The pha-4 locus encodes a forkhead box A (FoxA/HNF3) transcription factor homolog that specifies organ identity for C. elegans pharyngeal cells. Microarrays were used to identify pharyngeal genes, and those genes were analyzed to determine which were direct PHA-4 targets. The data suggest that PHA-4 directly activates most or all pharyngeal genes. Furthermore, the relative affinity of PHA-4 for different TRTTKRY (R = A/G, K = T/G, Y = T/C) elements modulates the onset of gene expression, providing a mechanism to activate pharyngeal genes at different developmental stages. It is suggested that direct transcriptional regulation of entire gene networks may be a common feature of organ identity genes (Gaudet, 2002).
Growth and development of the C. elegans foregut (pharynx) depends on coordinated gene expression, mediated by pharynx defective (PHA)-4/FoxA in combination with additional, largely unidentified transcription factors. Whole genome analysis was used to establish clusters of genes expressed in different pharyngeal cell types. An expectation maximization algorithm was created to identify cis-regulatory elements that activate expression within the pharyngeal gene clusters. One of these elements mediates the response to environmental conditions within pharyngeal muscles and is recognized by the nuclear hormone receptor (NHR) DAF-12. These data suggest that PHA-4 and DAF-12 endow the pharynx with transcriptional plasticity to respond to diverse developmental and physiological cues. This combination of bioinformatics and in vivo analysis has provided a powerful means for genome-wide investigation of transcriptional control (Ao, 2004).
FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth. The AAA+ ATPase homolog ruvb-1 has been identified as a potent suppressor of pha-4 mutations. This study shows that ruvb-1 is a component of the Target of Rapamycin (TOR) pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity. This study found that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan, but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations. In conclusion this data suggests that pha-4 and the CeTOR pathway antagonize one another to regulate postembryonic development and adult longevity. A model is suggested in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity, or disease (Sheaffer, 2008).
Xenopus winged helix factors XFD-13' and XFD-13 have been cloned. XFD-13/13' are regarded as pseudo-alleles and, based upon a comparison of sequences and genomic structures, represent the Xenopus orthologs to mammalian FREAC-1/HFH-8. XFD-13/13' genes are not transcribed during oogenesis: zygotic transcription starts at late gastrula/early neurula and transcripts persist throughout embryogenesis. Expression is found within head derived neural crest cells and the dorsolateral plate (DLP). At later developmental stages, cell populations of the DLP migrate to the ventral region but exclude the most posterior part. Since they are subsequently found to accumulate in vessel like structures, it has been suggested that these cells represent hematopoietic/endothelial progenitor cells (Koster, 1999).
Development of the visceral mesoderm is a critical process in the organogenesis of the gut. Elucidation of function and regulation of genes involved in the development of visceral mesoderm is therefore essential for an understanding of gut organogenesis. One of the genes specifically expressed in the lateral plate mesoderm, and later in its derivative, the visceral mesoderm, is the Fox gene FoxF1. Its function is critical for Xenopus gut development, and embryos injected with FoxF1 morpholino display abnormal gut development. In the absence of FoxF1 function, the lateral plate mesoderm, and later the visceral mesoderm, does not proliferate and differentiate properly. Region- and stage-specific markers of visceral mesoderm differentiation, such as Xbap and alpha-smooth muscle actin, are not activated. The gut does not elongate and coil. These experiments provide support for the function of FoxF1 in the development of visceral mesoderm and the organogenesis of the gut. At the molecular level, FoxF1 is a downstream target of BMP4 signaling. BMP4 can activate FoxF1 transcription in animal caps and overexpression of FoxF1 can rescue twinning phenotypes, which results from the elimination of BMP4 signaling. The cis-regulatory elements of FoxF1 are located within a 2 kb DNA fragment upstream of the coding region. These sequences can drive correct temporal-spatial expression of a GFP reporter gene in transgenic Xenopus tadpoles. These sequences represent a unique tool, which can be used to specifically alter gene expression in the lateral plate mesoderm (Tseng, 2004).
The cDNA sequences are described for two human transcription factors, Forkhead RElated ACtivator (FREAC)-1 and -2, that belong to the forkhead family of eukaryotic DNA binding proteins. FREAC-1 and -2 are encoded by distinct genes, are almost identical within their DNA binding domains and in the COOH termini, but are otherwise divergent. Cotransfections with a reporter carrying FREAC binding sites have shown that both proteins are transcriptional activators, and deletions locate the activation domains to the COOH-terminal side of the forkhead domains. Expression of FREAC-1 and FREAC-2 is restricted to lung and placenta. The promoters of genes for lung-specific proteins such as pulmonary surfactant proteins A, B, and C (SPA, SPB, and SPC) and the Clara cell 10-kDa protein (CC10) contain potential binding sites for FREAC-1 and FREAC-2. DNaseI footprinting verified that FREAC proteins bind to the predicted sites in the CC10 and SPB promoters. While an SPB promoter construct could be transactivated by both FREAC-1 and FREAC-2, CC10 is only activated by FREAC-1. Efficient activation of CC10 by FREAC-1 has been shown to be specific for a lung cell line with Clara cell characteristics (H441) and to involve a region of the FREAC-1 protein unable to activate in other cell types (Hellqvist, 1996).
The hepatocyte nuclear factor 3/fork head homolog (HFH) proteins are members of an extensive family of transcription factors that share homology in the winged helix DNA binding domain. Members of the winged helix family have been implicated in cell fate determination during pattern formation, in organogenesis and in cell type-specific gene expression. In this study, in situ hybridization was used to identify the cellular expression pattern of the winged helix transcription factor, HFH-8, during mouse embryonic development. HFH-8 expression initiates during the primitive streak stage of mouse embryogenesis in the extraembryonic mesoderm and in the lateral mesoderm, which gives rise to the somatopleuric and splanchnopleuric mesoderm. During organogenesis, HFH-8 expression is found in the splanchnic mesoderm in close apposition to the gut endoderm, suggesting a role in mesenchymal-epithelial induction of lung and gut morphogenesis. HFH-8 expression continues in lateral mesoderm-derived tissue throughout mouse development. HFH-8 expression is observed in the mesenchymal cells of the oral cavity, esophagus, trachea, lung, intestine, dorsal aorta and intersomitic arteries, but not in the vasculature of the head, liver, kidney or heart. Consistent with these embryonic expression studies, adult HFH-8 expression is restricted to the endothelium and connective fibroblasts of the alveolar sac and in the lamina propria and smooth muscle of the intestine. Several adult endothelial cell lines maintain abundant HFH-8 expression. Furthermore, the HFH-8 consensus sequence was used to identify putative target genes expressed in pulmonary and intestinal mesenchymal cells. Cotransfection assays with one of these target promoters, P-selectin, demonstrate that HFH-8 expression was required for IL-6 stimulation of P-selectin promoter activity and suggest that HFH-8 is involved in mediating its cell-specific transcriptional activation in response to cytokines (Peterson, 1997).
The growing family of forkhead transcription factors plays many important roles during embryonic development. In situ hybridization was used to explore the expression pattern of the forkhead transcription factor gene FoxF2 (FREAC-2, LUN) during mouse and rat embryogenesis, postnatal development, and in adult tissues. FoxF2 is expressed in the mesenchyme adjacent to the epithelium in alimentary, respiratory, and urinary tracts, similar to FoxF1 (FREAC-1, HFH-8). FoxF2 mRNA was also observed in organs that do not express FoxF1 during embryogenesis, e.g., in the central nervous system, eye, ear, and limb buds. In organs that express both FoxF2 and FoxF1, these transcription factors may have similar functions in epithelio-mesenchymal cross-talk, but the fact that FoxF2 is more widely expressed than FoxF1 suggests that FoxF2 also has an independent role as a developmental regulator (Aitola, 2000).
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. Foxf1 has been disrupted; 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, indicates 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 (Mahlapuu, 2001a).
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 suggests 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. 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 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 it is speculated that this is mediated by transcriptional activation of epithelial Bmp4 (in the case of FGF10) and by inhibition of Shh expression for FGF7 (Mahlapuu, 2001b).
Forkhead-related activator 2 (FREAC-2) is a human transcription factor expressed in lung and placenta that binds to cis-elements in several lung-specific genes. The parts of FREAC-2 responsible for trans-activation have been identified; two functionally redundant activation domains have been found on the C-terminal side of the DNA binding forkhead domain. Activation domain 1 consists of the most C-terminal 23 amino acids of FREAC-2 and contains a sequence motif conserved in an activation domain of another forkhead protein, FREAC-1. Activation domain 2 is built up by three synergistic subdomains in the central part of the FREAC-2 protein. FREAC-2 has been shown to interact in vitro with TBP and TFIIB. The target site for FREAC-2 on TBP is localized to the N-terminal repeat in the core domain of TBP. TFIIB binds FREAC-2 close to the cleft between its two globular domains. The part of FREAC-2 that binds TBP maps to 21 amino acids in the C-terminal end of the forkhead domain. This sequence is well conserved among forkhead proteins, raising the possibility that interaction with TBP may be a general characteristic of this family of transcription factors. Overexpression of TFIIB potentiates activation by FREAC-2 in a manner dependent on the FREAC-2 activation domains. Nuclear localization of FREAC-2 depends on sequences from both ends of the forkhead domain (Hellqvist, 1998).
Decreased pulmonary expression of Forkhead Box f1 (Foxf1) transcription factor is associated with lethal alveolar hemorrhage in 55% of the Foxf1 +/- newborn mice. The severity of the pulmonary abnormalities correlates with the levels of Foxf1 mRNA. Defects in alveolarization and vasculogenesis are observed in subsets of the Foxf1 +/- mice with relatively low levels of expression from the normal Foxf1 allele. Lung hemorrhage is coincident with disruption of the mesenchymal-epithelial cell interfaces in the alveolar and bronchiolar regions of the lung parenchyma and is associated with increased apoptosis and reduced surfactant protein B (SP-B) expression. Finally, the lung defect associated with the Foxf1 +/- mutation is accompanied by reduced expression of vascular endothelial growth factor (VEGF), the VEGF receptor 2 (Flk-1), bone morphogenetic protein 4 (Bmp-4), and the transcription factors of the Brachyury T-Box family (Tbx2-Tbx5) and Lung Kruppel-like Factor. Reduction in the level of Foxf1 causes neonatal pulmonary hemorrhage and abnormalities in alveologenesis, implicating this transcription factor in the regulation of mesenchyme-epithelial interaction critical for lung morphogenesis (Kalinichenko, 2001).
The forkhead box f1 (Foxf1) transcription factor is expressed in the visceral (splanchnic) mesoderm, which is involved in mesenchymal-epithelial signaling required for development of organs derived from foregut endoderm such as lung, liver, gall bladder, and pancreas. Haploinsufficiency of the Foxf1 gene causes pulmonary abnormalities with perinatal lethality from lung hemorrhage in a subset of Foxf1+/- newborn mice. During mouse embryonic development, the liver and biliary primordium emerges from the foregut endoderm, invades the septum transversum mesenchyme, and receives inductive signaling originating from both the septum transversum and cardiac mesenchyme. Foxf1 is expressed in embryonic septum transversum and gall bladder mesenchyme. Foxf1+/- gall bladders are significantly smaller and have severe structural abnormalities characterized by a deficient external smooth muscle cell layer, reduction in mesenchymal cell number, and in some cases, lack of a discernible biliary epithelial cell layer. This Foxf1+/- phenotype correlates with decreased expression of vascular cell adhesion molecule-1 (VCAM-1), alpha(5) integrin, platelet-derived growth factor receptor alpha (PDGFRalpha) and hepatocyte growth factor (HGF) genes, all of which are critical for cell adhesion, migration, and mesenchymal cell differentiation (Kalinichenko, 2002).
The forkhead genes encode a transcription factor involved in embryogenesis and pattern formation in multicellular organisms. They are mammalian transcriptional regulators that bind DNA as a monomer through their forkhead domain. The Foxf2 (LUN) mRNA is expressed in the mesenchyme directly adjacent to the ectoderm-derived epithelium in the developing tongue and in the mesenchyme adjacent to the endoderm-derived epithelium in the gastrointestinal (GI) tract, lungs, and genitalia. To investigate the developmental role of the Foxf2 gene during embryogenesis, the Foxf2 gene was disrupted, and these mutant mice died shortly after birth. Mice lacking the Foxf2 gene were found to develop cleft palate and an abnormal tongue. In addition, it was found that the GI tract and the lungs of Foxf2-deficient newborn mice were normal in both morphology and function. These results suggest that the Foxf2 gene plays key roles in palatogenesis by reshaping the growing tongue (Wang, 2003).
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, the early embryonic expression of Foxf2 was analzyed and compared with that of the better investigated Foxf1. 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 (Ormestad, 2004).
Development of the vertebrate gut is controlled by paracrine crosstalk between the endodermal epithelium and the associated splanchnic mesoderm. In the adult, the same types of signals control epithelial proliferation and survival, which account for the importance of the stroma in colon carcinoma progression. This study shows that targeting murine Foxf1 and Foxf2, encoding forkhead transcription factors, has pleiotropic effects on intestinal paracrine signaling. Inactivation of both Foxf2 alleles, or one allele each of Foxf1 and Foxf2, cause a range of defects, including megacolon, colorectal muscle hypoplasia and agangliosis. Foxf expression in the splanchnic mesoderm is activated by Indian and sonic hedgehog secreted by the epithelium. In Foxf mutants, mesenchymal expression of Bmp4 is reduced, whereas Wnt5a expression is increased. Activation of the canonical Wnt pathway -- with nuclear localization of beta-catenin in epithelial cells -- is associated with over-proliferation and resistance to apoptosis. Extracellular matrix, particularly collagens, is severely reduced in Foxf mutant intestine, which causes epithelial depolarization and tissue disintegration. Thus, Foxf proteins are mesenchymal factors that control epithelial proliferation and survival, and link hedgehog to Bmp and Wnt signaling (Ormestad, 2006).
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. A combination of genetic and in vitro culture methods was used to investigate the role of hedgehogs and their targets in murine extraembryonic vasculogenesis. Bmps, in particular Bmp4, are crucial for vascular tube formation, Bmp4 expression in extraembryonic tissues requires the forkhead transcription factor Foxf1, and the role of hedgehog proteins in this process is to activate Foxf1 expression in the mesoderm. In the allantois. 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 (Astorga, 2006).
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