockdown (siFoxf1) in OVX mice in vivo. We also assayed the expression of Foxf1 in bone tissue derived from postmenopausal osteoporosis (PMOP) patients and its link with bone mineral density (BMD). Finally, we examined the effect of Foxf1 knockdown on the osteoblastic differentiation of human BMSCs. FINDINGS: Foxf1 expression was significantly increased in bone extract and BMSCs from OVX mice and gradually decreased during osteoblastic differentiation of BMSCs but did not differ significantly in OVX mouse-derived BMMs or during osteoclast differentiation. In vitro, Foxf1 knockdown markedly increased the expression of osteoblast specific genes, alkaline phosphatase (ALP) activity, and mineralisation. Moreover, siFoxf1 activated the Wnt/ß-catenin signalling pathway. The siFoxf1-induced increase in osteogenic differentiation was partly rescued by inhibitor of Wnt signalling (DKK1). In OVX mice, Foxf1 siRNA significantly reduced bone loss by enhancing bone formation. Foxf1 expression levels negatively correlated with reduced bone mass and bone formation in bone tissue from PMOP patients. Finally, Foxf1 knockdown significantly promoted osteogenesis by human BMSCs. INTERPRETATION: Our findings indicate that Foxf1 knockdown promotes BMSC osteogenesis and prevents OVX-induced bone loss. Therefore, Foxf1 has potential as a biomarker of osteogenesis and may be a therapeutic target for PMOP.