Foxf1 Expression Research Articles

Gene expression of forkhead transcription factors in the normal and diseased human prostate

OBJECTIVE To assess the expression of forkhead transcription factors (FOX) in normal prostate and prostate diseases, as since the first FOX was identified, its family members have been implicated in a variety of cellular processes, including embryonic development and disease. MATERIAL AND METHODS We analysed a set of 12 different FOX genes by quantitative reverse transcription-polymerase chain reaction in prostate zones, prostate cancer, lymph node metastases, benign prostatic hyperplasia (BPH), xenografts and several prostate cell lines. RESULTS There were striking differences among the expression of various FOX family members; most prominent were the high expression of FOXF1 and FOXF2 in the normal prostate transition zone and BPH, and their decreased expression in prostate cancer. Interestingly, although the FOXF genes are stroma-specific, some of the androgen-independent prostate cancer xenografts uniquely express these two genes. FOXD1 and FOXD2 were more highly expressed in prostate cancer and lymph node metastases. FOXA1 and FOXC1 have an opposite expression pattern for androgen-dependent growth of prostate cancer cell lines and xenografts. CONCLUSIONS Various members of the FOX family are differentially expressed in the zones of the normal prostate and in benign and malignant outgrowths. The expression profiles of FOXF1 and FOXF2 suggest a role in epithelial to mesenchymal transition, while FOXA1 and FOXC1 expression is linked to androgen-associated growth status of cancer.

FoxF1 and FoxL1 Link Hedgehog Signaling and the Control of Epithelial Proliferation in the Developing Stomach and Intestine

The hedgehog (Hh) signaling pathway is a key component of cross-talk during vertebrate gut development, involving endodermally secreted Sonic (Shh) and Indian hedgehog (Ihh) proteins that directly signal to adjacent mesoderm. Here we show that the closely linked mesenchymal forkhead transcription factors Foxf1 and Foxl1 are part of this signaling cascade. Analysis of conserved non-coding sequences surrounding Foxf1 and Foxl1 identified seven Gli binding sites, with two sites near Foxl1 being identical among mammalian, bird, fish, and amphibian species. In vitro experiments indicate that Gli2 binds to these Gli sites, several of which are critical for Gli2-mediated activation of a luciferase reporter in 293 cells. In addition, we demonstrate occupancy of one of these elements by Gli proteins in the intestine in vivo using chromatin immunoprecipitation. Furthermore, expression of both Foxf1 and Foxl1 is reduced in the Gli2/Gli3 mutant gut. These results provide compelling evidence that Foxf1 and Foxl1 are mediators of the Hh (endoderm) to mesoderm signaling pathway.

Pulmonary Mastocytosis and Enhanced Lung Inflammation in Mice Heterozygous Null for the Foxf1 Gene

The Forkhead Box f1 (Foxf1) transcriptional factor (previously known as HFH-8 or Freac-1) is expressed in endothelial and smooth muscle cells in the embryonic and adult lung. To assess effects of Foxf1 during lung injury, we used CCl(4) and butylated hydroxytoluene (BHT) injury models. Foxf1(+/-) mice developed severe airway obstruction and bronchial edema, associated with increased numbers of pulmonary mast cells and increased mast cell degranulation after injury. Pulmonary inflammation in Foxf1(+/-) mice was associated with diminished expression of Foxf1, increased mast cell tryptase, and increased expression of CXCL12, the latter being essential for mast cell migration and chemotaxis. After ovalbumin (OVA) sensitization and OVA challenge, increased lung inflammation, airway hyperresponsiveness to methacholine, and elevated expression of CXCL12 were observed in Foxf1(+/-) mice. During lung development, Foxf1(+/-) embryos displayed a marked increase in pulmonary mast cells before birth, and this was associated with increased CXCL12 levels in the lung. Expression of a doxycycline-inducible Foxf1 dominant-negative transgene in primary cultures of lung endothelial cells increased CXCL12 expression in vitro. Foxf1 haploinsufficiency caused pulmonary mastocytosis and enhanced pulmonary inflammation after chemically induced or allergen-mediated lung injury, indicating an important role for Foxf1 in the pathogenesis of pulmonary inflammatory responses.

Visualizing expression patterns of Shh and Foxf1 genes in the foregut and lung buds by optical projection tomography

Congenital malformations of the foregut are common in humans. The respiratory and digestive tubes are both derived by division of the foregut primordium. Sonic hedgehog (Shh) and Fork head box F1 (Foxf1) genes encode regulatory molecules that play a pivotal role in gut and lung morphogenesis and are therefore important candidate genes to be examined in models of foregut developmental disruption. Optical projection tomography (OPT) is a new, rapid and non-invasive technique for three-dimensional (3D) imaging of small biological tissue specimens that allows visualization of the tissue distribution of RNA in developing organs while also recording morphology. To explore the application of OPT in this context, we visualized Shh and Foxf1 gene expression patterns in the mouse foregut and lung buds at several stages of development. Time-mated CBA/Ca mice were harvested on embryonic days 9-12. The embryos were stained following whole mount in situ hybridization with labelled RNA probes to detect Shh and Foxf1 transcripts at each stage. The embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh and Foxf1 expression in the foregut and lung buds revealed extra details of the patterns not previously reported, particularly in the case of Foxf1 where gene expression was revealed in a changing pattern in the mesenchyme around the developing lung. Shh expression was also revealed in the epithelium of the lung bud itself. Both genes were detected in complementary patterns in the developing bronchi as late as E12, showing successful penetration of molecular probes and imaging at later stages. OPT is a valuable tool for revealing gene expression in an anatomical context even in internal tissues like the foregut and lung buds across stages of development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo context in genetic or teratogenic models of congenital malformations.

Hedgehog induction of murine vasculogenesis is mediated by Foxf1 and Bmp4

The first vasculature of the developing vertebrate embryo forms by assembly of endothelial cells into simple tubes from clusters of mesodermal angioblasts. Maturation of this vasculature involves remodeling, pruning and investment with mural cells. Hedgehog proteins are part of the instructive endodermal signal that triggers the assembly of the first primitive vessels in the mesoderm. We used a combination of genetic and in vitro culture methods to investigate the role of hedgehogs and their targets in murine extraembryonic vasculogenesis. We show that Bmps, in particular Bmp4, are crucial for vascular tube formation, that Bmp4 expression in extraembryonic tissues requires the forkhead transcription factor Foxf1 and that the role of hedgehog proteins in this process is to activate Foxf1 expression in the mesoderm. We show in the allantois that genetic disruption of hedgehog signaling (Smo(-/-)) has no effect on Foxf1 expression, and neither Bmp4 expression nor vasculogenesis are disturbed. By contrast, targeted inactivation of Foxf1 leads to loss of allantoic Bmp4 and vasculature. In vitro, the avascular Foxf1(-/-) phenotype can be rescued by exogenous Bmp4, and vasculogenesis in wild-type tissue can be blocked by the Bmp antagonist noggin. Hedgehogs are required for activation of Foxf1, Bmp4 expression and vasculogenesis in the yolk sac. However, vasculogenesis in Smo(-/-) yolk sacs can be rescued by exogenous Bmp4, consistent with the notion that the role of hedgehog signaling in primary vascular tube formation is as an activator of Bmp4, via Foxf1.

Differential epithelial expression of SHH and FOXF1 in usual and nonspecific interstitial pneumonia

Morphogenetic factors have been shown to play a role in embryogenesis and post-embryonic disease. Interstitial pulmonary fibrosis is a chronic and often progressive disorder that can lead to end-stage cystic lung. Its two major subtypes, usual interstitial pneumonitis (UIP) and nonspecific interstitial pneumonitis (NSIP) differ in their response to immunosuppressive regimens, with UIP having a significantly worse prognosis. The clinical and histologic overlap between these disorders is substantial, and there are no ancillary findings that can accurately distinguish them. We examined surgical and autopsy specimens of lung in 13 cases of patients with either UIP or the fibrotic variant of NSIP (NSIP-F) for their expression of Sonic hedgehog (Shh) and Foxf1 in situ. We identified a pattern of strong Shh expression with weak expression of Foxf1 in all cases of UIP and a complementary expression of Shh and Foxf1 in cases of NSIP-F. We conclude that morphogenetic genes may participate differentially in the pathogenesis of UIP and NSIP-F.

Sequence and expression of the rainbow trout winged helix/forkhead transcription factor FoxF1

FoxF1 is a member of the winged helix/forkhead transcription factor gene family. We have cloned the cDNA encoding a rainbow trout FoxF1 homologue, and examined its developmental gene expression pattern. By 7 days postfertilization (dpf at 14 °C), FoxF1 is expressed throughout the alimentary tract in the mesenchymal cells adjacent to the endodermal epithelium, with intense signals on the dorsal side of the oral cavity and in the primitive stomach. As ontogeny proceeds, expression is down-regulated in the oral cavity and esophagus, but persists in the pharynx, stomach, and intestine. Hybridization signals are also detected in the developing liver, and in the mesenchyme layer around the notochord. From 18 dpf onwards, dramatic changes occur in gene expression in the branchial region. As the gill filaments elongate from the branchial arches, FoxF1 begins to be expressed along the central cell cord running through each gill filament, and then switches over its rod-like expression to a repetitive pattern, alternating on either side along the proximal part of the filament. A signal is further localized to the primitive pillar cells as they form gill lamellae. In addition to illustrating the conserved FoxF1 expression pattern in the developing digestive tract and liver, the results indicate a close association of FoxF1 with the formation of the fish gills.

Differences in the embryonic expression patterns of mouse Foxf1 and -2 match their distinct mutant phenotypes.

Murine genes encoding the forkhead transcription factors Foxf1 and -2 are both expressed in derivatives of the splanchnic mesoderm, i.e., the mesenchyme of organs derived from the primitive gut. In addition, Foxf2 is also expressed in limbs and the central nervous system. Targeted mutagenesis of Foxf1 and -2 suggests that Foxf1 is the more important of the two mammalian FoxF genes with early embryonic lethality of null embryos and a haploinsufficiency phenotype affecting foregut-derived organs. In contrast, the only reported defect in Foxf2 null embryos is cleft palate. To investigate if the differences in mutant phenotype can be attributed to nonoverlapping expression patterns or if distinct functions of the encoded proteins have to be inferred, we analyzed the early embryonic expression of Foxf2 and compared it with that of the better investigated Foxf1. We find that in the early embryo, Foxf1 is completely dominating-in terms of expression-in extraembryonic and lateral plate mesoderm, consistent with the malformations and early lethality of Foxf1 null mutants. Along the developing gut, Foxf1 is highly expressed throughout, whereas Foxf2 expression is concentrated to the posterior part-fitting the foregut haploinsufficiency phenotypes of Foxf1 mutants. Foxf2, on the other hand, is more prominent than Foxf1 in mesenchyme around the oral cavity, as would be predicted from the cleft palate phenotype. The differences in expression pattern also highlight areas where defects should be sought for in the Foxf2 mutant, for example limbs, the posterior gut, genitalia, and derivatives of the neural crest mesenchyme.

The forkhead box F1 transcription factor is expressed in brain and head mesenchyme during mouse embryonic development

The forkhead box f1 (Foxf1) transcription factor is expressed in septum transversum mesenchyme and splanchnic (visceral) mesoderm, which give rise to mesenchymal cells of gut-derived organs such as liver, gall bladder, lung, stomach, and intestine. Foxf1 −/− embryos die in utero and haploinsufficiency of the Foxf1 gene resulted in a variety of developmental abnormalities of the lung, gall bladder, esophagus, and trachea and caused defects in liver and lung repair. In this study, we used Foxf1 +/− mouse embryos containing the β-Galactosidase (β-Gal) gene knocked into the Foxf1 locus to examine whether Foxf1 is expressed in the developing brain and head region. In addition to previously reported Foxf1 expression patterns, β-Gal staining was also found in the mesenchyme of developing pituitary gland, eye, pancreas, heart, notochord, genital tubercle, Meckel's cartilage, and cartilage primordia of nasal septum and vertebral bodies. In adult Foxf1 +/− mice, β-Gal staining was detected in anterior and posterior pituitary gland, astrocytes of the cerebellum and cerebral cortex, lens and retina of the eye, tracheal cartilage, parathyroid, cortical layer of thymus, capsule of the spleen, coronary arteries, and pancreatic blood vessels.

Wild-type levels of the mouse Forkhead Box f1 gene are essential for lung repair.

The Forkhead Box (Fox) family of transcription factors plays important roles in regulating expression of genes involved in cellular proliferation and differentiation. In a previous study, we showed that newborn foxf1(+/-) mice with diminished Foxf1 levels exhibited abnormal formation of pulmonary alveoli and capillaries and died postnatally. Interestingly, surviving newborn foxf1(+/-) mice exhibited increased pulmonary Foxf1 levels and normal adult lung morphology, suggesting that wild-type Foxf1 levels are required for lung development and function. The present study was conducted to determine whether adult foxf1(+/-) mice were able to undergo lung repair similar to that observed in wild-type mice. We demonstrated that adult foxf1(+/-) mice died from severe lung hemorrhage after butylated hydroxytoluene (BHT) lung injury and that this phenotype was associated with a 10-fold decrease in pulmonary Foxf1 expression and increased alveolar endothelial cell apoptosis that disrupted capillary integrity. Furthermore, BHT-induced lung hemorrhage of adult foxf1(+/-) mice was associated with a drastic reduction in expression of the Flk-1, bone morphogenetic protein-4, surfactant protein B, platelet endothelial cell adhesion molecule, and vascular endothelial cadherin genes, whereas the expression of these genes was either transiently diminished or increased in wild-type lungs after BHT injury. Because these proteins are critical for lung morphogenesis and endothelial homeostasis, their decreased mRNA levels are likely contributing to BHT-induced lung hemorrhage in foxf1(+/-) mice. Collectively, our data suggest that sustained expression of Foxf1 is essential for normal lung repair and endothelial cell survival in response to pulmonary cell injury.

Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations.

The murine Foxf1 gene, encoding a forkhead - or winged helix - transcription factor, is expressed in splanchnic mesenchyme during organogenesis. The concentration of expression to subepithelial mesenchyme suggested that Foxf1 is activated by paracrine signals from endodermal epithelia. Homozygous Foxf1-null mice die before embryonic day 10, owing to defects in extra-embryonic mesoderm, and do not provide any information about the role of Foxf1 in morphogenesis of endodermally derived organs. We show that, on CD1 genetic background, Foxf1 heterozygote perinatal mortality is around 90%. The haploinsufficiency causes a variable phenotype that includes lung immaturity and hypoplasia, fusion of right lung lobes, narrowing of esophagus and trachea, esophageal atresia and tracheo-esophageal fistula. Similar malformations are observed in mutants that are defective in the sonic hedgehog (Shh) signaling pathway, and we show that exogenous Shh activates transcription of Foxf1 in developing lung. Foxf1 mRNA is absent in the lungs, foregut and sclerotomes of Shh(-/-) embryos, but persists in tissues where indian hedgehog (Ihh) is expressed. In lung organ cultures, activation of Foxf1 by Shh is counteracted by bone morphogenetic protein 4 (BMP4). Fibroblast growth factor (FGF) 10 and FGF7 both decrease Foxf1 expression and we speculate that this is mediated by transcriptional activation of epithelial Bmp4 (in the case of FGF10) and by inhibition of Shh expression for FGF7.

The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm.

The murine Foxf1 gene encodes a forkhead transcription factor expressed in extra-embryonic and lateral plate mesoderm and later in splanchnic mesenchyme surrounding the gut and its derivatives. We have disrupted Foxf1 and show that mutant embryos die at midgestation due to defects in mesodermal differentiation and cell adhesion. The embryos do not turn and become deformed by the constraints of a small, inflexible amnion. Extra-embryonic structures exhibit a number of differentiation defects: no vasculogenesis occurs in yolk sac or allantois; chorioallantoic fusion fails; the amnion does not expand with the growth of the embryo, but misexpresses vascular and hematopoietic markers. Separation of the bulk of yolk sac mesoderm from the endodermal layer and adherence between mesoderm of yolk sac and amnion, indicate altered cell adhesion properties and enhanced intramesodermal cohesion. A possible cause of this is misexpression of the cell-adhesion protein VCAM1 in Foxf1-deficient extra-embryonic mesoderm, which leads to co-expression of VCAM with its receptor, alpha(4)-integrin. The expression level of Bmp4 is decreased in the posterior part of the embryo proper. Consistent with this, mesodermal proliferation in the primitive streak is reduced and somite formation is retarded. Expression of Foxf1 and the homeobox gene Irx3 defines the splanchnic and somatic mesodermal layers, respectively. In Foxf1-deficient embryos incomplete separation of splanchnic and somatic mesoderm is accompanied by misexpression of Irx3 in the splanchnopleure, which implicates Foxf1 as a repressor of Irx3 and as a factor involved in coelom formation.

Characteristic overexpression of the forkhead box transcription factor Foxf1 in Patched-associated tumors

Patients with nevoid basal cell carcinoma syndrome carry germline mutations in the tumor suppressor gene Patched 1 (PTCH1) and are predisposed to develop basal cell carcinoma (BCC), medulloblastoma (MB), and rhabdomyosarcoma (RMS). These tumors are also present in the murine model for Ptch1 deficiency, the Ptch1neo67/+ mouse. Previous studies, including those from our laboratory, have shown that the forkhead box transcription factor Foxf1 is highly expressed in RMS of human and murine origin. We report on a more common role of Foxf1 in Ptch1-associated tumorigenesis, since we found a striking up-regulation of Foxf1 expression in Ptch1-associated BCC and MB compared with the respective non-neoplastic tissue. This overexpression was accompanied by increased levels of the Hedgehog target gene Gli1 as well as the putative Foxf1 targets Bmi1 and Notch2 in these tumors. We also describe a striking Foxf1 activation in Ptch1 null embryos. In contrast, basal expression levels of Foxf1, Gli1, Bmi1 and Notch2 were detected in a variety of adult mouse tissues, such as liver, kidney, spleen, lung, heart and brain. In conclusion, our study suggests that Foxf1 expression is characteristically up-regulated in tumors with a constitutively activated Hedgehog signaling pathway thereby defining a key role for Foxf1 in Hedgehog-associated tumorigenesis.