brachyenteron/T-related gene


Brachyury in Tunicates and Cephalochordates

A notochord-specific enhancer has been characterized from the Brachyury promoter region of the ascidian (Tunicate: phylum Urochordata), Ciona intestinalis. A minimal, 434 bp enhancer from the Ci-Bra promoter region mediates the notochord-restricted expression of both GFP and lacZ reporter genes. This enhancer contains a negative control region that excludes Ci-Bra expression from inappropriate embryonic lineages, including the trunk mesenchyme and tail muscles. Evidence is presented that the enhancer is activated by a regulatory element which is closely related to the recognition sequence of the Suppressor of Hairless transcription factor, thereby raising the possibility that the Notch signaling pathway plays a role in notochord differentiation. In this case, cell-cell interaction involving the Notch pathway might be required for the induction of notochord fate. Preliminary studies suggest that a Ciona homolog of Drosophila snail gene is a potential Ci-Bra repressor (Corbo, 1997).

A minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra) is sufficient to direct a notochord-specific pattern of gene expression. Evidence is presented that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays have identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages. Repression appears to depend on tight linkage between sna1 and sna2 sites and Su(H) activator sites, located on the minimal enhancer. The insertion of spacer sequences between sna1 and Su(H)1 or between Su(H)2 and sna2 results in a severe derepression of Ci-Bra/lacZ transgenes in the tail muscles. Intact sna1 and sna2 sites appear to be required for the repression of Ci-Bra in the tail muscles. The function of Ci-Snail in creating a boundary between notochord and muscle is likened to the function of Snail in Drosophila in creating a boundary between neuroectoderm and mesoderm (Fujiwara, 1998).

Studies on ascidians provided the first evidence for localized determinants in animal development. The destruction of particular blastomeres leads to the specific loss of muscle derivatives. Lineage studies have established a tight correlation between the distribution of yellow crescent and muscle differentiation in Styela. The yellow crescent becomes localized to the vegetal cytoplasm shortly after fertilization and is ultimately inherited by the two B4.1 blastomeres that form most of the tail muscles in the ascidian tadpole. A new T-box gene resembling Drosophila Optomotor blind, CiVegTR, that fulfils the criteria of the classic muscle determinant, has been isolated in the ascidian Ciona intestinalis. CiVegTR maternal RNAs become localized to the vegetal cytoplasm of fertilized eggs and are incorporated into muscle lineages derived from the B4.1 blastomere. The CiVegTR protein binds to specific sequences within a minimal, 262-bp enhancer that mediates Ci-snail expression in the tail muscles. Mutations in these binding sites abolish expression from an otherwise normal lacZ reporter gene in electroporated embryos. In addition to the previously identified AC-core E-box sequences, T-box recognition sequences are conserved in the promoter regions of many genes expressed in B4.1 lineages in both Ciona and the distantly related ascidian Halocynthia. These results suggest that CiVegTR encodes a component of the classical muscle determinant that was first identified in ascidians nearly 100 years ago (Erives, 2000).

A Ciona ortholog of the Drosophila Snail repressor (Ci-sna) was found to repress a 434-bp notochord-specific enhancer in the promoter region of the Ciona Brachyury gene. Ci-sna is expressed in the developing tail muscles, where it is important for restricting Ci-Bra expression to the developing notochord. Ci-sna is activated early during muscle specification (32-cell stage), at the time when maternal determinants first activate zygotic genes. The present study identifies a 262-bp enhancer from the Ci-sna 5' flanking region that is sufficient to mediate expression in derivatives of the B4.1 blastomeres. This enhancer contains two conserved sequence motifs that are also present in the regulatory regions of muscle-specific genes in the distantly related ascidian Halocynthia. One of the motifs corresponds to a specialized E-box sequence (CAACTG), whereas the other contains conserved residues recognized by different T-box DNA binding proteins (GT-GNNA). Mutations in either motif diminish or abolish the expression driven by otherwise normal Ci-sna/lacZ transgenes (Erives, 2000).

Brachyury is a sequence-specific transcriptional activator that is essential for notochord differentiation in a variety of chordates. In vertebrates, Brachyury is expressed throughout the presumptive mesoderm, but becomes restricted to the notochord at later stages of development. In ascidians, such as Ciona intestinalis, Brachyury is expressed exclusively in the notochord and does not exhibit an early pan-mesodermal pattern. Subtractive hybridization screens have been used to identify potential Ciona Brachyury (Ci-Bra) target genes. Of the genes that were identified in this screen, one corresponds to a new member of the tropomyosin superfamily, Ciona tropomyosin (Ci-trop). Ci-trop is specifically expressed in the developing notochord beginning at gastrulation, and expression persists in the notochord during tailbud and tadpole stages. A 3 kb region of the Ci-trop 5'-flanking sequence was characterized via electroporation of lacZ fusion genes into fertilized Ciona eggs. A minimal 114 bp enhancer was identified that is sufficient to direct the expression of a heterologous promoter in the notochord. DNA binding assays indicate that this enhancer contains two sets of low-affinity Brachyury half-sites, which are bound in vitro by a GST/Ci-Bra fusion protein. Deletion of the distal sites inactivates the notochord-specific staining pattern mediated by an otherwise normal Ci-trop/lacZ transgene. These results suggest that Ci-trop is a direct target gene of Ci-Bra and that Brachyury plays an immediate role in the cellular morphogenesis of the notochord (Di Gregorio, 1999).

The notochord has two major roles during chordate embryogenesis -- as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. cDNA clones have been isolated for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells. Twenty of these have been characterized by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein: all 3 appear to be involved in the control of cytoskeletal architecture. Additionaly identified were genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, b4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals, as it is in vertebrates. In addition, the present information could be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes that are expressed in the notochord cells of vertebrate embryos (Hotta, 2000).

Two axial structures, a neural tube and a notochord, are key structures in the chordate body plan, and a closer look at these structures furthers understanding of the origin of chordates. The neural tube of ascidian larvae is composed of about 340 cells, and is divided into three regions along the anteroposterior axis, which are, from anterior to posterior, the sensory vesicle, the visceral ganglion and the caudal neural tube. The sensory vesicle is composed solely of the a-line (anterior-animal) cells. The visceral ganglion present at the junction between the trunk and tail consists of the A-line (anterior-vegetal) cells. The caudal neural tube running along the length of the tail consists of four (dorsal, ventral and two lateral) rows of ependymal cells: the lateral and ventral cells are of A-line origin and the dorsal cells are of b-line (posterior-animal) origin. Beneath the neural tube, a stack of exactly 40 notochord cells runs along the tail. The anterior 32 cells (primary notochord) and the posterior 8 cells (secondary notochord) are derived from A-line and b-line cells, respectively. To expand knowledge on mechanisms of development of the neural tube in lower chordates, isolation and characterization of HrzicN, a new member of the Zic family gene of the ascidian, Halocynthia roretzi, was undertaken. HrzicN expression is detected by whole-mount in situ hybridization in all neural tube precursors, all notochord precursors, anterior mesenchyme precursors and a part of the primary muscle precursors. Embryos injected with HrzicN morpholino ('HrzicN knockdown embryos') exhibit failure of neurulation and tail elongation, and develop into larvae without a neural tube and notochord. Analysis of mesodermal marker gene expression in HrzicN knockdown embryos revealed unexpected roles for this gene in the development of mesodermal tissues. HrzicN knockdown leads to loss of HrBra (Halocynthia roretzi Brachyury) expression in all of the notochord precursors: this may be the cause for notochord deficiency. Hrsna (Halocynthia roretzi snail) expression is also lost from all the notochord and anterior mesenchyme precursors. By contrast, expression of Hrsna and the actin gene is unchanged in the primary muscle precursors. These results suggest that HrzicN is responsible for specification of the notochord and anterior mesenchyme (Wada, 2002).

Homologues of the murine Brachyury (T) gene have been cloned from several vertebrates, and are implicated in mesoderm formation and in differentiation of the notochord. In contrast, the roles of the ascidian (Cephalochordata within the phylum Chordata) Brachyury gene may be restricted to presumptive notochord. To understand the evolution of Brachyury genes and their developmental roles, homologues have been sought in amphioxus, representing the third chordate subphylum and the probable closest relative of the vertebrates. There are two amphioxus cDNA clones with clear homology to Brachyury genes. These derive from separate loci resultant from a recent gene duplication. This finding represents an exception to the emerging consensus of an archetypal prevertebrate genome in amphioxus. The spatial and temporal distribution of Brachyury transcripts during amphioxus development is remarkably similar to vertebrate Brachyury, in presumptive mesoderm, posterior mesoderm and the notochord. Gene expression extends throughout the anteroposterior axis of the notochord, despite the most rostral regions being a more recent specialization; it also persists into larval stages, despite differentiation into contractile tissue. It is thought that roles of Brachyury in notochord differentiation are more ancient than roles in mesoderm formation, and that the latter are shared by cephalochordates and all vertebrates (Holland, 1995).

Appendicularia (Larvacea) is a subgroup of Urochordata (Tunicata) comprised of holoplanktonic organisms that retain their tailed architecture throughout their life history, while other tunicates, including ascidians and doliolids, resorb the tail after metamorphosis. In order to investigate the characteristics of the appendicularian unresorbed notochord, a partial genomic clone and a full-length cDNA sequence homologous to the mouse Brachyury (T) gene was isolated from the appendicularian Oikopleura longicauda. Brachyury is known to be predominantly expressed in the notochord cells and plays an important role in their differentiation in other chordates. While phylogenetic analysis robustly supports the orthology of the isolated Brachyury gene, the exon-intron organization found in the genomic clone was distinct from that well-conserved among other T-box genes. In addition to a detailed observation of notochord development in living specimens, whole-mount double in situ hybridization was carried out using a Brachyury probe along with a muscle actin probe. The Brachyury transcripts were found in the notochord of the tailbud embryos and persist into later stages. The present study highlights characteristics of notochord development in the appendicularian. Furthermore, these results provide basic knowledge for comprehensive understanding of the cellular- and molecular-based mechanisms needed to build the characteristic cytoarchitecture of notochord, which varies among tunicate species (Nishino, 2001).

The notochord is one of the characteristic features of the phylum Chordata. The vertebrate Brachyury gene is known to be essential for the terminal differentiation of chordamesoderm into notochord. In the ascidian, which belongs to the subphylum Urochordata, differentiation of notochord cells is induced at the late phase of the 32-cell stage through cellular interaction with adjacent endoderm cells as well as neighboring notochord cells. The ascidian Brachyury gene (As-T) is expressed exclusively in the notochord-lineage blastomeres, and the timing of gene expression at the 64-cell stage precisely coincides with that of the developmental fate restriction of the blastomeres. In addition, experimental studies have demonstrated a close relationship between the inductive events and As-T expression. Overexpression of As-T by microinjection of the synthesized As-T RNA results in the occurrence, without the induction, of notochord-specific features in the A-line presumptive notochord blastomeres. Overexpression of As-T RNA leads to ectopic expression of notochord-specific features in non-notochord lineages, including those of spinal cord and endoderm. These results strongly suggest that the developmental role of the Brachyury is conserved throughout chordates in notochord formation (Yasuo,1998).

The Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates, at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells. It has been suggested that nonnotochordal roles for T were acquired in the cephalochordate-vertebrate lineage after it split with Urochordata. To test this hypothesis, T was cloned from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos shows early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. It is concluded that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord (Bassham, 2000).

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).

In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) has been visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected sub-equatorially encircling the earliest (vegetal plate stage) gastrula. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast, all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed (Zhang, 1997).

The Brachyury genes of two divergent ascidians, As-T of Halocynthia roretzi and Ci-Bra of Ciona intestinalis, are expressed exclusively in notochord precursor cells. The notochord-specific expression of Ci-Bra is controlled by a minimal promoter that is composed of three distinct regions: a region responsible for repression of expression in non-notochord mesoderm cells; a region for activation of expression in notochord cells, and a region for activation of expression in non-notochord mesoderm cells, distal to proximal to the transcription initiation site, respectively. Various deletion constructs of the As-T/lacZ fusion gene were examined and a module between -289 and -250 bp of the 5' -flanking region has been demonstrated to be responsible for notochord-specific expression of the reporter gene. Gel-shift assays suggest the binding of nuclear protein(s) to this module. The 5'-flanking region of As-T contains a potential T-binding motif (-ACCTAGGT-) around -160 bp. Deletion of this motif from the p(-289)As-T/lacZ diminishes the reporter gene expression. In addition, coinjection of p(-289)As-T/lacZ and synthetic As-T mRNA results in ectopic expression of lacZ in non-notochord cells, suggesting that the T-binding motif is responsible for autoactivation of the gene. Thus, in contrast to CiBra, As-T seems to have a simple module for the notochord-specific expression. These findings reveal striking differences between the minimal promoters of As-T and Ci-Bra, with respect to their notochord-specific expression. Furthermore, reciprocal injections of reporter gene constructs, namely As-T/lacZ into Ciona eggs and Ci-Bra/lacZ into Halocynthia eggs, suggest alterations in the cis-regulatory elements and trans-activation factors that have occurred during evolution of the two ascidian species (Takahashi, 1999).

This study reports the isolation of a recessive ENU-induced short-tailed mutant in the ascidian Ciona intestinalis that is the product of a premature stop in the brachyury gene. Notochord differentiation and morphogenesis are severely disrupted in the mutant line. At the larval stage, variable degrees of ectopic endoderm staining were observed in the homozygous mutants, indicating that loss of brachyury results in stochastic fate transformation. In post-metamorphosis mutants, a uniform defect in tail resorption was observed, together with variable defects in digestive tract development. Some cells misdirected from the notochord lineage were found to be incorporated into definitive endodermal structures, such as stomach and intestine (Chiba, 2008).

The manipulation of factors upstream of notochord specification, including bFGF, β-catenin, ZicN and FoxD, demonstrates the potential for presumptive notochord cells to assume neural and endodermal fates in ascidians. However, morpholino knockdown of brachyury in Ciona savignyi resulted in a short tail phenotype, but with no reported ectopic endoderm. The mutant line described here offers a stable, non-mosaic background in which to study the effects of loss of brachyury function. On examination of the pan-neural gene ETR-1 and muscle actin, there was no indication of notochord cells transfating to neural or endodermal tissues in bra-/- larvae. By contrast, the examination of alkaline phosphatase (AP) activity, as a marker of endoderm, revealed definitive, but highly variable, transfating of notochord to endoderm-like cells in the bra-/- larvae. Approximately one-half of the bra-/- larvae had ectopic AP staining in the core of the tail. Because of the variability in AP staining, the genotypes of individual larvae were determined by genomic PCR and dideoxy sequencing. Identical results were obtained by in situ hybridization using an endoderm-specific marker, kyotograil2005.572.7.1. Finally, AP activity colocalized with brachyury promoter-driven GFP fluorescence in bra-/- larvae, but not in wild-type larvae, indicating that the notochord cells were transfated to endoderm in the homozygous mutant (Chiba, 2008).

The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. This study identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, this study demonstrated that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, the organization of the transcription factor binding sites within the 155-bp CRM was investigated. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function (Passamaneck, 2009).

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).

Brachyury in fish

To analyse the roles of FGF activity and brachyury during gastrulation, the consequences of inhibition of FGF-receptor signaling was compared with the phenotype of the zebrafish brachyury homolog mutant, no tail. Expression of ntl is regulated by FGF and inhibition of FGF receptor-signaling leads to complete loss of the trunk and tail. Since the ntl mutant lacks the tail and notochord but has an otherwise normal trunk, this demonstrates that trunk development is dependent upon an unidentified gene, or set of genes, referred to as no trunk (ntk) which is regulated by FGF. Expression of eve1 and cad1 is also regulated by FGF activity, suggesting that during gastrulation FGF activity is normally restricted to the germ ring where these genes, and ntl, are expressed. Taken together these data suggest that the germ ring acts as a posteriorizing center during AP patterning, mediated by FGF activity in this tissue (Griffin, 1995).

Using fate mapping techniques, development of cells of the dorsal marginal region in wild-type and mutant zebrafish have been analyzed. There is a domain in the early gastrula that is located just at the margin and centered on the dorsal midline, in which most cells generate clones that develop exclusively as notochord. The borders of the notochord domain are sharp at the level of single cells, and coincide almost exactly with the border of the expression domain of the homeobox gene floating head (flh; zebrafish homologue of Xnot), a gene essential for notochord development. In flh mutants, cells in the notochord domain generate clones of muscle cells. In contrast, notochord domain cells form mesenchyme in embryos mutant for no tail (ntl; zebrafish homologue of Brachyury). A minority of cells in the notochord domain in wild-type embryos develop as unrestricted mesoderm, invariably located in the tail, suggesting that early gastrula expression of flh does not restrict cellular potential to the notochord fate. The unrestricted tail mesodermal fate is also expressed by the forerunner cells, a cluster of cells located outside the blastoderm, adjacent to the notochord domain. Cells can leave the dorsal blastoderm to join the forerunners, suggesting that relocation between fate map domains might respecify notochord domain cells to the tail mesodermal fate. An intermediate fate of the forerunners is to form the epithelial lining of Kupffer's vesicle, a transient structure of the teleost tailbud. The forerunners appear to generate the entire structure of Kupffer's vesicle, which also develops in most flh mutants. Although forerunner cells are present in ntl mutants, Kupffer's vesicle never appears, which is correlated with the later severe disruption of tail development (Melby, 1996).

The spatial and temporal expression pattern of zebrafish wnt11 and the regulation of the expression during zebrafish early development was examined, focusing on the interaction with the no tail (ntl) gene, a zebrafish ortholog of mouse Brachyury (T). Zygotic expression of wnt11 is first detected at the late blastula stage in the blastoderm margin, a presumptive mesoderm region. wnt11 expression coincides with mesoderm induction, and the expression is induced by mesoderm inducers such as the yolk cell or FGFs, indicating that, like ntl, wnt11 is one of the immediate-early genes in mesoderm induction. Initial expression domains of wnt11 and ntl overlap, and these genes show a similar response to mesoderm inducers. However, analysis of the ntl mutant embryos suggests that wnt11 and ntl are placed in distinct genetic pathways; the ntl mutation has no effect on wnt11 expression in the blastoderm margin. This is further supported by the result of RNA injection experiments showing that overexpression of Wnt11 does not affect ntl expression in the margin. Thus, wnt11 and ntl expression are induced and maintained independently in their initial phase of expression. In later stages, wnt11 is expressed in various organs, such as the somites, particularly in the developing notochord. Since no wnt gene has been reported to be expressed in the axial mesoderm, which is known to act as a signaling source that patterns the neural tube and somites, zebrafish wnt11 is the first wnt gene expressed in the notochord. Furthermore, in contrast to early expression, wnt11 expression in the notochord depends on Ntl activity. In the ntl mutant in which somite patterning is severely affected, wnt11 expression is completely lost, while another signaling molecule, sonic hedgehog, is expressed in the mutant notochord precursor cells. wnt11 expression in the somite also shows a characteristic pattern, correlated with the migration and differentiation of slow muscle precursors. These observations suggest a role for wnt11 in patterning the somites (Makita, 1998).

LIM class homeobox genes code for a family of transcriptional regulators that encode important determinants of cell lineage and cell type specificity. The lim3 gene from the zebrafish (see Drosophila Apterous), Danio rerio, is highly conserved in sequence and expression pattern, as compared to its homologs in other vertebrates. Immunocytochemical analysis of Lim3 protein expression was performed in the pituitary, pineal, hindbrain, and spinal cord of the embryo, revealing an asymmetrical, lateral and late program of pituitary development in zebrafish, distinct from the pattern in other vertebrates. Lim3 expression was studied in no tail, floating head, and cyclops mutant embryos, all of which have midline defects, with special reference to spinal cord differentiation (where Lim3 marks mostly motoneurons). cyclops embryos show essentially normal Lim3 expression in the hindbrain and spinal cord despite the absence of the floor plate, while no tail (Drosophila homolog: brachyenteron) mutant embryos, which lack a differentiated notochord, display an excess of Lim3-expressing cells in a generally normal pattern. In contrast, Lim3-positive cells largely disappear from the posterior spinal cord in floating head (coding for a homeodomain protein) mutants, except in patches that correlate with remnants of apparent floor plate cells. These results support the view that either notochord or floor plate signaling can specify Lim3-positive motoneurons in the spinal cord (Glasgow, 1997).

An investigation was carried out of the role of cAMP-dependent protein kinase A (PKA) in the induction of the early mesodermal marker genes goosecoid and no tail by activin in zebrafish embryos. Upon treatment with activin, zebrafish blastula cells exhibit a rapid and transient increase in PKA activity. In these cells, activin rapidly induces the expression of the immediate early response genes goosecoid and no tail. Stimulation and inhibition of PKA by activin, respectively, enhances and reduces the induction of goosecoid and no tail mRNA expression. Similar effects of PKA stimulation and inhibition on the induction by activin of a 1.8 kb zebrafish goosecoid promoter construct are observed. The induction by activin of a fragment of the zebrafish goosecoid promoter that mediates an immediate early response to activin is blocked by inhibition of PKA. Activation of PKA alone has no effect in these experiments. Finally, inhibition of PKA in whole embryos by overexpression of a dominant negative regulatory subunit of PKA reduces the expression of no tail and goosecoid, whereas the expression of even-skipped1 remains unaltered. Overexpression of the catalytic subunit of PKA in embryos does not affect expression of goosecoid, no tail or even-skipped1. These data show that in dissociated blastulae, PKA is required, but not sufficient for activin signalling towards induction of goosecoid and no tail. In intact zebrafish embryos, PKA contributes to induction of goosecoid and no tail, although it is neither required nor sufficient (Joore, 1998).

T-box genes encode transcriptional regulators that control many aspects of embryonic development. The mesodermally expressed zebrafish spadetail (spt)/VegT and no tail (ntl)/Brachyury T-box genes are semi-redundantly and cell-autonomously required for formation of all trunk and tail mesoderm. Despite the lack of posterior mesoderm in spt;ntl embryos, dorsal-ventral neural tube patterning is relatively normal, with the notable exception that posterior medial floor plate is completely absent. This contrasts sharply with observations in single mutants, as mutations singly in ntl or spt enhance posterior medial floor plate development. ntl function is required to repress medial floor plate and promote notochord fate in cells of the wild-type notochord domain, and spt and ntl together are required non cell-autonomously for medial floor plate formation, suggesting that an inducing signal present in wild-type mesoderm is lacking in spt;ntl embryos (Amacher, 2002).

The results suggest that spt/VegT and ntl/Brachyury can substitute for each other for a crucial early function, the specification of all posterior mesoderm. However, the same genes are required individually for what may be later functions, promoting development of distinct mesodermal types. Because both Spt and Ntl are both T-box transcription factors, they might be able to activate common transcriptional target genes, suggesting that they can functionally substitute for one another, at least partially, in regions of the embryo where they are co-expressed. In vitro binding site selection experiments show that Brachyury binds to a specific palindromic sequence, and crystallography structure analysis confirms that the Brachyury T-box domain can bind DNA as a dimer. Binding site selection experiments demonstrate that Brachyury, VegT and Eomesodermin all recognize pairs of the same core sequence, but that spacing and orientation of paired sites differs for each protein. However, no promoter analyzed to date contains sites that are perfect matches to the in vitro selected sites. At least three types of X. laevis direct T-box target genes whose promoters have been analyzed, namely Bix genes, fgfs and nodal-related genes of the TGFß family, are expressed in the blastoderm margin and are potential candidates for mesoderm specification genes that might be activated by either spt or ntl. FGFs and the TGFß family member Derriére are particularly intriguing candidates because of their proposed roles in posterior mesoderm development (Amacher, 2002 and references therein).

The lack of posterior mesoderm in spt;ntl embryos is very similar to the phenotype of zebrafish and frog embryos in which FGF signaling has been disrupted. To date, several zebrafish FGF genes (fgf8, fgf3, gfgf, fgf4) have been isolated that are expressed (at least transiently) in mesodermal precursors. Gene expression analyses in ntl, spt and fgf8/ace single mutants and compound heterozygotes indicate that zebrafish T-box genes and fgf8 are involved in a regulatory loop, similar to the auto-regulatory loop described for X. laevis Brachyury and eFGF. The X. laevis TGFß family member Derriére is involved in mesoendoderm development and appears to function in posterior regions of the embryo. It has been proposed that Derriére, zygotic VegT and Brachyury operate in an FGF-dependent regulatory loop in the early gastrula to specify posterior mesoderm development. A zebrafish derriére homolog has not yet been described, but may prove to be an important spt and/or ntl target gene (Amacher, 2002 and references therein).

Early embryonic development in many organisms relies upon maternal molecules deposited into the egg prior to fertilization. A maternal T-box gene in the zebrafish, eomesodermin (eomes), has been cloned and characterized. During oogenesis, the eomes transcript becomes localized to the cortex of the oocyte. After fertilization during early cleavage stages, eomes is expressed in a vegetal to animal gradient in the embryo, whereas Eomesodermin protein is distributed cytoplasmically throughout the blastoderm. Strikingly, following midblastula transition, nuclear-localized Eomesodermin is detected on the dorsal side of the embryo only. Overexpression of eomes results in Nodal-dependent and nieuwkoid/dharma independent ectopic expression of the organizer markers goosecoid (gsc), chordin and floating head (flh) and in the formation of secondary axes. The same phenotypes are observed when a VP16-activator construct is injected into early embryos, indicating that eomes acts as a transcriptional activator. In addition, a dominant-negative construct and antisense morpholino oligonucleotides leads to a reduction in gsc and flh expression. Together these data indicate that eomes plays a role in specifying the organizer (Bruce, 2003).

The heart, brain, and gut develop essential left-right (LR) asymmetries. Specialized groups of ciliated cells have been implicated in LR patterning in mouse, chick, frog, and zebrafish embryos. In zebrafish, these ciliated cells are found in Kupffer's vesicle (KV) and are progeny of dorsal forerunner cells (DFCs). However, there is no direct evidence in any vertebrate that the genes involved in LR development are specifically required in ciliated cells. By using a novel method in zebrafish, the function of no tail (ntl, homologous to mouse brachyury) was knocked down in DFCs without affecting its expression in other cells in the embryo. The Ntl transcription factor is found to function cell autonomously in DFCs to regulate KV morphogenesis and LR determination. This is the first evidence that loss-of-gene function exclusively in ciliated cells perturbs vertebrate LR patterning. These results demonstrate that the ciliated KV, a transient embryonic organ of previously unknown function, is involved in the earliest known step in zebrafish LR development, suggesting that a ciliary-based mechanism establishes the LR axis in all vertebrate embryos (Amack, 2004).

The T box transcription factor Brachyury is essential for the formation of the posterior body in all vertebrates, although its critical transcriptional targets have remained elusive. Loss-of-function studies of mouse Brachyury and the zebrafish Brachyury ortholog Ntl indicated that Brachyury plays a more significant role in higher vertebrates than lower vertebrates. A second zebrafish Brachyury ortholog (Bra) has been identified; a combined loss of Ntl and Bra recapitulates the mouse phenotype, demonstrating an ancient role for Brachyury in patterning all but the most anterior somites. Using cell transplantation, it was shown that the only essential role for Brachyury during somite formation is non-cell autonomous; Ntl and Bra are required for and can induce expression of the canonical Wnts wnt8 and wnt3a. It is proposed that a positive autoregulatory loop between Ntl/Bra and canonical Wnt signaling maintains the mesodermal progenitors to facilitate posterior somite development in chordates (Martin, 2008).

The zebrafish genes spadetail (spt) and no tail (ntl) encode T-box transcription factors that are important for early mesoderm development. Although much has been done to characterize these genes, the identity and location of target regulatory elements remain largely unknown. This study surveyed the genome for downstream target genes of the Spt and Ntl T-box transcription factors. Evidence was found for extensive additive interactions towards gene activation and limited evidence for combinatorial and antagonistic interactions between the two factors. Using in vitro binding selection assays to define Spt- and Ntl-binding motifs, target regulatory sequence were sought via a combination of binding motif searches and comparative genomics. Regulatory elements were identified for tbx6 and deltaD, and, using chromatin immunoprecipitation, in vitro DNA binding assays and transgenic methods, evidence was provided that both are directly regulated by T-box transcription factors. deltaD is directly activated by T-box factors in the tail bud, where it has been implicated in starting the segmentation clock, suggesting that spt and ntl act upstream of this process (Garnett, 2009).

During early zebrafish development the nodal signalling pathway patterns the embryo into three germ layers, in part by inducing the expression of T-box transcription factor no tail (ntl), which is essential for correct mesoderm formation. When nodal signalling is inhibited ntl fails to be expressed in the dorsal margin, but ventral ntl expression is unaffected. These observations indicate that ntl transcription is under both nodal-dependent and nodal-independent regulation. Consistent with these observations and with a role for ntl in mesoderm formation, some somites form within the tail region of embryos lacking nodal signalling. In an effort to understand how ntl is regulated and thus how mesoderm forms, the elements responsible for nodal-dependent and nodal-independent expression of ntl in the margin of the embryo were mapped. This work demonstrates that expression of ntl in the margin is the consequence of two separate enhancers, which act to mediate different mechanisms of mesoderm formation. One of these enhancers responds to nodal signalling, and the other to Wnt and BMP signalling. The nodal-independent regulation of ntl is essential for tail formation. Misexpression of Wnt and BMP ligands can induce the formation of an ectopic tail, which contains somites, in embryos devoid of nodal signalling, and this tail formation is dependent on ntl function. Similarly, nodal-independent tail somite formation requires ntl. At later stages in development ntl is required for notochord formation, and the analysis has also led to the identification of the enhancer required for ntl expression in the developing notochord (Harvey, 2010).

Formation of the early vertebrate embryo depends on a Brachyury/Wnt autoregulatory loop within the posterior mesodermal progenitors. This study shows that exogenous retinoic acid (RA), which dramatically truncates the embryo, represses expression of the zebrafish brachyury ortholog no tail (ntl), causing a failure to sustain the loop. It was found that Ntl functions normally to protect the autoregulatory loop from endogenous RA by directly activating cyp26a1 expression. Thus, the embryonic mesodermal progenitors uniquely establish their own niche - with Brachyury being essential for creating a domain of high Wnt and low RA signaling - rather than having a niche created by separate support cells (Martin, 2010).

Common examples of stem or progenitor cell niches in both embryonic and adult organisms consist of at least two general cell types: the stem/progenitor cells and the support cells, which provide the physical and molecular environment necessary for the maintenance of the stem/progenitor cells. These data provide evidence of a unique type of progenitor cell niche consisting of only one cell type, in which mesodermal progenitor cells of the zebrafish tailbud act as their own support cells. Mesodermal progenitors express ntl, wnt3a, wnt8, and cyp26a1, all of which are required within the progenitor population as a whole, but none of which are required by individual progenitor cells in a wild-type environment. This demonstrates that the wild-type mesodermal progenitor cells act as support cells for the genetically deficient progenitors and can sustain them by creating an environment of high Wnt and low RA signaling. The primary function of Ntl, therefore, is to create the mesodermal progenitor niche through direct regulation of canonical wnt ligands and cyp26a1. While this analysis focused specifically on zebrafish, the common phenotypes of brachyury loss of function and RA treatment in different vertebrates, as well as the conservation of expression patterns of brachyury, wnts, and cyp26a1, indicates that the same mechanism is common to all vertebrates. Thus, it is suggested that expression of brachyury in the progenitor domain was a vertebrate adaptation that allowed the progenitor cells to be sustained during the long process of somitogenesis, which in some species can last for many days. This unique function of Brachyury is particularly relevant, given that recent molecular analysis of various human cancers has demonstrated that brachyury is commonly up-regulated in tumors. The up-regulation of brachyury may, in effect, be creating a cancer cell niche that maintains high Wnt signaling and low RA signaling, both of which have been extensively demonstrated to be key components of cancer growth (Martin, 2010).

FGF and canonical Wnt signaling cooperate to induce paraxial mesoderm from tailbud neuromesodermal progenitors through regulation of a two-step EMT
Mesoderm induction begins during gastrulation. Recent evidence from several vertebrate species indicates mesoderm induction continues after gastrulation in neuromesodermal progenitor cells (NMPs) within the posterior-most embryonic structure called the tailbud. It is unclear to what extent the molecular mechanisms of mesoderm induction are conserved between gastrula and post-gastrula stages of development. Fibroblast growth factor (FGF) signaling is required for mesoderm induction during gastrulation through positive transcriptional regulation of the t-box transcription factor brachyury (ntla in zebrafish and Byn in Drosophila). This study found that FGF is continuously required for paraxial mesoderm (PM) induction in zebrafish post-gastrula NMPs, but has the opposite effect on brachyury expression. FGF signaling represses brachyury and the NMP marker sox2 (see Drosophila SoxNeuro) through regulation of tbx16 and msgn1, thereby committing cells to a PM fate. FGF mediated PM induction in NMPs functions in tight coordination with canonical Wnt signaling during the epithelial to mesenchymal transition from NMP to mesodermal progenitor. Wnt signaling initiates the EMT, while FGF signaling terminates this event. These results indicate that germ layer induction in the tailbud is not a simple continuation of gastrulation events (Goto, 2017).

Table of contents

brachyenteron/T-related gene: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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