brachyenteron/T-related gene

EVOLUTIONARY HOMOLOGS

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).

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).

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