Table of contents

Forkhead homologs from other invertebrates

Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. This study identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head (the anterior regeneration pole) and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. A model is suggested in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue (Vogg, 2014).

Echinoderms, like chordates and hemichordates, are deuterostomes that lack a notochord or any structure homologous with it. Brachyury and HNF-3beta homologs were investigated to gain insight into genes implicated in notochord formation. The sea urchin ortholog of forkhead (Hphnf3) is first detected at the swimming blastula stage and accumulates maximally at the gastrula and prism-embryo stages, decreasing at the pluteus-larva stage. It is found in cells of the vegetal plate of the swimming blastula. During gastrulation, intense staining is evident in cells surrounding the blastopore, whereas weak staining is detected in the invaginating archenteron (an endomesodermal tissue). At the prism-embryo stage, the entire archenteron stains intensely; then, at pluteus stage, the larval staining decreases in intensity. Hphnf3 and the Brachyury ortholog, HpTa begin to be expressed almost simultaneously in the vegetal plate at the late blastula stage. However, after the onset of gastrulation Hphnf3 is expressed in the posterior part of the archenteron, whereas HpTa is expressed in the secondary mesenchyme founder cells, which occupy the tip of the archenteron. Hphnf3 may contribute to specification of embryonic cells as archenteron, and the role of HpTa may be directed towards specification of mesodermal founder cells. Except for the basal character of expression in endoderm and endomesoderm, these transcription factors are clearly utilized differently in chordates (Harada, 1996).

A member of the HNF-3/forkhead gene family in ascidians (Tunicates: phylum Urochordata) has been isolated as a means to determine the role of winged-helix genes in chordate development. The MocuFH1 gene, isolated from a Molgula oculata cDNA library, exhibits a forkhead DNA-binding domain most similar to the zebrafish gene axial and the rodent gene HNF-3beta. The protein is a homolog of Drosophila Forkhead. MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome. The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages. During gastrulation, MocuFH1 expression occurs in the prospective endoderm cells, which invaginate at the vegetal pole, and in the presumptive notochord and mesenchyme cells, which involute over the anterior and lateral lips of the blastopore, respectively. However, this gene is not expressed in the presumptive muscle cells, which involute over the posterior lip of the blastopore. MocuFH1 expression continues in the same cell lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brain and spinal cord. The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduce MocuFH1 transcript levels during gastrulation. Embryos treated with antisense ODNs cleave normally and initiate gastrulation. However, gastrulation is incomplete: some of the endoderm and notochord cells do not enter the embryo and therefore do not undergo subsequent movements. Accordingly, axis formation in these cases is abnormal. In contrast, the prospective muscle cells, which do not express MocuFH1,do undergo involution, and later express muscle actin and acetylcholinesterase, markers of muscle cell differentiation. The results suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and axis formation. The effects of inhibiting MocuFH1 expression on embryonic axis formation in ascidians are similar to those reported for knockout mutations of HNF-3beta in the mouse, suggesting that HNF-3/forkhead genes have an ancient and fundamental role in organizing the body plan in chordates (Olsen, 1997).

The notochord and dorsal ectoderm induce dorsoventral compartmentalization of the vertebrate neural tube through the differential regulation of genes such as HNF-3beta, Pax3, Pax6 and snail. The expression of HNF-3beta (Drosophila homolog: forkhead) and snail (Drosophila homolog: snail) homologs were examined in the ascidian, Ciona intestinalis, a member of the subphylum Urochordata, the earliest branch in the chordate phylum. Ciona Snail is composed of 584 amino acids; the terminal stretch of 153 amino acids has five zinc fingers. The Ci-fkh homolog is has 587 amino acids and is highly conserved. The Ciona HNF-3beta homolog is expressed in the ventralmost ependymal cells of the neural tube. It is likely that Ci-fkh has a conserved function in the specification of an ascidian floor plate. Ciona snail homolog is expressed at the junction between the invaginating neuroepithelium and dorsal ectoderm, similar to the patterns seen in vertebrates. The snail expressing lateral border of the neural plate forms the neural crest and the dorsal roof of the neural tube. Ci-sna is also apparent in muscle/trunk mesenchyme precursors. These findings provide evidence that dorsoventral compartmentalization of the chordate neural tube is not an innovation of the vertebrates. It is propose that precursors of the floor plate and neural crest were present in a common ancestor of both vertebrates and ascidians (Corbo, 1997).

The Ciona forkhead/HNF-3beta gene (Ci-fkh) is expressed in the primary axial tissues of the developing tadpole, including the notochord, endoderm, and rudimentary floor plate of the CNS. In an effort to determine the basis for this complex pattern of expression a detailed analysis of the Ci-fkh 5'-regulatory region was carried out. Different 5' sequences were attached to a lacZ reporter gene and analyzed in electroporated Ciona embryos. A short regulatory sequence (AS) located ~1.7 kb upstream of the transcribed region has been shown to be essential for expression in all three axial tissues. The proximal 20 bp of the AS contains overlapping Snail repressor elements and a T-box motif. Deleting these sequences causes the loss of reporter gene expression in the endoderm, as well as expanded expression in the neural tube. These results suggest that a T-box gene such as Ci-VegTR activates Ci-fkh expression in the endoderm, while the Ci-Sna repressor excludes expression from the lateral ependymal cells and restricts the Ci-fkh pattern to the rudimentary floor plate in ventral regions of the neural tube. Evidence is presented for Ci-fkh positive autofeedback, whereby the Ci-Fkh protein binds to critical activator sites within the Ci-fkh 5'-regulatory region and helps maintain high levels of expression (Di Gregorio, 2001).

The mouse HNF-3beta gene and the Ciona Ci-fkh gene exhibit strikingly similar patterns of expression. Both genes are expressed in all three axial tissues: the floor plate, notochord, and gut. In mice, separate enhancers mediate expression in the node/notochord and floor plate. These enhancers are located far upstream and downstream of the HNF-3beta transcription unit. In contrast, the regulation of Ci-fkh expression depends on tightly linked cis-elements in the 5'-flanking region. Indeed, the 44-bp AS element is important for all aspects of the normal expression pattern. Despite the compaction of the Ci-fkh cis-regulatory elements, there are similarities with the regulation of HNF-3beta in mice. Expression in the endoderm and notochord depends on the distal AS element, whereas CNS expression is mediated, at least in part, by a separate element located in a more proximal region of the 5'-flanking sequence. Moreover, the detailed dissection of the 21.77 to 21.63 kb interval of the Ci-fkh promoter region results in the partial uncoupling of expression in the notochord and endoderm. The notochord/node and CNS enhancers are separated by nearly 20 kb in mice, whereas apparently comparable cis-regulatory elements are located within 1 kb of one another in Ciona. This streamlining in the organization of cis-elements is reminiscent of the situation encountered in the pufferfish genome. The entire Ciona genome is only ~1/20 as large as mammalian genomes. One reason for this dramatic difference is that Ciona has not undergone the genome-wide duplication events seen in vertebrates, so there are many fewer genes. This study provides evidence that the small genome size also reflects the tight packaging of cis-regulatory elements. Ciona should be useful for the rapid identification and characterization of cis elements that specify basic chordate tissues (Di Gregorio, 2001).

The expression pattern of a class I fork head/HNF-3 gene (HrHNF3-1) of the ascidian Halocynthia roretzi has been characterized. Zygotic HrHNF3-1 expression is detectable as early as the 16-cell stage; the transcript is evident in blastomeres of the endoderm, notochord and mesenchyme lineages of the early embryos. After the late gastrula stage, HrHNF3-1 is also expressed in the presumptive spinal cord cells and some brain cells. The spinal cord of the ascidian tadpole consists of four layers of cells: the dorsal layer, two lateral layers and the ventral layer; the latter of these simply lies on the notochord. Cross-sections of in situ hybridized specimens show that HrHNF3-1 is expressed in cells of the ventral layer, reminiscent of the floor plate of vertebrate embryos. Autonomy is found in the initiation of early HrHNF3-1 expression, because the gene is expressed in blastomeres continuously dissociated from the first cleavage until the 16-cell stage (Shimauchi, 1997).

The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail); however, its sister species, Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. The role of FH1 in modifying the larval body plan in the tailless species has been examined. FH1 function in tailless species×tailed species hybrids, in which the otolith, notochord, and tail are restored, were also examined. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. Antisense FH1 ODNs inhibit endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affect gastrulation in the tailless species, although the effects are less severe than in the tailed species, and an anural larva is formed. In hybrid embryos, antisense FH1 ODNs block restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development (Olsen, 1999).

The embryonic development of amphioxus (Cephalochordate) has much in common with that of vertebrates, suggesting a close phylogenetic relationship between the two chordate groups. To gain insight into alterations in the genetic cascade that accompanied the evolution of vertebrate embryogenesis, the formation of the chordamesoderm in amphioxus embryos was investigated using the genes Brachyury and fork head/HNF-3 as probes. Am(Bb)Bra1 and Am(Bb)Bra2 isolated from the amphioxus Branchiostoma belcheri are homologs of the mouse Brachyury gene. Molecular phylogenetic analysis suggests that the genes are independently duplicated in the amphioxus lineage. Both genes are initially expressed in the involuting mesoderm of the gastrula, then in the differentiating somites of neurulae, followed by the differentiating notochord and finally in the tail bud of ten-somite stage embryos. Amphioxis Brachyury genes are expressed in the three germ layers. In contrast, Am(Bb)fkh/HNF3-1, an amphioxus (B. belcheri) homolog of the fork head/HNF-3 gene, is initially expressed in the invaginating endoderm and mesoderm, then later in the differentiating notochord and in the tail bud. With respect to these two types of genes, the formation of the notochord and tail bud in amphioxus embryos shows both similarity and dissimilarity with that of the notochord and tail bud in vertebrate embryos. In amphioxus, Brachyury gene expression in the presumptive notochord of the gastrulae is not seen, whil in the middle of vertebrate gastrulation, expression is seen in involuting cells and in the presumptive notochord. Amphioxus HNF3beta expression is not detected in the anterior region of the archenteron, although the stage at which Xenopus HNF3beta expression begins is later than that of amphioxus. Whereas vertebrate Brachyury expression in the anterior notochord quickly disappears, in amphioxus Am(Bb)Bra2 expression remains. Am(Bb)fkh/HNF3-1 is not expressed in in the neural tube in amphioxus, but the homolog is expressed in the vertebrate neural tube (Terazawa, 1997).

Genomic cis-regulatory networks in the early Ciona intestinalis embryo

Precise spatiotemporal gene expression during animal development is achieved through gene regulatory networks, in which sequence-specific transcription factors (TFs) bind to cis-regulatory elements of target genes. Although numerous cis-regulatory elements have been identified in a variety of systems, their global architecture in the gene networks that regulate animal development is not well understood. This determined the structure of the core networks at the cis-regulatory level in early embryos of the chordate Ciona intestinalis by chromatin immunoprecipitation (ChIP) of 11 TFs. The regulatory systems of the 11 TF genes examined were tightly interconnected with one another. By combining analysis of the ChIP data with the results of previous comprehensive analyses of expression profiles and knockdown of regulatory genes, it was found that most of the previously determined interactions are direct. Focus was placed on cis-regulatory networks responsible for the Ciona mesodermal tissues by examining how the networks specify these tissues at the level of their cis-regulatory architecture. Many interactions were found that had not been predicted by simple gene knockdown experiments, and a significant fraction of TF-DNA interactions were found to make major contributions to the regulatory control of target gene expression (Kubo, 2010).

The developmental fates of blastomeres in the Ciona embryo have been determined by the gastrula stage. A comprehensive study has revealed that 53 TF genes are zygotically expressed and regulate one another in complex networks before gastrulation begins. To dissect the architecture of these networks at the level of protein-DNA interactions, focus was placed on 11 TF genes that play core roles in gene regulatory networks for endomesoderm specification: Brachyury, FoxA-a, FoxD, MyoD, Neurogenin, Otx, Snail, SoxC, Tbx6b, Twist-like1 and ZicL. Because the Ciona genome contains multiple copies of FoxD, Tbx6b and ZicL as gene clusters and their precise copy numbers have not yet been determined, these genes are collectively referred to FoxD, Tbx6b and ZicL in this paper. Likewise, there are two copies of Twist-like1, which are highly similar to each other, and these are collectively referred to as Twist-like1 (Kubo, 2010).

Eleven gene-fusion constructs were prepared that encode GFP-tagged TFs expressed under the control of their own promoters (e.g. a fusion gene that encodes GFP-tagged Brachyury driven by the Brachyury promoter). When these constructs were introduced into eggs, the resultant embryos expressed the fusion genes at the same time and in the same blastomeres as the endogenous genes. Exceptions were the Twist-like1 and the Snail constructs. Twist-like1 is normally expressed in three cell lineages (A7.6, B7.7 and B8.5), but the construct drove Twist-like1-GFP expression only in the B7.7 and B8.5 lines. Snail expression in the notochord lineage is normally very weak. The Snail construct did not recapitulate this expression in the notochord lineage but did drive Snail-GFP expression in the remaining lineages (Kubo, 2010).

Expression of these genes did not affect embryonic morphology at the stage when the embryos were fixed. The fixed embryos were subjected to ChIP using anti-GFP antibodies, and subsequently to microarray analysis. To define significant regions, two programs were used employing totally different algorithms. DNA segments regarded as positive by both programs were defined as significant. To confirm that this approach successfully identified TF binding sites, the sequences of ZicL and Tbx6b binding regions defined with three different false discovery rates (FDRs) were analyzed, as the consensus binding motifs of these two TFs are known. The frequencies of matches to the consensus binding sequences for ZicL and Tbx6b around peaks in 0.1% FDR were generally better than in 0.01% and 1% FDRs. As expected, the frequencies of the consensus binding sequences for ZicL and Tbx6b were markedly higher around peaks in the identified regions, suggesting that the method was able to successfully identify the TF binding regions (Kubo, 2010).

Brachyury and Ci-tropomyosin-like are the only known direct targets of ZicL and Brachyury, respectively. As an independent confirmation, the TF binding sites of these genes was expected. The ZicL ChIP profile showed a sharp peak around two known strong ZicL binding sites. The Brachyury ChIP profile also showed a peak around the known Brachyury binding site in the Ci-tropomyosin-like promoter. These peaks were included in significant regions identified with all the FDRs described above. ChIP-qPCRs were performed for these two known interactions. The ChIP-qPCR results showed excellent agreement with the ChIP-chip results (Kubo, 2010).

Next, the promoters were examined of genes that were identified in previous studies as likely direct targets of one of the 11 TFs on the basis of expression assays and gene knockdown assays. Among 29 interactions that had been found in the gene knockdown assays and for which both the source and target genes are expressed in the same cells, 28, 23 and 19 interactions were indicated to be direct under the FDRs of 1%, 0.1% and 0.01%, respectively. The remainder of the interactions were not regarded as direct. Otx expression in the A-line lineage requires a cis-regulatory module that includes Fox binding sites and is suppressed in FoxA-a morphants. The FoxA-a binding to this cis-regulatory element was counted with FDRs of 1% and 0.1%, but not with the most stringent FDR (0.01%). Similarly, several lines of evidence have suggested that MyoD is directly regulated by ZicL. First, MyoD expression is suppressed in ZicL morphants. Second, MyoD and ZicL are both expressed in presumptive muscle cells and the time windows of their expression overlap. Lastly, there is a putative ZicL binding site near to the peaks found in the MyoD upstream region. This putative binding was observed under the FDRs of 1% and 0.1%, but not under the most stringent FDR of 0.01%. On the basis of the above observations, in the following sections the results obtained at an FDR of 0.1% are generally described (Kubo, 2010).

The frequencies of the consensus sequences for ZicL and Tbx6b binding were markedly higher around peaks in the identified regions. Since the consensus binding motifs of the other nine TFs had not been determined previously, similar analyses was performed with motifs of homologs in other animals. The frequencies of the consensus binding motifs for six of the TFs, but not FoxD, SoxC or Twist-like1, were markedly higher around peaks. Because the position weight matrices (PWMs) for FoxD, SoxC and Twist-like1 gave higher background, no significant changes were seen. However, the number of matches to the motifs was markedly higher around peaks than in flanking regions and the background. These observations suggested that the method was able to successfully identify the TF binding regions (Kubo, 2010).

As has been reported in other animals, it was found that the regions bound by Brachyury, MyoD, Neurogenin, Snail, Tbx6b, Twist-like1 and ZicL, especially around the peaks, showed a marked GC bias. This bias is likely to be related to the consensus sequences, because the consensus sequences for these TFs are generally more GC-rich than those of the remaining TFs. The observed enrichment of recognition sequences was unlikely to be an artifact of GC bias because even if background sequences were picked with a base composition comparable to the averaged GC content of the bound regions (the difference between the average GC content of the bound and background regions was less than 0.8%), matches to the PWMs were enriched around peaks versus each of the GC-adjusted backgrounds (Kubo, 2010).

Next, attempts were made to discover overrepresented motifs in the regions (360 bp) around the peaks identified by each ChIP experiment using the Trawler program. It was found that overrepresented motifs were similar to the PWMs that were determined experimentally (Tbx6b and ZicL) or to those of homologs in other animals (the remaining nine TFs). This further supported the conclusion that the results of the ChIP experiments were of high quality (Kubo, 2010).

It is generally believed that TFs tend to bind near promoters, although many examples are known in which TFs bind to enhancers far from promoters. The distributions of peaks in all experiments, except Snail ChIP, were higher around transcription start sites. The reason why Snail binding sites were not enriched around transcription start sites is unclear, but this does not necessarily indicate that the results of the Snail ChIP were of low quality. Altogether, these observations support the conclusion that all of the ChIP experiments revealed in vivo occupancies of the TFs (Kubo, 2010).

TF genes were significantly enriched among the target genes of the 11 TFs. Among 670 potential TF genes in the Ciona genome, at least 607 encode proteins with known TF motifs or proteins with two or more zinc-finger motifs that potentially bind to DNA. A significantly greater number of TF genes were found among the targets than would be expected from random sampling. This enrichment indicates that the TFs examined bind targets selectively and not randomly (Kubo, 2010).

The ChIP data was compared with the results of the comprehensive gene knockdown experiments of a previously study. Among 76 interactions previously found in the early embryo, the ChIP assays indicated that 58 are direct. In addition, 251 novel interconnections were found. Among 121 (11×11) possible interconnections, 84 were observed in the present study. The data indicate that these genes are tightly interconnected with one another (Kubo, 2010).

Because the gene regulatory network model previously constructed from comprehensive expression profiles and comprehensive knockdowns of regulatory genes is of single-cell resolution, the ChIP data was interpred into this network by assuming that the examined TFs bind to the targets wherever their mRNAs are expressed. The reconstructed networks had a complex architecture (Kubo, 2010).

The reconstructed regulatory networks allow tracing of development at the single-cell level. Figs S8 and S9 in the supplementary material show the interconnections among the core 11 TFs in A-line and B-line blastomeres, which give rise to endomesodermal tissues, from the 8-cell to the early gastrula stage. Two of the three mesenchymal lineages (B-line mesenchymal cells) and 28 out of 36 muscle cells (B-line muscle cells) in the tadpole larvae are derived from B4.1 blastomeres at the 8-cell stage. Thirty-two and eight notochord cells are derived from A4.1 and B4.1 blastomeres, respectively. Previous studies demonstrated that Twist-like1, MyoD and Brachyury are essential for specification of the mesenchyme, muscle and notochord, respectively (Kubo, 2010).

Twist-like1 is expressed exclusively in the mesenchymal lineage and is regulated by FoxA-a, Otx and ZicL, as indicated by the fact that knockdown of any of these three genes results in loss or reduction of Twist-like1 expression. No direct binding was detected of FoxA-a to the Twist-like1 promoter, but it was found that FoxA-a binds to the upstream regions of Otx and ZicL, and that ZicL and Otx bind to the promoter of Twist-like1. Therefore, it is highly likely that FoxA-a mainly activates Twist-like1 indirectly through activating Otx and ZicL (Kubo, 2010).

Twist-like1 expression begins in B7.7 (the posterior B-line mesenchyme) at the 64-cell stage and in B8.5 (the anterior B-line mesenchyme) at the early gastrula stage. These two mesenchymal lines contribute to distinct adult tissues after metamorphosis. ZicL might be associated with the differences between these two lineages because the contribution of ZicL to Twist-like1 activation is weaker than that of Otx. To confirm this idea, a mutant Twist-like1 promoter was tested, from which a 150 bp segment containing the identified ZicL binding region was deleted. Because the Otx ChIP result indicated that the Otx binding region is distinct from the ZicL binding region, Otx was expected to bind to this mutant promoter. When introduced into fertilized eggs by electroporation, the wild-type promoter (1550 bp) drove reporter expression in 65% of the embryos, whereas the mutant promoter drove reporter expression in 36% of the embryos. In addition to the significant decrease in the number of embryos expressing the reporter, the overall fluorescence was weaker and the posterior B-line mesenchyme did not appear to express the reporter in the mutant construct. To confirm this observation, the experimental embryos were cleavage-arrested at the 110-cell stage. Cells in the arrested embryos cannot divide further, but the developmental programs proceed as in normal embryos. The mutant construct failed to drive reporter expression in the posterior B-line mesenchyme. These results suggest that ZicL contributes to the difference between these two lineages (Kubo, 2010).

A previous study showed that nine mesenchyme-specific non-regulatory genes are under the control of Twist-like1. None of these genes was identified as a direct target in the present study. Even when applied with an FDR of 1%, only one gene was identified as a direct target. Therefore, it is likely that Twist-like1 regulates the expression of mesenchyme-specific genes through its downstream regulatory gene circuit, although there is a possibility that Twist-like1 binds to the regulatory elements of these genes at later stages (Kubo, 2010).

The B6.2 and B6.4 cell pairs in the 32-cell embryo have the potential to give rise to mesenchyme and muscle. At the 64-cell stage, these cells divide, and one of the daughter cells becomes specified to give rise to the muscle cells. Previous functional assays showed that ZicL, Tbx6b and MyoD are essential for specification of muscle cells. Tbx6b begins to be expressed at the 16-cell stage, and cells expressing Tbx6b give rise not only to muscle cells but also to mesenchyme cells. Tbx6b expression declines to undetectable levels before the tailbud stage. ZicL starts to be expressed at the 32-cell stage in a variety of cells, including those with developmental fates of muscle, mesenchyme, notochord and neurons. ZicL expression in the muscle lineage disappears before the late gastrula stage. MyoD expression begins at the 44-cell stage exclusively in the muscle lineage under the control of Tbx6b and ZicL. The present study showed that ZicL, Tbx6b and MyoD constituted a tightly interconnected gene circuit that is responsible for this specification: (1) ZicL bound to the promoters of MyoD and Tbx6b; (2) Tbx6b bound to the promoters of MyoD and ZicL; and (3) MyoD bound to the promoter of Tbx6b and to its own promoter. All of these interactions, except MyoD binding to the Tbx6b promoter, have been confirmed by functional assays (Kubo, 2010).

To understand how this gene circuit regulates downstream muscle-specific genes, the promoters were examined of 13 muscle structural genes that are well annotated and known to be expressed in the larval tail muscle. Of these, ten were directly bound by MyoD and Tbx6, one by MyoD and ZicL, one by Tbx6b and ZicL, and one by MyoD alone (Kubo, 2010).

Both MyoD and Tbx6 bound to the promoters of more than three-quarters of the muscle genes examined. To test the action of this feed-forward loop comprising MyoD and Tbx6b in the regulation of muscle-specific gene expression, the expression patterns of genes under the control of this circuit were examined. Of the 155 genes under the direct control of MyoD and Tbx6b, 50 (including the above ten) were already known to be expressed in muscle cells. From the remaining 105 genes, 20 were randomly chosen, and 15 were found to be expressed in muscle cells, suggesting that this circuit is widely used for the regulation of genes expressed in muscle cells, and also that this circuit might not necessarily be sufficient for driving expression of the target (Kubo, 2010).

Brachyury is activated at the 64-cell stage exclusively in the notochord lineage, and this expression specifies the notochord fate. ZicL directly binds to the Brachyury promoter and activates its expression. It has also been shown that FoxD and FoxA-a are required for Brachyury expression, probably through regulating ZicL expression, and that FGF signaling is also required for Brachyury expression. The present assays showed that not only ZicL, but also FoxD binds to the Brachyury promoter. Although FoxD mRNA is not present in the notochord lineage at the 32-cell and 64-cell stages, when ZicL and Brachyury are activated, respectively (FoxD is expressed in the ancestors of cells in which ZicL and Brachyury are expressed), the ChIP assay indicated that FoxD binds to the promoters of ZicL and Brachyury. Because knockdown of FoxD eliminates ZicL and Brachyury expression and because the FoxD-GFP fusion protein exists in the notochord lineage at the 32-cell stage, it is likely that FoxD protein exists in these cells and binds to the promoters of ZicL and Brachyury when these two genes begin to be expressed (Kubo, 2010).

FoxA-a binding to the Brachyury promoter was not identified under 0.1% FDR. There was, however, a small peak that was counted as significant under 1% FDR. The possibility could not be ruled out that FoxA-a binds weakly to the Brachyury promoter. It is also possible that FoxA-a could bind weakly to a FoxD binding site because the FoxA-a binding peak coincided with that of FoxD. Even if this weak binding occurs in vivo, the regulation of Brachyury by FoxA-a would largely be achieved indirectly through FoxD and ZicL, since strong binding was found of FoxA-a to the promoters of FoxD and ZicL (Kubo, 2010).

Next, 14 non-regulatory genes were examined that are known to be expressed in the notochord under the control of Brachyury. Among them, 11 were identified here as direct targets of Brachyury. The present results suggest that the remaining three genes are regulated indirectly through a gene circuit under the control of Brachyury, although it cannot be ruled out that Brachyury binds to the regulatory elements of these three genes at later stages (Kubo, 2010).

The present study found many interactions between TFs and genomic DNA that were unexpected from preceding gene knockdown assays. Similar observations were also reported in preceding ChIP studies. To estimate what proportion of the binding makes a major contribution to gene regulation in Ciona embryos, MyoD mRNA or an MO against MyoD was injected into eggs, and their effects were analyzed on the expression of the same targets that were analyzed at the gastrula stage or at the tailbud stage, respectively. The mRNA levels of 14 targets, ten of which were expressed in muscle, were significantly increased (>2-fold) in embryos injected with MyoD mRNA, and MyoD MO injection significantly reduced the mRNA levels of three of these targets. The mRNA level of one target (KH.C12.38), which was weakly expressed in muscle at the tailbud stage, was significantly decreased in embryos injected with MyoD mRNA, whereas the mRNA level of one target (KH.C9.27), which was expressed in muscle at the gastrula stage, was significantly increased in embryos injected with the MyoD MO. In total, the mRNA levels of 16 targets were significantly altered by MyoD mRNA overexpression or gene suppression. The remaining four were not significantly affected, although three of these were expressed in muscle, implying that MyoD binding makes a relatively small contribution to activating these target genes. It was also found that eight of 15 Brachyury targets and seven of 12 Twist-like1 targets were significantly affected in the embryos by overexpression or knockdown of Brachyury or Twist-like1, respectively. Therefore, it is estimated that more than half of TF binding makes a major contribution to the regulatory control of gene expression (Kubo, 2010).

The transcription factor FoxB mediates temporal loss of cellular competence for notochord induction in ascidian embryos

In embryos of the ascidian Halocynthia roretzi, the competence of isolated presumptive notochord blastomeres to respond to fibroblast growth factor (FGF) for induction of the primary notochord decays by 1 hour after cleavage from the 32- to 64-cell stage. This study analyzes the molecular mechanisms responsible for this loss of competence and provides evidence for a novel mechanism. A forkhead family transcription factor, FoxB, plays a role in competence decay by preventing the induction of notochord-specific Brachyury (Bra) gene expression by the FGF/MAPK signaling pathway. Unlike the mechanisms reported previously in other animals, no component in the FGF signal transduction cascade appeared to be lost or inactivated at the time of competence loss. Knockdown of FoxB functions allowed the isolated cells to retain their competence for a longer period, and to respond to FGF with expression of Bra beyond the stage at which competence was normally lost. FoxB acts as a transcription repressor by directly binding to the cis-regulatory element of the Bra gene. These results suggest that FoxB prevents ectopic induction of the notochord fate within the cells that assume a default nerve cord fate, after the stage when notochord induction has been completed. The merit of this system is that embryos can use the same FGF signaling cascade again for another purpose in the same cell lineage at later stages by keeping the signaling cascade itself available. Temporally and spatially regulated FoxB expression in nerve cord cells was promoted by the ZicN transcription factor and absence of FGF/MAPK signaling (Hashimoto, 2011).

Differential temporal control of Foxa.a and Zic-r.b specifies brain versus notochord fate in the ascidian embryo

In embryos of an invertebrate chordate, Ciona intestinalis, two transcription factors, Foxa.a (see Drosophila Foxa) and Zic-r.b, (see Drosophila Odd-paired) are required for specification of the brain and the notochord, which are derived from distinct cell lineages. In the brain lineage, Foxa.a and Zic-r.b are expressed with no temporal overlap. In the notochord lineage, Foxa.a and Zic-r.b are expressed simultaneously. In the present study found that the temporally non-overlapping expression of Foxa.a and Zic-r.b in the brain lineage was regulated by three repressors, Prdm1-r.a and Prdm1-r.b ) (see Drosophila Hamlet) and Hes.a (see Drosophila Hairy). In morphant embryos of these three repressor genes, Foxa.a expression was not terminated at the normal time, in addition to precocious expression of Zic-r.b Consequently, Foxa.a and Zic-r.b were expressed simultaneously, which led to ectopic activation of Brachyury (see Drosophila Brachyury) and its downstream pathways for notochord differentiation. Thus, temporal controls by transcriptional repressors are essential for specifying the two distinct fates of brain and notochord by Foxa.a and Zic-r.b. Such a mechanism might enable the repeated use of a limited repertoire of transcription factors in developmental gene regulatory networks (Ikeda, 2016).

Non-mammalian vertebrate Forkhead homologs

Most vertebrate HNF-3 genes show several conserved sites of expression during development, inluding the dorsal lip/Hensen's node, notochord and floor plate, all structures known to organize adjacent tissues. Amphioxus has two HNF-3 genes, named AmHNF-3-1 and AmHNF-3-2, both derived from an independent duplication in the cephalochordate lineage. The expression of both genes in early development appears to be identical and shows striking similarites to that of vertebrates. In neurulae, transcripts of both genes are detected in the presumed organiser, endoderm, and notochord. This supports morphological and embryological evidence that these genes are homologous between vertebrates and amphioxus. This expression pattern overlaps that of amphioxus brachyury, suggesting that the functional relationship between these genes in vertebrates is conserved in amphioxus. Expression of both genes is maintained in the endoderm and notochord up to the 6 somite stage. After the 6 somite stage no expression of AmHNF-3-2 is detected and expression of AmHNF-3-1 begins to decrease in the notochord, so that by the 10 somite stage transcripts are only detected in the terminal regions. However, at this stage a column of AmHNF-3-1 expressing cells is detected at the ventral midline of the neural tube, a position occupied by the floor plate in vertebrates. This is the first evidence that amphioxus has a floor plate that is specified by a mechanism conserved in vertebrates (Shimeld, 1997).

The segregation of cells into germ layers is one of the earliest events in the establishment of cell fate in the embryo. In the zebrafish, endoderm and mesoderm are derived from cells that involute into an internal layer, the hypoblast, whereas ectoderm is derived from cells that remain in the outer layer, the epiblast. In this study, the origin of the zebrafish endoderm and its separation from the mesoderm are examined. By labeling individual cells located at the margin of the blastula, it has been demonstrated that all structures that are endodermal in origin are derived predominantly from the more dorsal and lateral cells of the blastoderm margin. Frequently marginal cells give rise to both endodermal and mesodermal derivatives, demonstrating that these two lineages have not yet separated. Cells located farther than 4 cell diameters from the margin give rise exclusively to mesoderm, and not to endoderm. Following involution, a variety of cellular changes are seen indicating the differentiation of the two germ layers. Endodermal cells gradually flatten and extend filopodial processes forming a noncontiguous inner layer of cells against the yolk. At this time, they also begin to express Forkhead-domain 2 protein, the zebrafish homolog of HNF3beta. Mesodermal cells form a coherent layer of round cells separating the endoderm and ectoderm. In cyclops-mutant embryos (cyclops codes for a nodal-related signaling molecule), which have reduced mesodermal anlage, it has been demonstrated that by late gastrulation not only mesodermal but also endodermal cells are fewer in number. This suggests that a common pathway initially specifies two germ layers together before a progressive sequence of determinative events segregates endoderm and mesoderm into morphologically distinct germ layers. A hierarchical sequence of determinative events is hypothesized: it is suggested that a cell is first specified to either the epiblast or hypoblast and, subsequently, within the hypoblast, a specification to either the endoderm or mesoderm. Before this latter separation, cells appear to be part of a uniform population of migrating cells, and no distinguishing characteristics between the endodermal and mesodermal precursors can be seen (Warga, 1999).

Axial, a member of the fork head/HNF3 gene family, begins to be expressed just before gastrulation in a narrow region on the dorsal side of the embryo, the fish equivalent of the amphibian organizer. Expression can be detected in the involuted cells comprising the mesendoderm of the developing axis. At the end of gastrulation expression is turned on in the ventral neural plate in cells adjacent to the Axial-expressing mesodermal cells. Thus, Axial appears to be a target of both mesoderm induction and neural induction, leading to expression in cells of all three germ layers along the developing axis. Like the Brachyury gene. Axial is strongly induced by activin A, suggesting a role for endogenous activins in specifying the overlapping domains of expression for these two genes along the axis. Axial-expressing cells in the neuroectoderm include those of the future floor plate and cells of the ventral forebrain. In embryos homozygous for the cyclops mutation, expression is normal in mesendodermal cells but is absent from the ventral neural tube. The primary defects of cyclops mutants (lack of floor plate, deficiencies in the brain and cyclopia) correlate well with the expression domain of the Axial gene in wild-type neuroectoderm. The lack of Axial expression in mutant cyclops neuroectoderm suggests that activation of Axial may be an immediate response to cyclops gene activity. Taken together, these data suggest that Axial plays a crucial role in specification of both the axial mesendoderm and the ventral central nervous system (Strahle, 1993).

The signaling molecule Sonic hedgehog is involved in a multitude of distinct patterning processes during vertebrate embryogenesis. In the nascent body axis of the zebrafish embryo, sonic hedgehog is co-expressed with axial (HNF3ß in mammals), a transcription regulator of the winged helix family. Misexpression of axial leads to ectopic activation of sonic hedgehog expression in the zebrafish, suggesting that axial is a regulator of sonic hedgehog transcription. The sonic hedgehog gene was cloned from zebrafish and its promoter was characterized with respect to activation by axial. Expression of axial or rat HNF3ß in HeLa cells results in activation of co-transfected sonic hedgehog promoter-CAT fusion genes. This effect is mediated by two Axial (HNF3beta) recognition sequences. A retinoic acid response element (RARE) was identified in the sonic hedgehog upstream region which can be bound by retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers in vitro. This confers retinoic acid inducibility to the sonic hedgehog promoter in the HeLa cell system. These results suggest that both Axial (HNF3beta) and retinoic acid receptors are direct regulators of the sonic hedgehog gene (Chang, 1997).

The roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish have been examined. Through cloning of monorail (mol), which encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, it has been shown that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. These results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurons of the raphé nucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurons ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins (Norton, 2005).

A zebrafish caudal-related homeobox (cdx1b) gene shares syntenic conservation with both human and mouse Cdx1. Zebrafish cdx1b transcripts are maternally deposited. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to the 1-2s stages and can be identified in the retinas, brain and somites during 18-22 hpf stages. After 28 hours of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino oligonucleotide-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function. Hypoplastic development of the liver and pancreas and intestinal abnormalities were observed in 96 hpf cdx1b morphants. In 85% epiboly cdx1b morphants, twofold decreases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1-binding motifs were identified in both gata5 and foxa2 genes by interspecific sequence comparisons. Cdx1b can bind to the Cdx1-binding motif located in intron 1 of the foxa2 gene based on an electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with the cdx1b MO rescued the expression domains of ceruloplasmin in the liver of 53 hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression, thus affecting early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs compared with its mammalian homologs (Cheng, 2008).

One of the Xenopus forkhead domain family proteins, XFKH1/Pintallavis/XFD1, has been shown to be involved in axial formation. The expression patterns of the other family members suggest that they too play a major role in the initial steps of patterning and axial organization. XFKH3 is expressed in developing somites but not notochord, XFKH4 in forebrain, anterior retina, and neural crest cells, and XFKH5 in a subset of epidermal cells and the neural floor plate. Transcripts of XFKH6 are seen in neural crest-derived cranial ganglia. At least some of the zebrafish forkhead genes might serve a comparable function. Zebrafish zf-FKH1 has a similar expression pattern as Xenopus XFKH1/Pintallavis/XFD1. It is transcribed in the notochord and neural floor plate. The polster or "pillow" also shows very high levels of zf-FKH1 mRNA (Dirksen, 1995).

Transforming growth factor (TGF)-beta family members play a central role in mesoderm induction during early embryogenesis in Xenopus. Although a number of target genes induced as an immediate-early response to activin-like members of the family have been described, little is known about the molecular mechanisms involved. Systematic analysis of the activin induction of the target gene XFKH1 reveals two regions that mediate activin-responsive transcription: one, in the first intron, is targeted directly by the activin-signalling pathway; the other, in the 5' flanking sequences, responds to activin indirectly, possibly being required for the maintenance of gene expression. A 107 bp region of the XFKH1 first intron acts as an enhancer and confers activin inducibility onto a minimal uninducible promoter in the absence of new protein synthesis. It bears little sequence similarity to other activin responsive sequences. Overexpression of a constitutively active derivative of Xenopus Smad2 (XSmad2), which has been implicated as a component of the activin signaling pathway, is sufficient for direct activation of transcription via this enhancer. XSmad2 acts indirectly on the proximal promoter element induced by activin via an indirect mechanism. These results establish the XFKH1 intron enhancer as a direct nuclear target of the activin signaling pathway in Xenopus embryos, and provide strong new evidence that XSmad2 is a transducer of activin signals (Howell, 1997).

Ectopic expression of the ventralizing morphogen BMP-4 in the dorsal lip (Spemann organizer) of Xenopus embryos blocks transcription of dorsal-lip-specific early response genes. The molecular mechanism underlying the BMP-4-induced inhibition of the fork head gene XFD-1' (pintallavis) was examined. The promoter of XFD-1' contains a BMP-triggered inhibitory element (BIE) that prevents gene activation at the ventral/vegetal side of the embryo in vivo. BMP-4-induced inhibition is not direct but indirect, and is mediated by Xvent homeobox proteins. Xvent proteins have no known Drosophila homolog(s). Micro-injections of Xvent-1 RNA and XFD-1' promoter deletion mutants demonstrate that Xvent-1 mimics the effect of BMP-4 signaling, not only by suppression of the XFD-1' gene, but also by utilizing the BIE. Suppression can be reverted using a dominant-negative Xvent-1 mutant. The repressor domain localizes to the N-terminal region of the protein. Gel-shift and footprint analyses prove that Xvent-1 binds to the BIE. PCR-based target-site selection for the Xvent-1 homeodomain confirms distinct motifs within the BIE as preferential binding sites. Thus, biological and molecular data suggest that Xvent-1 acts as a direct repressor for XFD-1' transcription and mediates BMP-4-induced inhibition (Friedle, 1998).

Different types of endoderm, including primitive, definitive and mesendoderm, play a role in the induction and patterning of the vertebrate head. These three types of endoderm are defined in order to compare the mechanism of head induction in model vertebrate organisms. (1) The primitive endoderm is a prospective extraembryonic tissue present in the mouse and the chick, whereas amphibia generate no extraembryonic tissue at all. This endoderm is not a product of gastrulation, and its fate is to become the stalk of the yolk sac. (2) The definitive anterior endoderm develops into the foregut and the liver. In amphibia, it also comprises yolky cells outside the epithelial lining. (3) The precordal mesendoderm as an organizer-derived tissue migrates anteriorly to lie under the developing forebrain. The formation of the anterior neural plate has been studied in chick embryos using the homeobox gene GANF as a marker. GANF is a member of the 'Anf' (anterior neural folds) family, from which a single member has been found in vertebrates, such as fish (Danf), amphibia (Xanf), chick (GANF), mice (Hesx1/Rpx) and human (HANF/HESX1), but has not been found in Drosophila. GANF is first expressed after mesendoderm ingression from Hensen's node. After transplantation, neither the avian hypoblast nor the anterior definitive endoderm is capable of GANF induction, whereas the mesendoderm (young head process, prechordal plate) exhibits a strong inductive potential. GANF induction cannot be separated from the formation of a proper neural plate, which requires an intact lower layer and the presence of the prechordal mesendoderm. It is inhibited by BMP4 and promoted by the presence of the BMP antagonist Noggin. In order to investigate the inductive potential of the mammalian visceral endoderm, use was made of rabbit embryos which, in contrast to mouse embryos, allow the morphological recognition of the prospective anterior pole in the living, pre-primitive-streak embryo. The anterior visceral endoderm from such rabbit embryos induces neuralization and independent, ectopic GANF expression domains in the area pellucida or the area opaca of chick hosts. In terms of the timing and the location of the head organizer, the chick is more similar to the frog, where the signaling comes from the organizer and its derivative, the mesendoderm. In contrast, head-inducing signals in mammals originate from the anterior visceral endoderm. Hence, mouse embryos begin the patterning of the head long before the mesendoderm ingresses, whereas chick head development occurs only after endoderm formation. Only mammals have shifted the head-inducing signals into the primitive endoderm, and they begin the induction and patterning process of the head long before (about 24 hours in mice and rabbits) the mesendoderm ingresses. Several genes are expressed in the independent primitive endoderm domain (the anterior visceral endoderm) in the mammalian head organizer before or at the onset of primitive streak formation, prior to their expression in the axial mesendoderm or the node during gastrulation. These include the homeobox genes Hesx1 (Rpx), Goosecoid, Lim1, Hex and Otx2; the forkhead gene HNF3beta; the nuclear protein gene mrg1; the growth factor gene Nodal, and the antigen VE-1. Thus, the signals for head induction reside in the anterior visceral endoderm of mammals whereas, in birds and amphibia, they reside in the prechordal mesendoderm, indicating a heterochronic shift of the head inductive capacity during the evolution of mammalia (Knoetgen, 1999).

Table of contents

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

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