Table of contents

Mammalian Forkhead homologs: Other forkhead family members

The gene mfh1 encodes a winged helix/forkhead domain transcription factor. mfh1 belongs to a class of winged helix/forkhead superfamily members distinct from those to which the Drosophila forkhead family members (Forkhead, Crocodile and Sloppy paired) belong. mfh1 is expressed in a dynamic pattern in paraxial and presomitic mesoderm and developing somites during mouse embryogenesis. Expression later becomes restricted to condensing mesenchyme of the vertebrae, head, limbs, and kidney. A targeted disruption of the gene was generated by homologous recombination in embryonic stem cells. Most homozygous mfh1 null embryos die prenatally but some survive to birth, with multiple craniofacial and vertebral column defects. The initial formation and patterning of somites occurs normally in mutants. Differentiation of sclerotome-derived cells also appears unaffected, although a reduction of the level of some markers (e.g., mtwist, mf1, scleraxis, and alpha1(II) collagen) is seen in the anterior of homozygous mutants. The most significant difference is a marked reduction in the proliferation of sclerotome-derived cells. This proliferation defect is also seen in micromass cultures of somite-derived cells treated with transforming growth factor ß1 and fibroblast growth factors. These findings establish a requirement for a winged helix/forkhead domain transcription factor in the development of the paraxial mesoderm (Winnier, 1997).

A model is proposed for the role of mfh1 in regulating the proliferation and differentiation of cell lineages giving rise to the sclerotome. mfh1 is expressed in unsegmented presomitic mesoderm. The sclerosome lineage arises from multipotent stem cells in the epithelial somite, as a result of inductive signals from the notochord. Cells migrate medially, ventrally, and laterally from the early somite. Those that condense around the notochord give rise to the cartilage primordium of the ventral body and centrum, whereas the medial and lateral condensations give rise to the neural arches and pedicles. Sclerotomal cells also give rise to intravertebral discs and connective tissue around the spinal ganglia and nerves. Vertebral formation involves an ordered progression of differentiated and progenitor cell populations, leading finally to the appearance of postmitotic chondrocytes and osteocytes. It is proposed the mhf1 plays a role in regulating clonal expansion, and possibly also progression along the differentiation pathway. A similar role for mfh1 may also occur in formation of the appendicular skeleton (Winnier, 1997).

Mesenchyme Fork Head-1 (MFH-1) is a forkhead transcription factor defined by a common 100-amino acid DNA-binding domain. MFH-1 is expressed in non-notochordal mesoderm in the prospective trunk region and in cephalic neural-crest and cephalic mesoderm-derived mesenchymal cells in the prechordal region of early embryos. Subsequently, strong expression is localized in developing cartilaginous tissues, kidney and dorsal aortas. To investigate the developmental roles of MFH-1 during embryogenesis, mice lacking the MFH-1 locus were generated by targeted mutagenesis. MFH-1-deficient mice die embryonically and perinatally, and exhibit interrupted aortic arch and skeletal defects in the neurocranium and the vertebral column. Strong expression is seen in the mesenchymal condensation around the optic vesicle, the mesenchyme underlying the midbrain and hindbrain. Thus, MFH-1 is expressed ubiquitously in the head mesoderm and subsequently localized in the mesenchymal condensations giving rise to the floor and wall of the neurocranium, palatine and Meckel's cartilage. Interruption of the aortic arch seen in the mutant mice is the same as in human congenital anomalies. These results suggest that MFH-1 plays crucial roles during the extensive remodeling of the aortic arch in neural-crest-derived cells and in skeletogenesis in cells derived from the neural crest and the mesoderm (Iida, 1997).

During axial skeleton development, the notochord is essential for the induction of the sclerotome and for the subsequent differentiation of cartilage forming the vertebral bodies and intervertebral discs. These functions are mainly mediated by the diffusible signaling molecule Sonic hedgehog. The products of the paired-box-containing Pax1 and the mesenchyme forkhead-1 (Mfh1) genes are expressed in the developing sclerotome and are essential for the normal development of the vertebral column. Mfh1 expression, like Pax1 expression, is dependent on Sonic hedgehog signals from the notochord, and Mfh1 and Pax1 act synergistically to generate the vertebral column. In Mfh1/Pax1 double mutants, dorsomedial structures of the vertebrae are missing, resulting in extreme spina bifida accompanied by subcutaneous myelomeningocoele, and the vertebral bodies and intervertebral discs are missing. The morphological defects in Mfh1/Pax1 double mutants strongly correlate with the reduction of the mitotic rate of sclerotome cells. Thus, both the Mfh1 and the Pax1 gene products cooperate to mediate Sonic hedgehog-dependent proliferation of sclerotome cells. The insufficient allocation of sclerotome cells in the dorsomedial region of the sclerotome could be the basis for the novel synergistic phenotype in Mfh1/Pax1 double mutants (Furumoto, 1999).

The mouse fkh-2 gene encodes a protein of 48 kDa with high similarity to other winged helix transcription factors within the DNA binding region, but unique potential transactivation domains. The gene is encoded by a single exon and is expressed in headfold stage embryos in the notochord, the anterior neuroectoderm, and a few cells of the definite endoderm. This expression becomes restricted to the anteriormost portions of the invaginating foregut and the developing midbrain. From day 11.5 of gestation onward, fkh-2 transcripts are restricted to the midbrain and become progressively localized to the red nuclei as the sole site of expression. The fkh-2 gene is a candidate gene for the mouse mutation mdf (muscle-deficient) which is characterized by nervous tremors and degeneration of the hindlimb muscles. Although the expression patterns of the fkh-2 gene and another winged helix protein, HNF-3 beta, are overlapping in early stages of gestation and although the promoter of the fkh-2 gene contains a HNF-3 binding site, the activation of the fkh-2 gene is independent of HNF-3 beta (Kaestner, 1995).

Mf1 encodes a forkhead/winged helix transcription factor expressed in many embryonic tissues, including prechondrogenic mesenchyme, periocular mesenchyme, meninges, endothelial cells, and kidney. Homozygous null Mf1lacZ mice die at birth with hydrocephalus, eye defects, and multiple skeletal abnormalities identical to those of the classical mutant, congenital hydrocephalus. Congenital hydrocephalus involves a point mutation in Mf1, generating a truncated protein lacking the DNA-binding domain. Mesenchyme cells from Mf1lacZ embryos differentiate poorly into cartilage in micromass culture and do not respond to added BMP2 and TGFbeta1. The differentiation of arachnoid cells in the mutant meninges is also abnormal. The human Mf1 homolog FREAC3 is a candidate gene for ocular dysgenesis and glaucoma mapping to chromosome 6p25-pter, and deletions of this region are associated with multiple developmental disorders, including hydrocephaly and eye defects (Kume, 1998).

The murine Mf1 and Mfh1 genes have overlapping patterns of expression in the embryo and encode forkhead/winged helix transcription factors with virtually identical DNA binding domains. Previous studies have shown that Mfh1 null mutants have severe cardiovascular defects, including interruptions and coarctations of the aortic arch and ventricular septal defects. Mf1(lacZ) homozygous null mutants also have a similar spectrum of cardiovascular abnormalities. Moreover, most embryos doubly heterozygous for Mfh1(tm1) and Mf1(lacZ) die before birth with interruptions and coarctations of the aortic arch, dysgenesis of the aortic and pulmonary valves, ventricular septal defects, and other cardiac anomalies. This nonallelic noncomplementation and the similar patterns of expression of the two genes in the mesenchyme and endothelial cells of the branchial arches, outflow tract, and heart suggest that Mf1 and Mfh1 play interactive roles in the morphogenesis of the cardiovascular system. Implications for the development of human congenital heart defects are discussed (Winnier, 1999).

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs reveals an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is closely related to Bf2, with only one amino acid difference within the winged helix domain; the protein is also closely related to two other mouse forkhead proteins, Hfh2 and Fkh2. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system, Mf2 is transcribed in restricted regions of the midbrain and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing Sonic hedgehog (Shh). In explant cultures of presomitic mesoderm, Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH (Wu, 1998a).

In order to study forebrain determination and patterning in the zebrafish Danio rerio, zebrafish homologs of two neural markers were isolated: odd-paired-like (opl), which encodes a zinc finger protein, and fkh5, which encodes a forkhead domain protein. At mid-gastrula, expression of these genes defines a very early pattern in the presumptive neurectoderm, with opl later expressed in the telencephalon, and fkh5 in the diencephalon and more posterior neurectoderm. Using in vitro explant assays, it was shown that forebrain induction had occurred even earlier, by the onset of gastrulation (shield stage). Signaling from the early gastrula shield, previously shown to be an organizing center, is sufficient for activation of opl expression in vitro. In order to determine whether the organizer is required for opl regulation, either the presumptive prechordal plate, marked by goosecoid (gsc) expression, or the entire organizer, marked by chordin (chd) expression was removed from late blastula stage embryos. opl is correctly expressed after removal of the presumptive prechordal plate; consistently, opl is correctly expressed in one-eyed pinhead (oep) mutant embryos, where the prechordal plate fails to form. However, after removal of the entire organizer, no opl expression is observed, indicating that this region is crucial for forebrain induction. Continued organizer function is required for forebrain induction, since beads of BMP4, which promote ventral fates, also prevent opl expression when implanted during gastrulation. These data show that forebrain specification begins early during gastrulation, and that a wide area of dorsal mesendoderm is required for its patterning (Grinblat, 1998).

The mouse Mf3 gene, also known as Fkh5 and HFH-e5.1, encodes a winged helix/forkhead transcription factor. In the early embryo, transcripts for Mf3 are restricted to the presomitic mesoderm and anterior neurectoderm and mesoderm. By 9.5 days post coitum, expression in the nervous system is predominantly in the diencephalon, midbrain and neural tube. After midgestation, the highest level of mRNA is in the mammillary bodies, the posterior-most part of the hypothalamus. Mice homozygous for a deletion of the mf3 locus on a [129 x Black Swiss] background display variable phenotypes consistent with a requirement for the gene at several stages of embryonic and postnatal development. Approximately six percent of the mf3-/- embryos show an open neural tube in the diencephalon and midbrain region, and another five percent show a severe reduction of the posterior body axis; both these classes of affected embryos die in utero. Surviving homozygotes have an apparently normal phenotype at birth. Postnatally, however, mf3-/- pups are severely growth retarded and approximately one third die before weaning. This growth defect is not a direct result of lack of circulating growth hormone or thyrotropin. Mice that survive to weaning are healthy, but they show an abnormal clasping of the hindfeet when suspended by the tail. Although much smaller than normal, the mice are fertile. However, mf3-/- females cannot eject their milk supply to feed their pups. This nursing defect can be corrected with interperitoneal injections of oxytocin. These results provide evidence that Mf3 is required for normal hypothalamus development and suggest that Mf3 may play a role in postnatal growth and lactation. Several winged helix genes are expressed in the presomitic mesoderm and/or somitic tissues, including Mfh1, Mf1 and Mf2. There are at least four wing helix genes in addition to Mf3 expressed in the developing mammillary region of the embryonic hypothalamus: Bf1, Bf2, Fkh4 and Mf2. It is therefore likely that the expression of these genes partially or completely compensate for the lack of MF3 in the presomitic mesoderm and neurectoderm in the less affected prenatal lethal and surviving mf3 knockouts (Labosky, 1997).

The murine winged helix gene Fkh5 is specifically expressed in the developing central nervous system (CNS). Early embryonic Fkh5 expression is restricted to the mammiliary body region of the caudal hypothalamus, midbrain, hindbrain and spinal cord. Postnatally, signals persist in specific nuclei of the mammillary body and in the midbrain. Fkh5 deficient mice were generated by homologous recombination to assess its in vivo function. At birth, Fkh5-deficient mice are viable and indistinguishable from wild-type and Fkh5 heterozygous littermates. However, about one third die within the first two days and another fifth before weaning. Surviving Fkh5-deficient mice become growth retarded within the first week and remain smaller throughout their entire life span. Fkh5-deficient females on 129Sv x C57BL/6 genetic background are fertile, but do not nurture their pups. More detailed analysis of Fkh5-deficient brains reveals distinct alterations in the CNS. In the midbrain, mutant mice exhibit reduced inferior colliculi and an overgrown anterior cerebellum. The hypothalamic mammillary body of Fkh5-deficient brains lacks the medial mammillary nucleus. These results suggest that Fkh5 plays a major role during CNS development (Wehr, 1997).

Three inductive interactions result in the regionalization of the mouse forebrain: (1) medial (ventral) patterning signals originating from the notochord and the more anterior precordal plate induce the primordia of the basal plate; (2) local signals arising from the anterior neural ridge (ANR), including Fgf8, induce expression of BF1 (a Forkhead related protein in a distinct sloppy-paired related subfamily, more distantly related to Drosophila Forkhead than is HNF3ß) , which regulates the development of specific forebrain structures such as telencephalic and optic vesicles, and (3) lateral (dorsal) patterning signals (BMPs) that arise from the non-neural ectoderm flanking the neural plate induce expression of Msx1 and patterning of the alar plate. This paper deals with the first two of these inductive interactions. Molecular properties of the medial neural plate are regulated by signals originating from the prechordal plate perhaps through the action of Sonic Hedgehog. Sonic induces homeobox gene Nkx2.1 (a homolog of Drosophila vnd) in the medial part of the mouse prosencephalic neural plate as early as the 3-somite stage, and Pax6 is expressed more laterally at similar or slighty later times. HNF3ß and not Nkx2.1 is expressed in posterior parts of explants, demonstrating that this tissue responses to Sonic and is not competent to express Nkx2.1. This suggests that the forebrain employs the same medial-lateral (ventral-dorsal) patterning mechanisms present in the rest of the central nervous system (Shimamura, 1997).

Gene expression in the antero-lateral neural plate (the anterior neural ridge is the junction between the anterior neural plate and anterior non-neural ectoderm) is regulated by non-neural ectoderm and bone morphogenetic proteins. BF1 expression is first detectable as early as the 3-somite-stage in the non-neural ectoderm underlying the anterior margin of the neural plate. By the 8-somite-stage, the expression is also detectable in the anterolateral neural plate. BF-1 expression in the developing brain is restricted to the telencephalic neuroepithelium and the nasal half of the retina and optic stalk. Its expression domain is adjacent to that of BF-2, which is restricted to the rostral diencephalon and the temporal half of the retina and optic stalk. Thus, the anterior neural ridge regulates patterning of the anterior neural plate, through a mechanism that is distinct from those that regulate general medial-lateral patterning. The anterior neural ridge is essential for expression of BF1; this neural ridge expresses Fgf8. Recombinant FGF8 protein is capable of inducing BF1, suggesting that FGF8 regulates the development of anterolateral neural plate derivatives (Shimamura, 1997).

The neural plate is subdivided into distinct anterior-posterior domains that have different responses to inductive signals from the prechordal plate, Sonic Hedgehog, the anterior neural ridge and FGF8. For example, Engrailed 2 is induced by beads placed more posteriorly than those that induce BF1. The induced BF1-expression domain is delineated posteriorly by a sharp boundary, which may be orthogonal to the long axis of the explants. The posterior boundary of BF1 and the anterior boundary of En2 are nearly adjacent. In sum, these results suggest that regionalization of the forebrain primordia is established by several distinct patterning mechanisms: (1) anterior-posterior patterning creates transverse zones with differential competence within the neural plate; (2) patterning along the medial-lateral axis generates longitudinally aligned domains and (3) local inductive interactions, such as a signal(s) from the anterior neural ridge, further define the regional organization (Shimamura, 1997).

Fkhl0 is a member of the forkhead family of winged helix transcriptional regulators. Genes encoding forkhead proteins are instrumental during embryogenesis in mammals, in particular during development of the nervous system. Mice with a targeted disruption of the Fkh10 locus exhibit circling behaviour, poor swimming ability and abnormal reaching response -- all common findings in mice with vestibular dysfunction. These animals also fail to elicit a Preyer reflex in response to a suprathreshold auditory stimulation, as seen in mice with profound hearing impairment. Histological examination of the inner ear reveals a gross structural malformation of the vestibulum as well as the cochlea. These structures have been replaced by a single irregular cavity in which neither proper semicircular ducts nor cochlea can be identified. At 9.5 days post coitum (dpc), Fkh10 is exclusively expressed in the otic vesicle. These findings implicate Fkh10 as an early regulator necessary for development of both cochlea and vestibulum and identify its human homolog FKHL10 as a previously unknown candidate deafness gene at 5q34 (Hulander, 1998).

Xlens1 is a novel Xenopus member of the fork head gene family, named for its nearly restricted expression in the anterior ectodermal placode, presumptive lens ectoderm (PLE), and anterior epithelium of the differentiated lens. The temporal and spatial restriction of its expression suggests that: (1) Xlens1 is transcribed initially at neural plate stages in response to putative signals from the anterior neural plate that transform lens-competent ectoderm to lens-biased ectoderm; (2) further steps in the process of lens-forming bias restrict Xlens1 expression to the presumptive lens ectoderm (PLE) during later neural plate stages; (3) interactions with the optic vesicle maintain Xlens1 expression in the lens placode; and (4) Xlens1 expression is downregulated as committed lens cells undergo terminal differentiation. Induction assays demonstrate that pax6 induces Xlens1 expression, but unlike Xlens1, pax6 cannot induce the expression of the lens differentiation marker beta-crystallin. In the whole embryo, overexpression of Xlens1 in the lens ectoderm causes it to thicken and maintain gene expression characteristics of the PLE. Also, this overexpression suppresses differentiation in the lens ectoderm, suggesting that Xlens1 functions to maintain specified lens ectoderm in an undifferentiated state. Misexpression of Xlens1 in other regions causes hypertrophy of restricted tissues but only occasionally leads ectopic sites of gamma-crystallin protein expression in select anterior head regions. These results indicate that Xlens1 expression alone does not specify lens ectoderm. Lens specification and differentiation likely depends on a combination of other gene products and an appropriate level of Xlens1 activity (Kenyon, 1999).

The hepatocyte nuclear factor-3 (HNF-3)/fork head homolog (HFH) proteins are an extensive family of transcription factors, which share homology in the winged helix DNA binding domain. Members of the HFH/winged helix family have been implicated in cell fate determination during pattern formation, in organogenesis, and in cell-type-specific gene expression. A full-length HFH-3 cDNA clone has been isolated from a human kidney library. It encodes a 351-amino acid protein containing a centrally located winged helix DNA binding domain. HFH-3 is a potent transcriptional activator requiring 138 C-terminal residues for activity. HFH-3 expression is restricted to the epithelium of the renal distal convoluted tubules. Putative HFH-3 target genes include the Na/K-ATPase, Na/H and anion exchangers, E-cadherin, and mineralocorticoid receptor genes as well as genes for the transcription factors HNF-1, vHNF-1, and HNF-4 (Overdier, 1997).

Members of the TGF-beta superfamily of signaling molecules work by activating transmembrane receptors with phosphorylating activity (serine-threonine kinase receptors); these in turn phosphorylate and activate SMADs, a class of signal transducers (see Drosophila Mad). Activins are growth factors that act primarily through Smad2, possibly in partnership with Smad4, which forms heteromeric complexes with different ligand-specific SMADs after activation. In frog embryos, Smad2 participates in an activin-responsive factor (ARF), which then binds to a promoter element of the Mix.2 gene. The principal DNA-binding component of ARF is FAST-1 (Forkhead activin signal transducer 1), a transcription factor with a novel winged-helix structure. The forkhead domain of FAST-1 is as similar to known members of the forkhead family as these are to one another. Smad4 is present in ARF, and FAST-1, Smad4 and Smad2 co-immunoprecipitate in a ligand-regulated fashion. The site of interaction between FAST-1 and Smad2/Smad4 has been mapped to a novel carboxy-terminal domain of FAST-1, and overexpression of this domain specifically inhibits activin signaling. In a yeast two-hybrid assay, the FAST-1 carboxy terminus interacts with Smad2 but not Smad4. Deletion mutants of the FAST-1 carboxy terminus that still participate in ligand-regulated Smad2 binding no longer associated with Smad4 or ARF. These results indicate that Smad4 stabilizes a ligand-stimulated Smad2-FAST-1 complex as an active DNA-binding factor (Chen, 1997).

A Xenopus TGF-ß responsive immediate-early response gene, Mix.2, encodes a homeobox gene expressed in prospective mesoderm and endoderm just after the mid-blastula transition. An activin-response factor (ARF) binds specifically to a 50-bp Mix.2 promoter element. The ARF complex contains XMAD2, a Xenopus homolog of the Drosophila MAD protein. A second component of ARF has been identified as forkhead activin signal transducer-1 (FAST-1) which contains a domain clearly related to the winged-helix domain of the forkhead/HNF3ß family of transcription factors. FAST-1 mRNA is present in oocytes and in early embryos until shortly after gastrulation. It is concluded that FAST-1 and XMAD2 are partners in the coactivation of Mix.2 (Chen, 1996).

A mammalian forkhead domain protein, FAST2, has been identifed that is required for induction of the goosecoid (gsc) promoter by TGF beta or activin signaling. FAST2 binds to a sequence in the gsc promoter, but efficient transcriptional activation and assembly of a DNA-binding complex of FAST2, Smad2, and Smad4 requires an adjacent Smad4 site. Smad3 is closely related to Smad2 but suppresses activation of the gsc promoter. Inhibitory activity is conferred by the MH1 domain, which unlike that of Smad2, binds to the Smad4 site. Through competition for this shared site, Smad3 may prevent transcription by altering the conformation of the DNA-binding complex. Thus, a mechanism is described whereby Smad2 and Smad3 positively and negatively regulate a TGF beta/activin target gene (Labbe, 1998).

Many cell-cycle-specific events are supported by stage-specific gene expression. In budding yeast, at least three different nuclear factors seem to cooperate in the periodic activation of G2/M-specific genes. Chromatin immunoprecipitation polymerase chain reaction assays have been used to show that a positive regulator, Ndd1, becomes associated with G2/M promoter regions in a manner that depends on the stage of the cell cycle. The recruitment of Ndd1 depends on a permanent protein-DNA complex consisting of the MADS box protein, Mcm1, and a recently identified partner, Fkh2, a forkhead/winged helix related transcription factor. The lethality of Ndd1 depletion is suppressed by fkh2 null mutations, which indicates that Fkh2 may also have a negative regulatory role in the transcription of G2/M-induced RNAs. It is concluded that Ndd1-Fkh2 interactions may be the transcriptionally important process targeted by Cdk activity (Koranda, 2000).

Forkhead transcription factors have been implicated in many developmental processes. They are necessary for proliferative responses and cell differentiation, and have been identified as targets of signal transduction systems. Their connection to a well-defined cell-cycle-specific program in yeast has been established. In comparison with some other systems involving forkhead transcription factors, the function of the yeast factors Fkh1 and Fkh2 does not seem to be determined by nucleo-cytoplasmic shuttling. Fkh1 and Fkh2 are constitutively bound to promoters and seem to function by providing a permanent platform for further regulatory inputs such as Ndd1 recruitment. How does the mitotic Clb/Cdk kinase impact on this system? In principle, two mechanisms are converging on Ndd1: (1) Ndd1 might be stabilized in a cell-cycle-specific manner either directly as a substrate of the kinase or indirectly through regulation of the ubiquitination/degradation machinery; (2) one could imagine that phosphorylation events control the interaction between Ndd1 and the Mcm1-Sff complex. Conclusive evidence for regulated phosphorylation of Ndd1 or Fkh2 has been elusive; notably, however, both Fkh1 and Fkh2 contain a so-called 'FHA' (forkhead-associated) domain that is thought to act as docking site for phospho-serine or phospho-threonine motifs (Koranda, 2000).

The hepatocyte nuclear factor 3/fork head homolog (HFH) proteins are 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. HFH-8 winged helix motif exhibits considerable amino acid differences from the orignial HNF-3 and Forkhead proteins. HFH-8, however, possesses regions rich in glycine residues that are also present in the amino terminus of Fkh and HNF-3gamma and demonstrates conservation with the region II transcriptional activation motif, a sequence found in nimerous HNF-3/Fkh proteins. HFH-8 exhibits homology with the HNF-3/FKH region III activation motif. In situ hybridization was used to identify the cellular expression pattern of 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 of 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. The determined HFH-8 consensus sequence identifies 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 is 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).

Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 most likely triggers apoptosis by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene (Brunet, 1999).

The floor plate is a morphologically distinct structure of epithelial cells situated along the midline of the ventral spinal cord in vertebrates. It is a source of guidance molecules directing the growth of axons along and across the midline of the neural tube. In the zebrafish, the floor plate is about three cells wide and composed of cuboidal cells. Two cell populations can be distinguished by the expression patterns of several marker genes, including sonic hedgehog and the fork head-domain gene fkd4: a single row of medial floor plate (MFP) cells, expressing both shh and fkd4, is flanked by rows of lateral floor plate (LFP) cells that express fkd4 but not shh. Systematic mutant searches in zebrafish embryos have identified a number of genes, mutations that visibly reduce the floor plate. In these mutants either the MFP or the LFP cells are absent, as revealed by the analysis of the shh and fkd4 expression patterns. MFP cells are absent, but LFP cells are present, in mutants of cyclops, one-eyed pinhead, and schmalspur, wherein development of midline structures is affected. LFP cells are absent, but MFP cells are present, in mutants of four genes: sonic you, you, you-too, and chameleon, collectively called the you-type genes. This group of mutants also shows defects in patterning of the paraxial mesoderm, causing U-shaped instead of V-shaped somites. One of the you-type genes, sonic you, encodes the zebrafish Shh protein, suggesting that the you-type genes encode components of the Shh signaling pathway. In the zebrafish shh is required for the induction of LFP cells, but not for the development of MFP cells. This conclusion is supported by the finding that injection of shh RNA causes an increase in the number of LFP, but not MFP cells. Embryos mutant for iguana, detour, and umleitung share the lack of LFP cells with you-type mutants, while somite patterning is not severely affected. In mutants that fail to develop a notochord, MFP cells may be present, but are always surrounded by LFP cells. These data indicate that shh, expressed in the notochord and/or the MFP cells, induces the formation of LFP cells. In embryos doubly mutant for cyclops (cyc) and sonic you, both LFP and MFP cells are deleted. The number of primary motor neurons is strongly reduced in cyc;syu double mutants, while almost normal in single mutants, suggesting that the two different pathways have overlapping functions in the induction of primary motor neurons (Odenthal, 2000).

The anterior heart field (AHF) mediates formation of the outflow tract (OFT) and right ventricle (RV) during looping morphogenesis of the heart. Foxh1 is a forkhead DNA binding transcription factor in the TGFß-Smad pathway. Foxh1−/− mutant mouse embryos form a primitive heart tube, but fail to form OFT and RV and display loss of outer curvature markers of the future working myocardium, similar to the phenotype of Mef2c−/− mutant hearts. Further, Mef2c is shown to be a direct target of Foxh1, which physically and functionally interacts with Nkx2-5 to mediate strong Smad-dependent activation of a TGFß response element in the Mef2c gene. This element directs transgene expression to the presumptive AHF, as well as the RV and OFT, a pattern that closely parallels endogenous Mef2c expression in the heart. Thus, Foxh1 and Nkx2-5 functionally interact and are essential for development of the AHF and its derivatives, the RV and OFT, in response to TGFß-like signals (von Both, 2004).

Foxh1, a Smad DNA-binding partner, mediates TGFbeta-dependent gene expression during early development. Few Foxh1 targets are known. This study describes a genome-wide approach that couples systematic mapping of a functional Smad/Foxh1 enhancer (SFE) to Site Search, a program used to search annotated genomes for composite response elements. Ranking of SFEs that are positionally conserved across species yielded a set of genes enriched in Foxh1 targets. Analysis of top candidates, such as Hesx1, Lgr4, Lmo1, Fgf8, and members of the Aldh1a subfamily, revealed that Foxh1 initiates a transcriptional regulatory network within the developing anterior neuroectoderm. The Aldh1a family is required for retinoic acid (RA) synthesis, and, in Foxh1 mutants, expression of Aldh1a1, -2, and -3 and activation of a RA-responsive transgenic reporter is abolished in anterior structures. Integrated mapping of a developmental transcription factor network thus reveals a key role for Foxh1 in patterning and initiating RA signaling in the forebrain (Silvestri, 2008).

The formation of locomotor circuits depends on the spatially organized generation of motor columns that innervate distinct muscle and autonomic nervous system targets along the body axis. Within each spinal segment, multiple motor neuron classes arise from a common progenitor population; however, the mechanisms underlying their diversification remain poorly understood. This study shows that the Forkhead domain transcription factor Foxp1 (closest Drosophila BLAST hit: CG16899) plays a critical role in defining the columnar identity of motor neurons at each axial position. Using genetic manipulations, it was demonstrated that Foxp1 establishes the pattern of LIM-HD protein expression and accordingly organizes motor axon projections, their connectivity with peripheral targets, and the establishment of motor pools. These functions of Foxp1 act in accordance with the rostrocaudal pattern provided by Hox proteins along the length of the spinal cord, suggesting a model by which motor neuron diversity is achieved through the coordinated actions of Foxp1 and Hox proteins (Rousso, 2008).

The precision with which motor neurons innervate target muscles depends on a regulatory network of Hox transcription factors that translates neuronal identity into patterns of connectivity. A single transcription factor, FoxP1, coordinates motor neuron subtype identity and connectivity through its activity as a Hox accessory factor. FoxP1 is expressed in Hox-sensitive motor columns and acts as a dose-dependent determinant of columnar fate. Inactivation of Foxp1 abolishes the output of the motor neuron Hox network, reverting the spinal motor system to an ancestral state. The loss of FoxP1 also changes the pattern of motor neuron connectivity, and in the limb motor axons appear to select their trajectories and muscle targets at random. These findings show that FoxP1 is a crucial determinant of motor neuron diversification and connectivity, and clarify how this Hox regulatory network controls the formation of a topographic neural map (Dasen, 2008).

Cardiomyocyte proliferation is high in early development and decreases progressively with gestation, resulting in the lack of a robust cardiomyocyte proliferative response in the adult heart after injury. Little is understood about how both cell-autonomous and nonautonomous signals are integrated to regulate the balance of cardiomyocyte proliferation during development. This study shows that a single transcription factor, Foxp1, can control the balance of cardiomyocyte proliferation during development by targeting different pathways in the endocardium and myocardium. Endocardial loss of Foxp1 results in decreased Fgf3/Fgf16/Fgf17/Fgf20 expression in the heart, leading to reduced cardiomyocyte proliferation. This loss of myocardial proliferation can be rescued by exogenous Fgf20, and is mediated, in part, by Foxp1 repression of Sox17. In contrast, myocardial-specific loss of Foxp1 results in increased cardiomyocyte proliferation and decreased differentiation, leading to increased myocardial mass and neonatal demise. Nkx2.5 is a direct target of Foxp1 repression, and Nkx2.5 expression is increased in Foxp1-deficient myocardium. Moreover, transgenic overexpression of Nkx2.5 leads to increased cardiomyocyte proliferation and increased ventricular mass, similar to the myocardial-specific loss of Foxp1. These data show that Foxp1 coordinates the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation of Fgf ligand and Nkx2.5 expression (Zhang, 2010).

This work reports the characterization and functional analysis of disrupted in schizophrenia 1 (disc1), a well-documented schizophrenia-susceptibility gene, in zebrafish cranial neural crest (CNC). The data demonstrated that disc1 is expressed in zebrafish CNC cells. Loss of Disc1 resulted in persistent CNC cell medial migration, dorsal to the developing neural epithelium, and hindered migration away from the region dorsal to the neural rod. General CNC cell motility was not affected by Disc1 knockdown, however, as the speed of CNC cells was indistinguishable from that of wild-type counterparts. The failure of CNC cells to migrate away from the neural rod correlates with the enhanced expression of two transcription factors, foxd3 and sox10. These transcription factors have many functions in CNC cells, including the maintenance of precursor pools, timing of migration onset, and the induction of cell differentiation. This work suggests that the perpetuation of expression of these factors affects several aspects of CNC cell development, leading to a loss of craniofacial cartilage and an expansion of peripheral cranial glia. Based on these data, a model is proposed in which Disc1 functions in the transcriptional repression of foxd3 and sox10, thus mediating CNC cell migration and differentiation (Drerup, 2009).

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. This study introduced these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, medium spiny neurons were found to have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans (Enard, 2009).

Foxp1 and Foxp2, which belong to the forkhead transcription factor family, are expressed in the developing and adult mouse brain, including the striatum, thalamus, and cerebral cortex. Recent reports suggest that FOXP1 and FOXP2 are involved in the development of speech and language in humans. Although both Foxp1 and Foxp2 are expressed in the neural circuits that mediate speech and language, including the corticostriatal circuit, the functions of Foxp1 and Foxp2 in the cerebral cortex remain unclear. To gain insight into the functions of Foxp1 and Foxp2 in the cerebral cortex, Foxp1- and Foxp2-expressing cells were characterized in postnatal and adult mice using immunohistochemistry. In adult mice, Foxp1 was expressed in neurons of layers III-VIa in the neocortex, whereas the expression of Foxp2 was restricted to dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32)(+) neurons of layer VI. In addition, Foxp2 was weakly expressed in the neurons of layer V of the motor cortex and hindlimb and forelimb regions of the primary somatosensory cortex. Both Foxp1 and Foxp2 were expressed in the ionotropic glutamate receptor (GluR) 2/3(+) neurons, and colocalized with none of GluR1, gamma-aminobutyric acid, calbindin, and parvalbumin, indicating that expression of Foxp1 and Foxp2 is restricted to projection neurons. During the postnatal stages, Foxp1 was predominantly expressed in Satb2(+)/Ctip2(-) corticocortical projection neurons of layers III-V and in Tbr1(+) corticothalamic projection neurons of layer VIa. Although Foxp2 was also expressed in Tbr1(+) corticothalamic projection neurons of layer VI, no colocalization of Foxp1 with Foxp2 was observed from postnatal day (P) 0 to P7. These findings suggest that Foxp1 and Foxp2 may be involved in the development of different cortical projection neurons during the early postnatal stages in addition to the establishment and maintenance of different cortical circuits from the late postnatal stage to adulthood (Hisaoka, 2010).

HEB and E2A function as SMAD/FOXH1 cofactors

Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. A new motif, termed SMAD complex-associated (SCA), was identified that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. Two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation (Yoon, 2011).

ChIP-seq was used to generate genome-wide occupancy maps for the Nodal signaling factors SMAD2/3, SMAD3, SMAD4, and FOXH1 in both hESCs and derived endoderm. This study sought to identify novel SMAD complex cofactors by performing de novo motif discovery on the SMAD/FOXH1 genomic targets. Three nonrepetitive motifs were identified that were consistently enriched in all data sets (SMAD2/3, SMAD3, SMAD4, and FOXH1) and in both cell types, hESCs and endoderm. The first and second motifs contain the canonical SMAD- and FOXH1-binding sites, respectively, confirming their genome-wide cooperativity in regulating Nodal signaling and further validating the antibodies used for ChIP. The third motif, CCTGCTG, has not previously been shown to associate with any of the SMAD/FOXH1 complex proteins.This element is referred to as the SCA (SMAD complex-associated) motif (Yoon, 2011).

This study presents strong genomic, biochemical, and functional evidence that E2A and HEB interact with SMAD2/3/4 and FOXH1 to regulate transcription of Nodal target genes. E2A and HEB associate with SMAD2/3 and FOXH1 at the SCA consensus site, which is functionally conserved between frogs and humans. The genomic identification of this site using the power of large sequence reads in multiple data sets provided inroads into testing the interaction of E2A, HEB, and the SMAD/FOXH1 complex. Using biochemical approaches, this study shows that these proteins interact in a DNA-independent manner, but then associate with similar target regions. Based on evidence presented in this study, it is hypothesized that a complex consisting of E2A, HEB, SMAD2/3, and FOXH1 forms within the nucleus in response to Nodal, but that maintenance of this complex is independent of continual Nodal signaling. Overall, it is suggested that E2A and HEB are key regulators of SMAD2/3-mediated transcriptional responses, and thus are fundamental Nodal cofactors that have not previously been implicated in this important developmental pathway (Yoon, 2011).

While genomic and biochemical association is suggestive of a key signaling role, the phenotypic effect of knocking down e2a in X. tropicalis embryos is highly reminiscent of phenotypes resulting from perturbation of other key Nodal signaling factors, such as overexpression of a dominant-negative Nodal receptor or of the Nodal antagonists Cerberus-short and Lefty. Furthermore, it was shown epistatically that e2a knockdown inhibits the ability of both Activin and Xnr1 to induce bottle cell formation, strongly suggesting a key downstream role in the pathway. In the mouse, the roles of HEB and E2A and their family member, E2-2, have been extensively characterized as essential factors in hematopoiesis. The phenotypes of single-gene knockout models for E2A and HEB demonstrated that E2A was the primary E-protein member driving B-cell development, but that both E2A and HEB were required for proper T-cell development. Interestingly, however, there is very strong evidence that these proteins are highly redundant due to their heterodimerizing abilities. Dominant-negative HEB, which can also disrupt E2A function through nonproductive heterodimer formation, causes a stronger phenotype than the heb-null mutation. In B-cell development, HEB, driven by the E2A promoter, can rescue E2A loss of function. These complex genetics and the associated lethality of some compound mutants have made investigation of the roles of these proteins in early embryonic development difficult, and a role for E2A or HEB in early embryogenesis or SMAD/FOXH1 signaling has never been identified. Conditional genetic approaches to ablate several family members during gastrulation will more accurately address the role of E2A and HEB during mammalian germ layer formation. It is noted with interest that loss of e2a function in X. tropcialis achieves an effect on gastrulation not seen in the mouse. It is hypothesized that the expansion of the Nodal pathway in frogs during evolution may have generated less redundancy between the E proteins; this is currently being tested by evaluating compound MOs. Overall, further investigation of the mechanisms used by E2A and HEB to modulate Nodal signal transduction will elucidate new insights into how this important pathway is diversified to induce cell lineages within distinct species (Yoon, 2011).

The human language-associated gene SRPX2 regulates synapse formation and vocalization in mice

Synapse formation in the developing brain depends on the coordinated activity of synaptogenic proteins, some which have been implicated in a number of neurodevelopmental disorders. This study shows that the sushi repeat-containing domain protein X-linked 2 (SRPX2) gene encodes a protein that promotes synaptogenesis in the cerebral cortex. In humans, SRPX2 is an epilepsy- and language-associated gene that is a target of the foxhead box protein P2 (FoxP2) transcription factor. It was also shown that FoxP2 modulates synapse formation through regulating SRPX2 levels, and that SRPX2 reduction impairs development of ultrasonic vocalization in mice. The results suggest FoxP2 modulates the development of neural circuits through regulating synaptogenesis and that SRPX2 is a synaptogenic factor that plays a role in the pathogenesis of language disorders (Sia, 2013).

This study has shown that SRPX2 is a sushi domain protein involved in synapse formation. In invertebrates, sushi domain proteins have been shown to cluster AChRs at synapses in C. elegans, and the Drosophila homolog, Hikaru Genki, is localized to the nascent synaptic cleft. In vertebrates, sushi domain proteins are primarily studied as regulators of the classical complement cascade. The current results suggest that sushi domain proteins may also play roles in regulating synaptic development and organization in vertebrates. In addition, as genes of the classical complement cascade have been shown to regulate synapse elimination, it is speculated that SRPX2 may act through modulation of components of the complement cascade (Sia, 2013).

To date, FoxP2 is the only gene that has been shown to be involved in a human monogenic language disorder, although the cellular mechanisms involved remain obscure. Previous studies have suggested that FoxP2 may regulate neurite growth, dendritic morphology and synaptic physiology of basal ganglia neurons. This study shows that FoxP2 can regulate synaptogenesis of excitatory synapses in cortical neurons through SRXP2. While activity-regulated transcription factors have been shown to regulate synaptogenesis, developmental synapse formation can occur in the complete absence of activity, and it is unclear whether such synapse formation is also regulated by activity-independent transcription factors. This study shows that FoxP2 is an activity-independent transcription factor that regulates synaptogenesis through SRPX2. In conclusion, this study suggests that FoxP2 can affect the development of language-related neural circuitry through regulating synaptogenesis, and that SRPX2 may be involved in the pathogenesis of language disorders (Sia, 2013).

A novel N-terminal motif is responsible for the evolution of neural crest-specific gene-regulatory activity in vertebrate FoxD3

The neural crest is unique to vertebrates and has allowed the evolution of their complicated craniofacial structures. During vertebrate evolution, the acquisition of the neural crest must have been accompanied by the emergence of a new gene regulatory network (GRN). To investigate the role of protein evolution in the emergence of the neural crest GRN, the neural crest cell (NCC) differentiation-inducing activity of chordate FoxD genes was examined. Amphioxus and vertebrate (Xenopus) FoxD proteins both exhibited transcriptional repressor activity in Gal4 transactivation assays and bound to similar DNA sequences in vitro. However, whereas vertebrate FoxD3 genes induced the differentiation of ectopic NCCs when overexpressed in chick neural tube, neither amphioxus FoxD nor any other vertebrate FoxD paralogs exhibited this activity. Experiments using chimeric proteins showed that the N-terminal portion of the vertebrate FoxD3 protein is critical to its NCC differentiation-inducing activity. Furthermore, replacement of the N-terminus of amphioxus FoxD with a 39-amino-acid segment from zebrafish FoxD3 conferred neural crest-inducing activity on amphioxus FoxD or zebrafish FoxD1. Therefore, fixation of this N-terminal amino acid sequence may have been crucial in the evolutionary recruitment of FoxD3 to the vertebrate neural crest GRN (Ono, 2014).

Table of contents

forkhead: Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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