Table of contents

Other Drosophila Forkhead domain proteins

Drosophila sloppy-paired 1 and 2 both have Forkhead domains (Hacker, 1992). Sloppy paired belongs to a different class of forkhead domain proteins than does Forkhead itself and HNF-3ß. Sloppy paired's closest homolog is BF-1. Another forkhead homolog in Drosophila has been discovered, the crocodile gene, required for the establishment of head structures. Crocodile's closest mammalian homolog is FD1 belonging to a different class of forkhead domain proteins than does Forkhead. Forkhead belongs to the same class as HNF-3alpha, HNF-3ß, HNF-3gamma, XFKH1/XFD1, and XFD1/pintallavis (Sasaki, 1993 and Hacker, 1995).

Forkhead homologs from other insects

To analyze how the silk glands of the lepidopteran Bombyx mori develop, two genes were cloned and identified that encode the homeodomain and its flanking regions identical to the corresponding regions of Drosophila Deformed and Sex combs reduced. The Bombyx silk gland is assumed to be an evolutionally homologous organ to the Drosophila salivary gland because both structures are formed in the labial segment and share some similar functions. Bombyx Deformed is expressed in the mandibular and maxillary segments, whereas expression of Bombyx Sex combs reduced is first limited to the labial segment and at later stages extended to the anterior part of the prothoracic segment. The expression of Bombyx Sex combs reduced then disappears from the invaginating placodes of silk glands where expression of Bombyx fork head/SGF-1 follows. In the mutant embryos, which lack the 3' end region of Bombyx Antennapedia, Bombyx Sex combs reduced is expressed ectopically in the thoracic and abdominal regions, in addition to expression in the labial segment. Bombyx fork head/SGF-1 is also ectopically expressed in the T1, T2, and T3 segments, resulting in the ectopic induction of the silk gland invaginations. These results suggest that Bombyx homeobox genes such as the Bombyx Deformed and Sex combs reduced are associated with determination of the segment identities and Bombyx Sex combs reduced is involved in the induction of silk gland development (Kokubo, 1997).

Transcription factor Forkhead regulates expression of antimicrobial peptides in the tobacco hornworm, Manduca sexta

Antimicrobial peptides (AMPs) play an important role in defense against microbial infections in insects. Expression of AMPs is regulated mainly by NF-κB factors Dorsal, Dif and Relish. Previous work has shown that both NF-kappaB and GATA-1 factors are required for activation of moricin promoter in the tobacco hornworm, Manduca sexta, and a 140-bp region in the moricin promoter contains binding sites for additional transcription factors. This study, we identified three forkhead (Fkh)-binding sites in the 140-bp region of the moricin promoter and several Fkh-binding sites in the lysozyme promoter, and demonstrated that Fkh-binding sites are required for activation of both moricin and lysozyme promoters by Fkh factors. In addition, Fkh mRNA was undetectable in Drosophila S2 cells, and M. sexta Fkh (MsFkh; see Drosophila Fkh) interacted with Relish-Rel-homology domain (RHD) but not with Dorsal-RHD. Dual luciferase assays with moricin mutant promoters showed that co-expression of MsFkh with Relish-RHD did not have an additive effect on the activity of moricin promoter, suggesting that MsFkh and Relish regulate moricin activation independently. These results suggest that insect AMPs can be activated by Fkh factors under non-infectious conditions, which may be important for protection of insects from microbial infection during molting and metamorphosis (Zhong, 2017).

Yeast forkhead domain proteins

In the yeast Saccharomyces cerevisiae, the MADS-box protein Mcm1, which is highly related to mammalian SRF (serum response factor), forms a ternary complex with SFF (Swi five factor) to regulate the cell cycle expression of genes such as SWI5, CLB2 and ACE2. The forkhead protein Fkh2 is a component of SFF and is essential for ternary complex formation on the SWI5 and ACE2 promoters. Fkh2 is essential for the correct cell cycle periodicity of SWI5 and CLB2 gene expression and is phosphorylated with a timing that is consistent with a role in this expression. Furthermore, investigation of the relationship between Fkh2 and a related forkhead protein, Fkh1, demonstrates that these proteins act in overlapping pathways to regulate cell morphology and cell separation. This is the first example of a eukaryotic transcription factor complex containing both a MADS-box and a forkhead protein, and it has important implications for the regulation of mammalian gene expression (Pic, 2000).

How is the Mcm1-SFF transcription factor complex regulated during the cell cycle? The Mcm1-SFF complex may be detectable in extracts prepared from G1 cells. This is consistent with a model where Mcm1-SFF binds to the SWI5 promoter throughout the cell cycle and that the Mcm1-SFF complex undergoes cell cycle regulation to activate transcription. In agreement with this, the Fkh2 protein can be detected in the nucleus of cells throughout the cell cycle. What other mechanism(s) could be responsible for the regulation of Mcm1-SFF? Previous studies have shown that the activity of Cdc28-Clb2 is required for the periodic activation of expression of the SWI5 and CLB2 genes, leading to the suggestion that a positive feedback loop acts to regulate G2 periodic gene expression. It is possible that the Mcm1-SFF transcription factor complex is phosphorylated directly by Cdc28-Clb2. However, although Mcm1 is known to be a phosphoprotein, it also regulates the expression of several genes in a non-cell cycle-dependent manner. Hence Fkh2 is a more likely target for specific cell cycle phosphorylation. Indeed, examination of the predicted sequence of the Fkh2 protein reveals several S/TP motifs, which may be potential targets for Cdc28-Clb2. Furthermore, Fkh2 becomes phosphorylated with a cell cycle timing that is consistent with the role of this protein in the activation of gene expression at G2/M. The molecular basis of this phosphorylation of Fkh2 is under investigation, but data suggest that the activity of the Mcm1-SFF transcription factor complex may be regulated by a cell cycle-regulated protein kinase(s) (Pic, 2000 and references therein).

The yeast Mcm1-SFF complex has been proposed to represent a functionally analogous complex to the mammalian SRF-TCF complex, which plays a pivotal role in regulating the induction of immediate-early genes such as the proto-oncogene c-fos in response to mitogenic stimuli. Thus the SRF-TCF complex is thought to function to promote cell cycle entry. Similarly, the Mcm1-SFF complex plays a role in regulating cell cycle progression, albeit at a later time point. Molecularly, their mechanisms of complex assembly and function appear to be similar, with both TCF (in mammals) and SFF (in yeast) being recruited to promoters by the highly related MADS-box proteins SRF and Mcm1. Both SFF and TCF are critical for the transcriptional activity of these complexes. The identification of Fkh2 as a component of SFF has uncovered a number of other intriguing parallels between these mammalian and yeast complexes. (1) While the TCFs and Fkh2 are members of different transcription factor families, ETS-domain and forkhead, respectively, their DNA-binding domains are related and form part of the larger winged helix-turn-helix family. Indeed, ETS-domain proteins appear unique to the metazoan lineage. (2) Both Fkh2 and TCF are modified by phosphorylation. In the case of Fkh2, there are numerous S/TP motifs in its C-terminal region, which is highly reminiscent of TCF's structure. Hence these similarities in structure and regulation suggest that studies of the mechanisms of regulation of activity and interaction between these highly conserved proteins will shed new light on the roles and activities of these proteins in eukaryotes (Pic, 2000 and references therein).

Hydra and sea anemone Forkhead homologs

Accumulating evidence indicates that a common set of genes and mechanisms regulates the developmental processes of a variety of triploblastic organisms, despite large variation in their body plans. To what extent these same genes and mechanisms are also conserved among diploblasts, which arose earlier in metazoan evolution, is unclear. A hydra homolog has been characterized for the fork head/HNF-3 class of winged-helix proteins, termed budhead. Budhead expression patterns suggest a role(s) similar to that found in vertebrates. The vertebrate HNF-3 beta homologs are expressed early in embryogenesis in regions that have organizer properties; later they play several roles, among these an important role in rostral head formation. In the adult hydra, where axial patterning processes are continuously active, budhead is expressed in the upper part of the head, which shows organizer properties. It is also expressed during the formation of a new axis as part of the development of a bud, hydra's asexual form of reproduction. Expression during later stages of budding, during head regeneration and the formation of ectopic heads, indicates a role in head formation. It is likely that budhead plays a critical role in head as well as axis formation in hydra. In hydra, initiation of axis formation begins with the commitment of tissue to head formation. Hence, budhead may have a single function that is related to head formation. In amphibian embryos, the organizer region consists of two parts, one organizing the head and the other the bud. Recent evidence suggests a similar situation in other vertebrate embryos as well. Vertebrate HNF-3 beta homologs are related to the head organizer and hence, as in hydra, are related to head formation. HNF-3 beta homologs are also expressed in the trunk organizer, where they have a role in notochord formation. Since diploblastic organisms have no mesoderm, the common element between hydra and vertebrates here is the role of the HNF-3 beta in the head organizer. These considerations raise the intriguing possibility that the molecular machinery for the head organizer, and hence head formation, originated in a common ancestor of all modern metzoans (Martinez, 1997).

The winged helix transcription factor Forkhead and the zinc finger transcription factor Snail are crucially involved in germ layer formation in Bilateria. A homolog of forkhead/HNF3 (FoxA/group 1) and of snail was isolated and characterized from a diploblast, the sea anemone Nematostella vectensis. Nematostella forkhead expression starts during late Blastula stage in a ring of cells that demarcate the blastopore margin during early gastrulation, thereby marking the boundary between ectodermal and endodermal tissue. snail, by contrast, is expressed in a complementary pattern in the center of forkhead-expressing cells marking the presumptive endodermal cells fated to ingress during gastrulation. In a significant portion of early gastrulating embryos, forkhead is expressed asymmetrically around the blastopore. While snail-expressing cells form the endodermal cell mass, forkhead marks the pharynx anlage throughout embryonic and larval development. In the primary polyp, forkhead remains expressed in the pharynx. The detailed analysis of forkhead and snail expression during Nematostella embryonic and larval development further suggests that endoderm formation results from epithelial invagination, mesenchymal immigration, and reorganization of the endodermal epithelial layer, that is, by epithelial-mesenchymal transitions (EMT) in combination with extensive morphogenetic movements. snail also governs EMT at different processes during embryonic development in Bilateria. These data indicate that the function of snail in Diploblasts is to regulate motility and cell adhesion, supporting the notion that the triggering of changes in cell behavior is the ancestral role of snail in Metazoa (Fritzenwanker, 2004).

In most Bilateria, the formation of the two inner germ layers, endoderm and mesoderm, is intimately linked during the process of gastrulation. In vertebrates, endodermal and mesodermal cells immigrate or invaginate together as endomesoderm and become separated morphologically only later during gastrulation. The evolutionary origin of the mesoderm is currently a matter of intense investigation, but still not clear. Some evidence from the two major diploblastic phyla, Cnidaria (also known as coelenterates) and Ctenophora, support the view of an endodermal origin of the mesoderm. However, other molecular data suggest that the third germ layer arose from the blastopore region with contributions from both ectoderm and endoderm (Fritzenwanker, 2004).

The evolution of the bilaterian foregut is also debated. Textbook knowledge postulates that foregut (and mouth) formation in Protostomes and Deuterostomes is fundamentally different and evolved convergently. However, similar expression of conserved transcription factors in the foregut anlage of basal deuterostome and protostome ciliary larva challenged this view and suggested a conserved molecular regulation of mouth development and homology of the foregut in Bilateria. The foregut is of great interest because it is the boundary between ectoderm and endoderm. In insects, both foregut (stomodeum) and hindgut (proctodeum) are regarded as an ectodermal derivative because in the adult these structures have a chitinized cuticula (Fritzenwanker, 2004).

One of the crucial conserved genes for mesoderm formation in Bilateria codes for the zinc finger transcription factor Snail. In insects, snail has been shown to repress the expression of neuroectodermal genes thereby marking the boundary between mesodermal and neurogenic region in the Drosophila embryo. In vertebrates, snail function has been implicated in epithelial-mesenchymal transitions of migrating cells of the developing mesoderm and of the neural crest. Snail has also been isolated from Podocoryne carnea, a hydrozoan cnidarian and from the coral Acropora millepora. Podocoryne snail is expressed in the entocodon of the developing medusa bud, suggesting a role in muscle development of the medusa, while Acropora snail is expressed in the endoderm during embryogenesis indicating a role in germ layer specification (Fritzenwanker, 2004).

A conserved marker gene for the foregut in Bilateria codes for the winged helix transcription factor Forkhead. The founder member forkhead is expressed in the foregut and hindgut anlage in Drosophila. Forkhead belongs to the group 1/HNF3/FoxA subfamily. In vertebrates, three highly related HNF3 genes, alpha, beta, and gamma, exist that differ by the timing and location of expression. In particular, HNF-3beta plays a crucial role during early vertebrate development. In mice and frogs, HNF-3beta is expressed in the organizer (node) and in the derivatives, the notochord but also the floor plate. HNF-3beta is involved in formation of the dorsoventral axis; HNF-3beta −/− mutant mice have defects in the DV patterning of the neural tube and of the dorsal mesoderm. This gene also has a conserved role in mesoderm formation in a dose-dependent manner and acts synergistically with brachyury to specify axial mesoderm in chordates (Fritzenwanker, 2004).

In insects, forkhead plays a conserved role in terminal patterning and formation of the foregut and hindgut anlage. The first forkhead homolog, from a diploblast, budhead, was isolated from the hydrozoan Hydra. budhead is expressed in the hypostome, the polyps' mouth and appears to have a role in axial patterning. The role of forkhead during cnidarian embryogenesis, however, is unknown. Since Hydra embryogenesis is highly derived and not easily accessible at all stages, a new model organism, the anthozoan Nematostella vectensis, was examined. Anthozoa are regarded as the basal group among Cnidaria and embryogenesis in Nematostella is inducible and readily accessible. This study reports the isolation and characterization of forkhead and snail homologs from Nematostella vectensis. The analysis shows that forkhead is expressed at the blastopore margin, that is, the boundary between ectoderm and endoderm and it marks the presumptive pharynx of the primary polyp. By contrast, snail has a virtually complementary expression pattern and marks all ingressing endodermal cells. The detailed analysis of forkhead and snail expression highlights that endoderm formation in this basal cnidarian is characterized by a relatively complex cellular behavior involving epithelial-mesenchymal transitions and morphogenetic movements (Fritzenwanker, 2004).

The evolution of the bilaterian gut has been studied by comparing the expression pattern of specific marker genes from a variety of organisms. It appears that a conserved cassette of developmental genes, mostly transcription factors, is expressed in fore- and hindgut primordia in all or most bilateria. These include the transcription factors caudal, brachyury, and forkhead and the signalling molecule wingless. All four genes are expressed in the blastopore in vertebrates and many insects, where they specify the derivatives of the blastopore, the foregut, and the hindgut (in Drosophila, the amnioproctodeal and the stomodeal invagination). While comparative data on caudal expression in different organisms are still scarce, at least brachyury, forkhead and wingless appear to have overlapping expression domains in most animals studied, hence they form an evolutionarily conserved synexpression group, suggesting that they might act in concert to specify a homologous structure in a wide range of animals. For instance, in the cnidarian Hydra, homologs of brachyury, Wnt3a, and forkhead have overlapping spatio-temporal expression domains in the hypostome, which correspond to the organizer of the polyp. Similarly, in Nematostella embryos, brachyury and forkhead are coexpressed at the ectodermal margin of the blastopore during gastrulation. A comparative analysis of expression patterns of these two genes shows that they are co-expressed in all animals analyzed. This suggests a close functional relationship of these two genes during animal development throughout metazoan evolution. Although a direct interaction of the two proteins has not been demonstrated to date, in Xenopus, they act synergistically to form dorsal mesoderm, in particular the notochord. Thus, forkhead and brachyury are an evolutionarily ancient synexpression group in Eumetazoa (Fritzenwanker, 2004).

Forkhead expression in particular is surprisingly conserved among metazoans. Together with brachyury, it marks the future blastopore and its derivatives, that is, foregut and hindgut. For instance, in hemichordates and echinoderms, forkhead is expressed in the vegetal plate cells before gastrulation, later in the involuting endoderm, and finally most strongly in the stomodeum anlage and the proctodeum of the Tornaria and Pluteus larva, respectively. Hence, in these lower deuterostomes, expression appears ectodermally restricted (if proctodeum and stomodeum are defined as ectodermal structures). Yet, at least in chordates, forkhead expression is not germ layer specific, but rather region- and organ-specific. In mice, the forkhead homolog HNF-3beta is expressed in the visceral endoderm, the node, (which gives rise to axial mesoderm, the notochord) and the floor plate . In the urochordates and protochordates (Amphioxus and Ascidians), the forkhead homolog is also expressed in gastrulating endoderm, the notochord and the floor plate. This suggests a close association of endoderm and the dorsal mesoderm (the notochord). In line with this, the notochord has been proposed to be a derivative of the archenteron roof in lower vertebrates, based on classical embryology (Fritzenwanker, 2004).

Much less information is available from Protostomes. However, in several species the expression domains are strikingly similar: in the Ecdysozoa, such as Drosophila, and Tribolium and in C. elegans, forkhead marks and is essential for the developing fore- and hind-gut before and during gastrulation. Among the Lophotrochozoa, expression of forkhead has been studied in the mollusc Patella vulgata. Strikingly, forkhead is expressed in the endoderm and anterior mesoderm, deriving from the anterior edge of the blastopore. At larval stages, forkhead is most strongly expressed in the stomodeum and somewhat weaker in the endoderm, reminiscent of the situation of vertebrates, where forkhead is also expressed in the prechordal plate. Based on expression data of forkhead in Nematostella vectensis, it is proposed that forkhead has an ancestral role in defining the blastopore and one derivative, the ectodermally derived pharynx. The evolutionary conservation of the synexpression group of brachyury, forkhead and several other genes suggest an establishment and coevolution of a cassette of conserved transcription factors in the blastopore during early metazoan evolution. Since forkhead and other node-specific genes also play important roles in dorsal-ventral axis formation in vertebrates, it is proposed that the blastopore evolved as an organizer for axis formation and mesoderm formation during early metazoan evolution (Fritzenwanker, 2004).

C. elegans Forkhead homologs

The DNA binding region of the C. elegans forkhead/HNF-3 homolog, Ce-fkh-1 is 75% - 78% identical to the fly and rat liver genes. Ce-fkh-3 is expressed in both pharynx and intestine of the embryo, beginning at the midproliferation stage. A second phase of expression occurs in cells of the larval somatic gonad. Whereas the Drosophila gene is expressed in salivary glands and in some cells of the central nervous system, Ce-fkh-3 is not. The MSaa and MSpa cells, which will migrate into the embryo interior to produce the posterior pharynx and whose descendents will express the Ce-fkh-1 gene, are in the some position relative to the involuting/ingressing endoderm as are the cells that give rise to the HNF-3-expressing axial structures like the notochord in frog embryos. The implication is that the relative spatial arrangements of the primary feature of gastrulating embryos have been conserved (Assaria, 1996).

The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death over a period of less than 3 weeks. In contrast, mutants with reduced activity of the gene daf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2 mutations requires the activity of the gene daf-16. daf-16 appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved (Lin, 1997).

The nematode pharynx is a large neuromuscular organ used for feeding. Like the foregut compartments of more complex animals, the pharynx is composed of many different cell types, such as muscles, glands, neurons, epithelia, and valves. Two early blastomeres, ABa and MS, generate the pharynx. Each blastomere gives rise to multiple cell types within the pharynx, and each uses a different genetic program to produce pharyngeal cells. The ABa-derived pathway depends on intercellular signaling mediated by the GLP-1 receptor, whereas pharynx production from MS appears to be cell autonomous. Two predicted transcription factors, skn-1 and pop-1, are necessary for the MS developmental program, including the production of pharyngeal cells. Recent studies have begun to elucidate the gene networks controlling glp-1, skn-1, and pop-1; however, the mechanisms that mediate pharyngeal organogenesis and act downstream of these genes are largely unknown (Horner, 1998 and references).

The genetic pathways that generate the ABa and MS cell lineages converge on the pha-4 locus. Animals lacking zygotic pha-4 activity fail to produce pharynx cells from either ABa or MS. In addition, these mutants lack a rectum. The rectum is descended from a third blastomere called ABp and does not derive from the pharyngeal cell lineages. Most other cells appear to be generated normally in pha-4 mutant embryos, including cells from ABa and MS that are not part of the pharynx. Thus, the pha-4 locus is critical to generate a group of cells related by function (the digestive tract) rather than by cell lineage (ABa, MS) or cell type (pharynx neuron, pharynx muscle, etc.). This phenotype reflects a transition during embryogenesis from maternal genes, like glp-1, skn-1, and pop-1, that control entire cell lineages and broad domains of axial patterning, to zygotic genes, like pha-4, that regulate the formation of specific organs, tissues, and cell types (Horner, 1998 and references).

PHA-4 is observed in the nuclei of cells destined to form the digestive tract. At the 4E stage, weak expression is detected in 8 MS great-granddaughters and 10 ABa descendants (ABaraaaa/p, ABaraapa/p, ABarapaa/p, ABalpaaa/p, ABalpapa/p) that each produce pharyngeal cells, as well as nonpharyngeal cells. Faint expression is also detected in the four midgut precursors. Brighter staining is observed in 6-8 MS-derived and 16-18 ABa-derived cells after the next cell division (MSaaaa/p, MSaapa/p, MSpaaa/p, MSpapa/p). Soon thereafter, at the 8-12E stage, Pha-4 is first observed in the rectal precursors. Expression is maintained in all pharyngeal, midgut, and rectal cells throughout embryogenesis. In summary, Pha-4 expression defines the digestive tract during embryogenesis. The initiation of bright expression at the 8E stage corresponds to the time when the pharyngeal precursors are born. These cells are destined to produce only pharyngeal cells. However, they are not restricted to any particular cell type within the pharynx, consistent with pha-4's proposed global role in specifying pharynx identity (Horner, 1998 and references).

The pha-4 (Ce-fkh-1) locus establishes organ identity for the Caenorhabditis elegans pharynx. In pha-4 mutants, pharyngeal cells are transformed into ectoderm. Conversely, ectopic pha-4 expression produces excess pharyngeal cells. pha-4 function is required for the earliest signs of pharyngeal development during embryogenesis. In the wild type, the pharyngeal precursors congregate at the ventral midline, where they ingress, beginning at the 4-8E stage. In pha-4 mutant embryos, ingression is delayed or aborted so that most of these cells remain on or near the ventral surface. In addition, whereas wild-type pharyngeal precursors cluster into a column as they gastrulate, this process usually fails in pha-4 mutants. All other embryonic blastomeres behave normally at this time, including midgut cell. Thus, pha-4 activity is required for the proper migration and possibly adhesion of the pharyngeal precursors by the 4-8E stage (Horner, 1998).

In the absence of pha-4 function, pharyngeal cells adopt an ectodermal fate. Staining with an antibody that recognizes LIN-26, a zinc-finger protein expressed in epidermis and neuronal support cells, shows that excess numbers of LIN-26+ cells are made in pha-4 mutants. To determine the source of these cells, individual blastomeres were allowed to develop after all other blastomeres were killed with a laser microbeam. This experiment demonstrates that excess LIN-26+ cells are made by isolated ABa and MS blastomeres, whereas their sister cells, ABp and P2, are unaffected. Therefore, cells that would become part of the pharynx in a wild-type embryo, appear to be transformed into LIN-26+ ectodermal cells in a pha-4 mutant. This conclusion was confirmed by following the late cell lineages of ABa and MS in pha-4 embryos. The terminal cell divisions and programmed cell deaths were altered in the cell lineages that would normally generate pharyngeal cells, suggesting that these cells no longer have pharynx identity. For example, in wild-type embryos, ABaraapapaa and ABaraapppaa divide to generate one daughter that dies and one pharyngeal neuron. In pha-4 embryos, ABaraapapaa and ABaraapppaa arrest without undergoing the terminal cell division. In addition, no programmed cell deaths were observed for ABalpaaapap, ABarapaaapp, or MSaaappa (Horner, 1998).

The new cell lineage patterns observed in pha-4 embryos do not resemble a different branch of the wild-type lineage. Normally, individual sublineages can be identified by the total number of cell divisions within the lineage, the pattern of programmed cell deaths and the morphology of individual cells. In pha-4 embryos, AB-derived cells divided nine times and have few programmed cell deaths, unlike any specific sublineage in wild-type embryos. Thus, pha-4 mutant cells lose their pharyngeal lineage characteristics without adopting a pattern that mimics another wild-type lineage. This result is consistent with pha-4's involvement in establishing pharyngeal fate rather than a particular cell lineage (Horner, 1998).

Expression of ectopic pha-4 is sufficient to confer pharynx identity to embryonic blastomeres. A heat shock promoter was used to express PHA-4 throughout the embryo. All manipulated embryos arrest with a large pharynx-like organ, bounded by a basement membrane. These embryos fail to undergo normal morphogenesis and contain fewer epidermal cells or body wall muscles. Most heat-treated embryos contain at least 50% more pharyngeal muscle cells and three times more pharyngeal marginal cells, when compared to the wild type. Conversely, the number of LIN-26+ epidermal and neuronal support cells is strongly reduced after heat shock, as is the number of body wall muscles (Horner, 1998).

How might pha-4 govern pharynx fate? One idea is that pha-4 regulates the proper positioning and migration of cells, and the new location of cells in pha-4 mutants interferes with normal pharyngeal development. Alternatively, pha-4 might specify pharynx fate directly, and the loss of pharyngeal identity in pha-4 mutants could lead to altered behaviors. Two lines of evidence support the latter hypothesis: (1) most embryos with severe defects in morphogenesis and cell placement still form a pharynx, and (2) the ectopic pharyngeal cells seen in heat shock experiments are often located in regions of the embryo that do not normally produce pharyngeal cells. Thus, normal patterns of cell migration, adhesion, and ingression depend on correct pharynx fate specification by pha-4. These studies suggest that the subdivision of the digestive tract into foregut, midgut, and hindgut is evolutionarily ancient and relies on conserved molecular mechanisms. pha-4 encodes an HNF-3 homolog necessary to specify pharyngeal and rectal precursors. In other organisms, HNF-3 proteins have been implicated in foregut and hindgut development, the equivalent of the nematode pharynx and rectum. In Drosophila fork head mutants, for example, foregut and hindgut cells are transformed into head sclerite structures, whereas in vertebrates, the absence of HNF-3beta leads to severe abnormalities in the foregut. The nematode nkx2.5 homolog ceh-22 is expressed in foregut muscle, similar to its murine and Drosophila counterparts, and end-1, a GATA factor involved in nematode midgut development, resembles the Drosophila midgut regulator serpent. The conserved patterns of expression and phenotype for these three genes strongly support the notion that the mechanisms that compartmentalize the digestive tract have been phylogenetically conserved (Horner, 1998 and references).

The C. elegans Ce-fkh-1 gene has been cloned on the basis of its sequence similarity to the winged-helix DNA binding domain of the Drosophila forkhead and mammalian HNF-3 alpha, beta and gamma genes. Mutations in the zygotically active pha-4 gene have been shown to block formation of the pharynx (and rectum) at an early stage in embryogenesis. Ce-fkh-1 and pha-4 have been shown to be the same gene. PHA-4 protein is present in the nuclei of essentially all five types of pharyngeal cells. PHA-4 protein first appears close to the point at which a cell lineage will produce only pharyngeal cells, independent of cell type. PHA-4 is shown to bind directly to a pan-pharyngeal enhancer element previously identified in the promoter of the pharyngeal myosin myo-2 gene; in transgenic embryos, ectopic PHA-4 activates ectopic myo-2 expression. Ectopic PHA-4 can activate ectopic expression of the ceh-22 gene, a pharyngeal-specific NK-2-type homeodomain protein (Drosophila homolog, Vnd) previously shown to bind a muscle-specific enhancer near the PHA-4 binding site in the myo-2 promoter. It is proposed that the combination of pha-4 and regulatory molecules (such as ceh-22) produce the specific gene expression patterns during pharynx development. Overall, pha-4 can be described as an organ identity factor, completely necessary for organ formation, present in all cells of the organ from the earliest stages, capable of integrating upstream developmental pathways (in this case, the two distinct pathways that produce the anterior and posterior pharynx) and participating directly in the transcriptional regulation of organ specific genes. The distribution of PHA-4 protein in C. elegans embryos is remarkably similar to the distribution of the Forkhead protein in Drosophila embryos: high levels in the foregut/pharynx and hindgut/rectum; low levels in the gut proper. pha-4 expression in the C. elegans gut is regulated by elt-2, a C. elegans gut-specific GATA-factor and possible homolog of the Drosophila gene serpent, which influences forkhead expression in the fly gut. Overall, these results provide evidence for a highly conserved pathway regulating formation of the digestive tract in all (triploblastic) metazoa (Kalb, 1998).

In mammals, insulin signaling regulates glucose transport together with the expression and activity of various metabolic enzymes. In the nematode Caenorhabditis elegans, a related pathway regulates metabolism, development and longevity. Wild-type animals enter the developmentally arrested dauer stage in response to high levels of a secreted pheromone, accumulating large amounts of fat in their intestines and hypodermis. Mutants in DAF-2 (a homolog of the mammalian insulin receptor) and AGE-1 (a homolog of the catalytic subunit of mammalian phosphatidylinositol 3-OH kinase) arrest development at the dauer stage. Animals bearing weak or temperature-sensitive mutations in daf-2 and age-1 can develop reproductively, but nevertheless show increased energy storage and longevity. Null mutations in daf-16 suppress the effects of mutations in daf-2 or age-1; lack of daf-16 bypasses the need for this insulin receptor-like signaling pathway. Thus, the principal role of DAF-2/AGE-1 signaling is to antagonize DAF-16. daf-16 is widely expressed and encodes three members of the Fork head family of transcription factors. Daf-16 is similar to human FKHR and AFX. There is a region at the N-terminal end of the protein that is 55% identical among DAF-16, FKHR and AFX; this region is not conserved between other Forkhead-related proteins. Thus, these proteins represent a distinct group of Forkhead-related proteins. The DAF-2 pathway acts synergistically with the pathway activated by a nematode TGF-beta-type signal, DAF-7, suggesting that DAF-16 cooperates with nematode SMAD proteins in regulating the transcription of key metabolic and developmental control genes. The probable human orthologues of DAF-16, FKHR and AFX, may also act downstream of insulin signaling and cooperate with TGF-beta effectors in mediating metabolic regulation. These genes may be dysregulated in diabetes (Ogg, 1997).

A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. Two C. elegans Akt/PKB homologs, akt-1 and akt-2, transduce insulin receptor-like signals that inhibit dauer arrest. (Protein kinase B [PKB], the serine/threonine kinase Akt, is otherwise known as ‘related to A and C’ protein kinase [RAC]). Both AKT-1 and AKT-2 signals are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1, revealed by a genetic screen, as well as increased dosage of wild-type akt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 and akt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation (Paradis, 1998).

The roles of AKT-1 and AKT-2 in regulating the metabolic shift and developmental arrest associated with dauer formation suggest the following model. Under normal growth conditions, an insulin-like molecule binds to the DAF-2 insulin receptor kinase inducing autophosphorylation and recruitment of AGE-1 PI3K. A parallel pathway (or pathways) from the DAF-2 insulin receptor-like protein is also activated. The AKT-1 and AKT-2 kinases, as well as molecules from the parallel pathway, negatively regulate DAF-16 activity, possibly via phosphorylation. Phosphorylated DAF-16 could be inactive, or function to activate genes required for reproductive growth and metabolism, or repress genes required for dauer arrest and energy storage. Other signaling molecules that are activated by DAF-2 must also converge downstream of AGE-1 (e.g., on DAF-16 or AKT-1/AKT-2) for proper regulation of metabolism and life span. The dauer arrest induced by loss of AGE-1 PI3K or AKT-1/AKT-2 activity implies that the loss of only one of these inputs to DAF-16 is sufficient to cause dauer arrest. Under dauer-inducing conditions, DAF-2, AGE-1, AKT-1/AKT-2, and other signaling pathways from DAF-2 are inactive and therefore DAF-16 is active, presumably because it is under-phosphorylated. Active DAF-16 either represses genes required for reproductive growth and metabolism or activates genes necessary for dauer arrest and energy storage (Paradis, 1998).

The DAF-16 Fork head protein has been suggested to interact with the DAF-3, DAF-8, or DAF-14 Smad proteins to integrate converging TGF--like neuroendocrine signals with insulin-like signals. DAF-16 may form a complex with the DAF-3 Smad protein under dauer-inducing conditions to regulate these downstream genes and AKT-1/AKT-2 phosphorylation of DAF-16 may inhibit the formation of a Smad/Fork head complex during reproductive development. Akt/PKB has been implicated in mammalian insulin receptor signaling that localizes glucose transporters to the plasma membrane and has been shown to regulate glycogen synthesis via direct phosphorylation of GSK3, two events that are not transcriptional. While there also may be such Akt/PKB outputs in C. elegans, the DAF-16 Fork head transcription factor represents the major output of DAF-2/AGE-1/AKT-1/AKT-2 insulin receptor-like signaling. Similarly, Akt/PKB action in the insulin/IGF-I anti-apoptotic pathway may also converge on transcription factors related to DAF-16. This model, based on genetic evidence that Akt/PKB couples insulin receptor-like signaling to transcriptional output via the DAF-16 Fork head transcription factor in C. elegans, predicts that Akt/PKB will have transcriptional outputs in insulin-like signaling across phylogeny. Human homologs of DAF-16 may be the pertinent downsteam effectors of insulin signaling. Two of the consensus Akt/PKB sites conserved in DAF-16 and its human homologs are located outside of the Fork head DNA-binding domain, and two sites are located in the highly basic W2 region of the Fork head domain that has been shown to mediate DNA phosphate backbone contacts and possibly nuclear localization (Paradis, 1998).

Caenorhabditis elegans responds to its complex chemical environment using a small number of chemosensory neurons. Each of these neurons exhibits a unique sensory response repertoire. The developmental mechanisms that generate this diversity of function are largely unknown. Many C. elegans chemosensory neurons, including the AWA and ASG neurons, arise as lineal sisters of an asymmetric division. The gene unc-130, which plays a role in the generation of the AWA and ASG neurons, is described. In unc-130 mutants, the ASG neurons adopt the fate of the AWA neurons. unc-130 encodes a member of the forkhead domain family of transcription factors, and is expressed in the precursors to AWA and ASG neurons. unc-130 sequence most closely resembles that of Drosophila fd59A. Misexpression of unc-130 in the AWA neurons is partly sufficient to repress the AWA fate, but not to promote ASG fate. unc-130 also plays a role in the development of additional chemosensory neurons. The ASG neurons share a developmental default state in common with three types of olfactory neurons. It is proposed that distinct cell fates and hence diversity of function in the chemosensory neurons of C. elegans are generated in a hierarchical manner, utilizing both lineage-dependent and independent mechanisms (Sarafi-Reinach, 2000).

Thus UNC-130 functions to regulate the asymmetric division that gives rise to the AWA and ASG chemosensory neurons. How might UNC-130 act? Daughter cells arising from the division of a precursor cell can adopt different fates through a combination of intrinsic and extrinsic mechanisms. Intrinsic mechanisms involve the asymmetric localization and segregation of cell fate determinants, resulting in the generation of daughter cells that differ from each other at birth. Extrinsic mechanisms involve the generation of asymmetry in initially symmetric cells, as a result of signaling between the cells or between the cells and the environment. Both types of mechanisms have been described in several organisms. In the central nervous system of Drosophila, determinants such as Numb and Prospero are asymmetrically localized to the basal pole of neuroblasts, and are segregated to only the GMC daughter cells. Similar asymmetric localization of determinants, and subsequent asymmetric cell division, has been well described in the early embryonic development of C. elegans, as well as in unicellular organisms such as Saccharomyces cerevisiae and Bacillus subtilis. Regulation of asymmetry by signaling mechanisms such as the Wnt/wingless pathway has also been characterized (Sarafi-Reinach, 2000 and references therein).

It is proposed that transcription mediated by UNC-130 is required for the correct expression and/or asymmetric segregation of putative cell fate determinants in the AWA/ASG precursor cells. Defects in the segregation of such determinants could result in the observed ASG cell fate transformation, as well as occasional loss of AWA-specific gene expression. Interestingly, a similar role has been proposed for the Jumeaux forkhead domain protein in Drosophila. Jumeaux is required for the correct localization and segregation of Numb in the precursor to the RP2 motor neuron. Homologs of Numb and Prospero have been identified in C. elegans, although their functions in the nervous system have not been elucidated (Sarafi-Reinach, 2000 and references therein).

Previous work has suggested that the AWC fate is the default developmental fate of olfactory neurons in C. elegans. Expression of genes such as odr-7 and lim-4 modifies this fate to result in the generation of the three types of olfactory neurons. Results presented in this study suggest that the AWC fate may be the default fate of other chemosensory neurons in addition to the olfactory neurons. In unc-130; odr-7 double mutants, the ASG neurons appear to exhibit AWC-like characteristics, such as expression of the AWC-specific str-2 marker. It is suggested that the AWC fate may be the default fate of most, if not all, chemosensory neurons. These chemosensory neurons could be related by overall function, or possibly by inclusion in a particular sense organ. Chemosensory neurons in the head are part of the amphid sense organ. Organ identity genes have been previously defined such as the C. elegans winged helix gene pha-4, which is required for the expression of pharyngeal organ identity. Amphid organ identity genes could function through the lineage or through the action of extracellular signals to induce the basal AWC fate in all cells that will form this organ. Alternatively, a common chemosensory neuron determinant could be induced in all chemosensory cell types, including chemosensory cells in other sense organs (Sarafi-Reinach, 2000 and references therein).

It is proposed that diversity of function in the chemosensory nervous system results from layers of identity, generated through the action of transcription factors. The basal layer in both the AWA and ASG neurons could be AWC-like. Expression of ODR-7 generates AWA identity in the AWA neurons. The ASG neurons are also capable of exhibiting AWA identity; however, this fate is repressed by the action of UNC-130, which also acts to promote ASG fate. Similarly, in the case of other chemosensory neurons, expression of genes combinatorially in a lineage-specific manner could layer distinct cell identities. Further cell type-specific differentiated features may be regulated by linear or parallel cascades of additional genes. A major challenge for the future is to identify the genes that act to define the identities and specific functional characteristics of each type of chemosensory neuron (Sarafi-Reinach, 2000).

Secreted proteins required for cellular movements along the circumference of the body wall in Caenorhabditis elegans include UNC-6/netrin and the novel TGF-beta UNC-129. Expression of these proteins is graded along the dorsoventral (D/V) axis, providing polarity information to guide migrations. Graded expression of UNC-129 in dorsal but not ventral body muscles depends on unc-130, which encodes a Forkhead transcription factor. The phenotype of unc-130 mutants closely mimics the reported effects of ectopically expressing unc-129 in both dorsal and ventral body muscles. This fits the present finding that unc-130 cell autonomously represses unc-129 expression in the ventral body muscles. Thus the cell-specific effects of unc-130 on ventral, but not dorsal, body muscle expression of unc-129 accounts for the D/V polarity information required for UNC-129-mediated guidance. Genetic interactions between unc-130 and other guidance genes show that several molecular pathways function in parallel to guide the ventral to dorsal migration of distal tip cells (DTCs) and axonal growth cones in C. elegans. Genetic interactions confirm that UNC-129 does not require the only known type II TGF-beta receptor in C. elegans (DAF-4) for its guidance functions. Also, unc-130 is partially required for male tail morphogenesis and for embryogenesis (Nash, 2000).

The C. elegans gene pes-1 encodes a transcription factor of the forkhead family and is expressed in specific cells of the early embryo. Despite this expression, which suggest that pes-1 has to have an important regulatory role in embryogenesis, inactivation of pes-1 causes no apparent phenotype. This lack of phenotype is a consequence of genetic redundancy. Whereas a weak, transitory effect is observed upon disruption of just T14G12.4 (renamed fkh-2: a homolog of Drosophila sloppy paired) gene function, simultaneous disruption of the activity of both fkh-2 and pes-1 results in a penetrant lethal phenotype. Sequence comparison suggests these two forkhead genes are not closely related and the functional association of fkh-2 and pes-1 was explored only because of the similarity of their expression patterns. Conservation of the fkh-2/pes-1 genetic redundancy between C. elegans and the related species C. briggsae has been demonstrated. Interestingly the redundancy in C. briggsae is not as complete as in C. elegans and this could be explained by alterations of pes-1 specific to the C. briggsae ancestry. With overlapping function retained on an evolutionary time-scale, genetic redundancy may be extensive and expression pattern data could, as here, have a crucial role in characterization of developmental processes (Molin, 2000).

While molecular phylogenetic analysis has revealed likely orthologs for many of the C. elegans forkhead genes in species outside the Nematoda, no ortholog of pes-1 has yet been identified beyond Caenorhabditis, not even in the substantially complete Drosophila genome sequence. In contrast, fkh-2 is quite a close homolog of the Drosophila segmentation gene sloppy-paired (slp) and of the chordate gene Brain factor 1 (BF-1). These observations might suggest that pes-1 is a phylum-specific gene and/or that a low level of selection on pes-1 in the evolutionary history of the common ancestor of C. briggsae and C. elegans (because of genetic redundancy) might have contributed to the divergence of the pes-1 gene. Indeed, pes-1 and fkh-2 may be related by a gene duplication event that, although predating the C. elegans/C. briggsae divergence, may be much more recent than the molecular phylogenetic analysis might suggest. In addition, the anterior expression of BF-1 in chordates and of slp in Drosophila could correlate with the expression of pes-1 and fkh-2 in the AB cell lineage in C. elegans. Curiously slp also demonstrates functional redundancy, in this case as an adjacent pair of more similar genes that therefore probably originated with a relatively recent, gene duplication event (Molin, 2000).

The C. elegans gut-specific esterase gene (Ce-ges-1) has the unusual ability to be expressed in different modules of the embryonic digestive tract (anterior pharynx, posterior pharynx, and rectum) depending on sequence elements within the Ce-ges-1 promoter. The expression of the ges-1 homolog (Cb-ges-1) from the related nematode Caenorhabditis briggsae has been studied. Cb-ges-1 also has the ability to switch expression between gut and pharynx + rectum. The control of this expression switch centers on a tandem pair of WGATAR sites in the Cb-ges-1 5'-flanking region, just as it does in Ce-ges-1. Sequence alignments and subsequent deletions have been used to identify a region at the 3'-end of both Ce-ges-1 and Ce-ges-1 that acts as the ges-1 cryptic pharynx enhancer whose activity is revealed by removal of the 5' WGATAR sites. This region contains a conserved binding site for PHA-4 (the C. elegans ortholog of forkhead/HNF3alpha, beta, gamma factors), which is expressed in all cells of the developing pharynx and a subset of cells of the developing rectum. A model is proposed in which the normal expression of ges-1 is controlled by the gut-specific GATA factor ELT-2. It is proposed that, in the pharynx (and rectum), PHA-4 is normally bound to the ges-1 3'-enhancer sequence but that the activation function of PHA-4 is kept repressed by a (presently unknown) factor binding in the vicinity of the 5' WGATAR sites. This control circuitry might be maintained in Caenorhabditis because pharyngeal expression of ges-1 is advantageous only under certain developmental or environmental conditions (Marshall, 2001).

Regulation of organogenesis by the Caenorhabditis elegans FoxA protein PHA-4

The pha-4 locus encodes a forkhead box A (FoxA/HNF3) transcription factor homolog that specifies organ identity for C. elegans pharyngeal cells. This study used microarrays to identify pharyngeal genes and analyzed those genes 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).

Cells acquire distinct fates during development in response to a host of regulators, many of which encode transcription factors. A long-standing issue has been the nature of the target genes controlled by these transcriptional regulators: Do they activate a few genes at the top of a hierarchy, or do they activate many genes that function at multiple stages throughout development? This question has been difficult to resolve because identification of downstream genes has relied largely on genetic approaches, which demand easily observed phenotypes. This study used a genomic approach to investigate how the predicted transcription factor PHA-4 promotes development of an organ, the C. elegans pharynx (Gaudet, 2002).

The C. elegans pharynx is a neuromuscular organ that pumps food in from the environment and initiates digestion. The precursors of the pharynx are generated by distinct genetic programs and cell lineages and are assembled into a 92-cell primordium that undergoes complex patterning and morphogenesis. Ultimately, the precursors acquire different fates within the pharynx, including muscles, neurons, glands, structural cells, valves, and epithelia (Gaudet, 2002).

The pha-4 loss-of-function phenotype and the expression pattern of PHA-4 reveal that all cells of the pharynx, regardless of their distinct ancestry or ultimate cell fate, share a common pharyngeal identity that is specified bypha-4. pha-4 encodes a protein that most closely resembles forkhead box A (FoxA) transcription factors, which have been implicated in gut development in other organisms (e.g., Drosophila Forkhead and mammalian FoxA2). Few target genes are known for FoxA members. For example, the only identified direct target of PHA-4 is the pharyngeal myosin gene myo-2, indicating that PHA-4 can function late in development. This study has identified and analyzed PHA-4 targets to study how FoxA proteins function during organogenesis (Gaudet, 2002).

To identify pharyngeally expressed genes, C. elegans microarrays were probed with labeled complementary DNA from embryos that produced either excess pharyngeal cells (par-1 mutants) or no pharyngeal cells (skn-1 mutants. Three independent experiments were performed with microarrays that contained 62% of the C. elegans genome (11,917 out of 19,099 predicted genes), and 240 candidate pharyngeal genes were identified with an average par-1/skn-1 ratio >=2.0 (Gaudet, 2002).

Three lines of evidence indicate that the microarray experiments selectively identified pharyngeally expressed genes. First, the microarrays contained 18 genes previously shown to be expressed in the embryonic pharynx. Fifteen of these genes were identified, including genes expressed at low levels (e.g., ceh-22). Second, a search of messenger RNA (mRNA) in situ hybridization data demonstrated that 59 out of 70 of the positives were pharyngeally expressed. Third, green fluorescent protein (GFP) reporters were constructed for 10 randomly chosen microarray positives, and 8 out of 10 were found to be expressed predominantly or exclusively in the pharynx. In total, the expression patterns of 96 of the microarray positives were characterized in this study or elsewhere, and 82% (79 out of 96) were selectively expressed in the pharynx (Gaudet, 2002).

Pharynx-specific expression of eight out of eight reporter constructs depended on TRTTKRY elements. For every promoter analyzed, elimination of one or more TRTTKRY elements resulted in either a complete loss of pharyngeal GFP or a substantial decrease in expression and a delay in the observed onset of expression. Moreover, pharyngeal expression of each of three additional genes [avr-15, H30A04.1/eat-20, and peb-1 was dependent on an intron containing two or more TRTTKRY elements. GFP reporters were also constructed for one additional microarray positive and one gene with a borderline par-1/skn-1ratio, neither of which contained any TRTTKRY elements in its upstream region. Neither reporter was expressed in the pharynx (Gaudet, 2002).

These findings suggest that expression in the pharynx is critically dependent on TRTTKRY elements for genes expressed either early (e.g., M05B5.2) or later (e.g., ZK816.4) in pharynx development. Because the reporter constructs relied on randomly selected genes, these findings suggest that many, perhaps all, genes selectively expressed in the pharynx depend on TRTTKRY elements (Gaudet, 2002).

Based on the known expression patterns of C. elegans Fox genes, PHA-4 is the best candidate to recognize the pharyngeal TRTTKRY elements. Previous studies showed that pha-4functions early in embryogenesis. If PHA-4 directly activates most or all pharyngeal genes, then PHA-4 should also be required during later pharynx development. To test this idea, a temperature-sensitive configuration ofpha-4[pha-4(ts)] was engineered, and these animals were shifted from permissive to restrictive temperatures at different developmental time points (Gaudet, 2002).

pha-4 activity was required throughout development for viability. Shiftingpha-4(ts) to a restrictive temperature any time after the formation of pharyngeal precursors, but before the pharynx was fully developed (early and late embryos), resulted in 100% lethality. Late embryos shifted to restrictive temperatures arrested as first stage (L1) larvae with a 'stuffed' pharynx phenotype that resembled that of animals with defects in pharyngeal pumping. Substantial lethality was observed among pha-4(ts) animals shifted as L1's, after pharynx development was complete. Adults that developed from these L1's were small and unhealthy, consistent with a feeding defect. These data demonstrate that pha-4 activity is required during both early and late times in embryonic development (Gaudet, 2002).

To characterize the temperature-sensitive phenotype further, expression of three of the reporters was analyzed. For D2096.6 and C44H4.1 reporters, a reduction in PHA-4 activity in the mature pharynx resulted in a reduction of pharyngeal GFP. Conversely, restoration of PHA-4 activity was sufficient to reactivate pharyngeal expression. This effect was specific because a reporter expressed in nonpharyngeal cells (coelomocytes) showed no change in expression when animals were shifted to a restrictive temperature (Gaudet, 2002).

The third pharynx reporter, myo-2::GFP, showed no appreciable decrease in expression. myo-2 expression is probably maintained by the combined actions of other transcription factors, including CEH-22 and PEB-1. This hypothesis is consistent with the finding that the PHA-4 site of myo-2 is important for initial expression of a myo-2reporter but not for later expression (Gaudet, 2002).

A second line of evidence that indicates that TRTTKRY elements are recognized by PHA-4 is that PHA-4 binds this consensus in vitro. The ability of four different TRTTKRY sequences to compete against a labeled TGTTTGC-bearing probe was tested for binding to PHA-4 in electrophoretic mobility shift assays (EMSAs). The TGTTTGC-bearing sequence represented a characterized PHA-4 site from the myo-2 promoter (myo2-WT). Three more sites identified in reporter assays were tested: TGTTGAC from M05B5.2 (myo2-M), TGTTTGT from D2096.6 (myo2-D), and TATTTGT from ceh-22(myo2-ceh). These sequences were placed within the context of myo2-wild-type (myo2-WT) flanking sequences to minimize any effects of sequences outside the TRTTKRY core. Each of these TRTTKRY sequences competed specifically against myo2-WT compared to a nonspecific competitor (myo2-non, TGAACAG) (Gaudet, 2002).

Examination of in vitro binding curves revealed that PHA-4 bound different TRTTKRY sequences with different affinities. Quantitative analyses demonstrated that myo2-ceh had four times less affinity for PHA-4 than did myo2-WT, whereas the myo2-WT, myo2-M, and myo2-D sites had comparable affinities. Differences were observed between the myo2-WT site (TGTTTGC) and the D site (TGTTTGT) when endogenous flanking sequence from D2096.6 was used rather thanmyo-2. EMSA was performed with labeled myo2-WT, as above, and two cold competitors: D2096-WT (wild-type D2096.6 sequence, which includes TGTTTGT) and D2096-mut (D2096.6 sequence mutated to include TGTTTGC) . In the context of endogenous D2096.6 promoter sequence, it was observed that D2096-mut had a twofold higher affinity than D2096-WT. The ability to detect this difference in the context of D2096.6, but not in themyo-2 sequence, suggests that sequences flanking TRTTKRY influence the binding affinity of PHA-4 (Gaudet, 2002).

The affinity of TRTTKRY elements for PHA-4 influenced the onset of expression of six out of six GFP reporters in a manner consistent with the in vitro binding data. 'Up' mutations (from a low- to high-affinity site) and 'down' mutations were generated in TRTTKRY elements of the GFP reporters. Predicted down mutations in M05B5.2 or T05E11.3 promoters (from TGTTGAC or TGTTTGC to TGTTTGT, respectively) resulted in a delay of 2 to 3 hours in the onset of pharyngeal expression as compared to unaltered reporters. Conversely, when predicted weaker elements were mutated to TGTTTGC, a shift of 1 to 3 hours was noted in the onset of expression to earlier stages. Despite differences in the onset of expression, alterations in the sequence of TRTTKRY elements did not affect the strength of GFP fluorescence in later stages (Gaudet, 2002).

If the affinity of PHA-4 for TRTTKRY elements regulates onset of expression, then a decrease in PHA-4 levels should mimic a down mutation of a PHA-4 binding site. This prediction was tested with the early-expressed, panpharyngeal M05B5.2 reporter in the pha-4(ts) strain. In animals grown at an intermediate temperature (20°C), the pharynx was formed, but was often disorganized, suggesting that PHA-4 was present at insufficient levels to promote proper pharynx development. Under these conditions, expression of the M05B5.2 reporter was delayed as compared to the same strain grown at a permissive temperature. Eventually, expression of this reporter reached a level equivalent to that of unshifted controls. This result is consistent with the model and suggests that PHA-4 is critical for controlling the onset of pharyngeal gene expression (Gaudet, 2002).

The effect of affinity on the onset of pharyngeal expression may be generally true. An analysis of the promoters of the microarray positives revealed a striking correlation between the rank of positive genes (according to their differential expression in microarray experiments) and the probability of a gene having a high-affinity PHA-4 binding site in its promoter. Genes that were highly ranked were more likely to contain a moderate- to high-affinity site than were lower ranked genes. Conversely, lower ranked genes were slightly more likely to contain only a low-affinity PHA-4 site. It is suggested that this correlation was revealed because RNA was isolated from embryos at mid-stages of pharyngeal development, when the abundance of a given mRNA reflected the onset of its expression (Gaudet, 2002).

It is proposed that (1) PHA-4 directly regulates a wide array of genes expressed at multiple stages of pharyngeal development, and (2) the affinity of TRTTKRY elements for PHA-4 regulates the relative onset of the expression of target genes. Two observations suggest that temporal regulation by PHA-4 is important in vivo. First, mutations that alter the affinity of TRTTKRY elements shift the onset of GFP expression by 1 to 3 hours, which is a substantial effect given the rapid development of C. elegans embryos (<14 hours). Second, using ourpha-4(ts) strain, a concordance was observed between the temperature at which lethality was first observed and the temperature at which delayed GFP expression was first detected (Gaudet, 2002).

It is proposed that high-affinity sites bind PHA-4 in early embryos whereas low-affinity sites may not bind until later, after PHA-4 levels increase. Consistent with this idea, PHA-4 is expressed at low levels at the ~50-cell stage of embryogenesis and increases as development progresses. Moreover, 15 out of 23 genes expressed during early pharyngeal development carry high-affinity sites, and the remaining 8 genes carry sites whose affinity has not been tested. This affinity model is reminiscent of the threshold response of genes to a gradient of bicoid along the anteroposterior axis or dorsal activity along the dorsoventral axis of the Drosophila embryo. PHA-4 target genes may demonstrate temporally, rather than spatially, restricted threshold responses so that genes required for early aspects of pharynx development (e.g., regulators) are expressed before those required for later aspects of development (e.g., differentiation markers) (Gaudet, 2002).

The affinity of a PHA-4 binding site does not correlate with the absolute onset of pharyngeal gene expression. The most striking example of this discrepancy is the different behavior of T05E11.3 andmyo-2 reporters. Both genes depend on a TGTTTGC site for expression in the pharynx, but T05E11.3 expression is first visible in early embryos, and myo-2 expression is not visible until ~3 to 4 hours later. These data suggest that a high-affinity PHA-4 site may be necessary but not sufficient for early activation of transcription. In some cases, the availability of other activators may determine the onset of expression. For example, other transcription factors that activate myo-2expression are expressed later in development than PHA-4 (Gaudet, 2002).

FoxA proteins in many species may directly regulate most or all genes within an organ. Both mammalian and Drosophila orthologs of PHA-4 are required for early aspects of digestive tract formation and also directly activate late-acting genes. Moreover, heterozygous Foxa/+ mice have numerous abnormalities, demonstrating that vertebrates are sensitive to the dosage of FoxA proteins, consistent with the model presented in this study. More generally, direct regulation of an entire genetic network may be a hallmark of organ identity factors. The selector gene scalloped directly activates genes that function at two distinct steps of the regulatory hierarchy leading to wing formation in Drosophila. Similarly, the paired-type homeodomain factor Pax6/eyeless is both necessary and sufficient for specification of eye formation in Drosophila imaginal discs, where it directly activates the expression of both early- and late-acting genes. These observations suggest that organ identity factors may use a strategy similar to that of PHA-4 to establish gene expression during organogenesis (Gaudet, 2002).

These findings suggest that a complex gene network regulated by PHA-4 could have evolved by serial recruitment of target genes. If the digestive tract of primitive organisms was a simple epithelial sac, then direct targets of ancestral PHA-4 may have been those that encoded digestive enzymes and those required to form epithelia. As additional genes, including transcription factors, fell under PHA-4 control, patterns of gene expression within the resulting organ structures may have increased in complexity. This model provides a possible explanation for the fact that FoxA proteins play a conserved role in foregut development in animals whose foreguts are morphologically and mechanistically different from one another. FoxA proteins may have acquired different target genes in different organisms, enabling new cell types or morphologies to emerge (Gaudet, 2002).

The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans

The let-7 microRNA is phylogenetically conserved and temporally expressed in many animals. C. elegans let-7 controls terminal differentiation in a stem cell-like lineage in the hypodermis, while human let-7 has been implicated in lung cancer. To elucidate let-7's role in temporal control of nematode development, sequence analysis and reverse genetics were used to identify candidate let-7 target genes. The nuclear hormone receptor daf-12 is a let-7 target in seam cells, while the forkhead transcription factor pha-4 is a target in the intestine. Additional likely targets are the zinc finger protein die-1 and the putative chromatin remodeling factor lss-4. Together with the previous identification of the hunchback ortholog hbl-1 as a let-7 target in the ventral nerve cord, these findings show that let-7 acts in at least three tissues to regulate different transcription factors, raising the possibility of let-7 as a master temporal regulator (Grosshans, 2005).

MicroRNAs mediate dietary-restriction-induced longevity through PHA-4/FOXA and SKN-1/Nrf transcription factors

Dietary restriction (DR) has been shown to prolong longevity across diverse taxa, yet the mechanistic relationship between DR and longevity remains unclear. MicroRNAs (miRNAs) control aging-related functions such as metabolism and lifespan through regulation of genes in insulin signaling, mitochondrial respiration, and protein homeostasis. A network analysis was conducted of aging-associated miRNAs connected to transcription factors PHA-4/FOXA and SKN-1/Nrf (homologs of Drosophila Forkhead and Cap'n'collar respectively), which are both necessary for DR-induced lifespan extension in Caenorhabditis elegans. This network analysis has revealed extensive regulatory interactions between PHA-4, SKN-1, and miRNAs and points to two aging-associated miRNAs, miR-71 and miR-228, as key nodes of this network. This study shows that miR-71 and miR-228 are critical for the response to DR in C. elegans. DR induces the expression of miR-71 and miR-228, and the regulation of these miRNAs depends on PHA-4 and SKN-1. In turn, PHA-4 and SKN-1 were shown to be negatively regulated by miR-228, whereas miR-71 represses PHA-4. This study has discovered new links in an important pathway connecting DR to aging. By interacting with PHA-4 and SKN-1, miRNAs transduce the effect of dietary-restriction-mediated lifespan extension in C. elegans. Given the conservation of miRNAs, PHA-4, and SKN-1 across phylogeny, these interactions are likely to be conserved in more-complex species (Smith-Vikos, 2014).

Nuclear mobility of transcription factor FoxA

FoxA proteins are pioneer transcription factors, among the first to bind chromatin domains in development and enable gene activity. The Fox DNA-binding domain structurally resembles linker histone and binds nucleosomes stably. Using fluorescence recovery after photobleaching, it was found that FoxA1 and FoxA2 move much more slowly in nuclei than other transcription factor types, including c-Myc, GATA-4, NF-1, and HMGB1. Slower nuclear mobility correlates with high nonspecific nucleosome binding, and point mutations that disrupt nonspecific binding markedly increase nuclear mobility. FoxA's distinct nuclear mobility is consistent with its pioneer activity in chromatin (Sekiya, 2009).

It is speculated that the markedly higher affinity of FoxA for chromatin, compared with the behavior of c-Myc, reflects the enhanced ability of FoxA, in the half-area FRAP assay, to scan both free DNA and nucleosomal target sequences. This has features similar to the role for nonspecific DNA interactions proposed to allow lac repressor and p53 to scan 'linearly' along free DNA (Kalodimos, 2004; McKinney, 2004). Due to lower nucleosome affinity and less stable binding, c-Myc can exist free in the nucleoplasm for longer periods than FoxA and hence move more quickly across the nucleus. By performing site-directed mutagenesis of the contact surface between FoxA1 and DNA, it was discovered that the movement of FoxA1 in the nucleus is restricted particularly by sequence-independent interactions with chromatin (Sekiya, 2009).

In cancer cell lines, FoxA1 preferentially associates with specific target sequences containing histone H3 that is dimethylated on Lys 4. This study found that FoxA binds with high affinity to nucleosomes made with recombinant histones lacking eukaryotic modifications and that the slow movement of FoxA in nuclei is limited by the factor's affinity toward nonspecific nucleosomal DNA. It is speculated that the slow movement allows FoxA to locate high-affinity target sites in highly compacted chromatin in vivo, such as occurs at silent genes in development. For example, a GATA-4 site is occupied adjacent to a FoxA site at the silent alb1 gene in undifferentiated endoderm. Yet GATA4 binding to nucleosomes is greatly facilitated by FoxA binding, and, as found in this study, GATA-4 is much more mobile in the nucleus than FoxA. It is suggested that slow nuclear mobility reflects FoxA's function as a pioneer factor: probing 'chromatinized' sites in nuclear DNA that many other TFs cannot access, followed by stable binding that allows other factors to engage the chromatin (Sekiya, 2009).

Table of contents

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

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