brachyenteron/T-related gene


Table of contents

Drosophila T-box proteins

optomotor blind is homologous to mouse Brachyury. The central region of OMB is homologous to the N-terminal half of the Brachyury protein. This conserved domain has a general DNA binding affinity but has no significant protein sequence similarity to recognized DNA binding motifs. Information about vertebrate Brachyury and its role in mesoderm formation is found at the Brachyenteron (T-related gene site). The T-box family has a slightly greater sequence affinity to Drosophila Optomotor blind than to Drosophila Brachyenteron and consequently information about the T-box family is found in the Omb site.

Brachyury in invertebrates

Podocoryne carnea is a typical representative of the class Hydrozoa (jellyfish). With few exceptions, hydrozoans are marine and exhibit a life cycle that consists of the free swimming planula larva, the sessile polyp and the sexual stage, the medusam which is formed from polyps through budding. In some hydrozoans, the medusa generation has become secondarily reduced. It is generally assumed that all cnidarians are formed of an outer and an inner layer of multifunctional myoepithelial cells. The other cell types, interstitial cells, nerve cells or nematocytes are interspersed in either of the two layers. Therefore, Cnidaria are classified as diploblasts or simple bilayered animals. While this classification accurately describes the basic tissue organization of the planula larva and the polyp, the anatomy of the medusa is more complex. Most of the differences are found in the medusa bell, which not only can carry complicated sense organs such as lens eyes, statocysts, and nerve rings but also consists of two nonmyoepithelial cell layers and additionally a third layer of epithelial mononucleated striated muscle cells. In the entire phylum, the planula larva and the polyp lack these medusa-specific cell types and sense organs (Spring, 2002 and references therein).

In bilaterians, the striated and smooth muscle tissues are in general a derivative of the third or middle germ layer, the mesoderm. In the hydrozoan jellyfish, the striated muscle is a derivative of the entocodon, a tissue layer that separates from the ectodermal layer early in medusa development. The entocodon is located between the distal ectodermal and the endodermal tissue, and is separated from both layers by an extracellular matrix. Entocodon cells in early bud stages are embryonic in appearance and highly proliferative. Later the entocodon forms a cavity, which finally connects to the outside by the developing velar opening. In older bud stages mitotic activity in the bell gradually stops and the outer wall of the entocodon differentiates into the striated muscle while the inner wall forms the smooth muscle of the feeding and sex organ of the animal (Spring, 2002 and references therein).

The histology and developmental pattern of muscle formation in medusa development has led to the idea that the entocodon could be a mesoderm-like layer. Striated myofilaments are usually found in cells derived from the mesoderm, however, exceptions are known. Tentacles of entoprocts contain flagellated ectodermal epithelia that contain striated myofilaments. This indicates that the appropriate structural genes can be activated independent of the germ layer and that the analysis of the striated muscle-specific structural genes alone would not be sufficient. The molecular analysis of muscle development in Podocoryne has demonstrated that the structural genes for a tropomyosin and a myosin heavy chain are structurally and functionally conserved and specific for the striated muscle tissue. Furthermore, the presence of the homeobox gene Otx, a head and gastrulation regulator in bilaterians, in jellyfish striated muscle and the basic helix-loop-helix (bHLH) factor Twist during the formation of the entocodon in medusa development, indicates that genes with specific roles in mesoderm patterning of bilaterians are already present in the common ancestor with bilaterians. Next to homeodomain and bHLH transcription factors, the best-studied regulatory genes are members of the T-box, MADS-box and zinc finger families, such as Brachyury, Mef2, and Snail, respectively. These three gene families are involved at different levels in the specification of the mesodermal and myogenic lineage of bilaterian animals from Drosophila to vertebrates (Spring, 2002 and references therein).

To investigate the hypothesis that the entocodon of jellyfish is homologous to the mesoderm of bilaterians, a Podocoryne homolog of each of the three gene families was isolated and structure and expression patterns were studied throughout the life cycle and specifically during muscle development. The results demonstrate that all three genes are expressed during myogenic differentiation. Additionally, as is true for their bilataterian cognates, they appear to have other functions as well. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. The data further strengthen the hypothesis that the common ancestor of cnidarians and bilaterians already used the same regulatory and structural genes and comparable developmental patterns to build muscle systems (Spring, 2002).

Homologues of the T-box gene Brachyury play important roles in mesoderm differentiation and other aspects of early development in all bilaterians. In the diploblast Hydra, the Brachyury homologue HyBra1 acts early in the formation of the hypostome, the location of the organiser in adult Hydra. This study reports the isolation and characterisation of a second Brachyury gene, HyBra2. Sequence analysis suggests that HyBra1 and HyBra2 are paralogues, resulting from an ancient lineage-specific gene duplication. Both paralogues acquired novel functions, both at the level of their cis-regulation as well as through significant divergence of the coding sequence. Both genes are expressed in the hypostome, but HyBra1 is predominantly endodermal, whereas HyBra2 transcripts are found primarily in the ectoderm. During bud formation, both genes are activated before any sign of evagination, suggesting an early role in head formation. During regeneration, HyBra1 is an immediate-early response gene and is insensitive to protein synthesis inhibition, whereas the onset of expression of HyBra2 is delayed and requires protein synthesis. The functional consequence of HyBra1/2 protein divergence on cell fate decisions was tested in Xenopus. HyBra1 induces mesoderm, like vertebrate Brachyury proteins. By contrast, HyBra2 shows a strong cement-gland and neural-inducing activity. Domain-swapping experiments show that the C-terminal domain of HyBra2 is responsible for this specific phenotype. These data support the concept of sub- and neofunctionalisation upon gene duplication and show that divergence of cis-regulation and coding sequence in paralogues can lead to dramatic changes in structure and function (Bielen, 2007).

C. elegans mab-9 mutants are defective in hindgut and male tail development because of cell fate transformations in two posterior blast cells, B and F. mab-9 has been cloned and is shown to encode a member of the T-box family of transcriptional regulators. T-box genes are characterized by having a large DNA-binding domain of ~200 amino acids, the T-box domain, which is highly conserved between T-box genes from different species. Outside the T-box domain there is very little sequence homology. Analysis of C. elegans T-box genes reveals that they are mostly highly diverged from those described previously from vertebrates, arthropods and ascidians (phylum Urochordata). However, four C. elegans T-box genes, mab-9 (T27A1.6), F21H11.3, H14A12.4, and ZK328.6, are much more similar to T-box genes described from other organisms. The closest relatives of mab-9 are the human gene TBX20 (63% identity across the T-box region) and the Drosophila gene H15 (54% identity); no functional information is as yet available about these genes. The genomic structure of the T-box domain of many T-box genes is also highly conserved across a wide range of species and typically contains four introns, the first, third, and fourth of which tend to be at identical positions. This conservation of genomic structure suggests a common and ancient evolutionary origin. The intron/exon structure of the T-box domain of mab-9 shares this conserved pattern, whereas other more divergent C. elegans T-box genes lack one or more of them or have them at different positions. MAB-9 localizes to the nucleus of B and F and to their descendents during development, suggesting that it acts cell autonomously in the posterior hindgut to direct cell fate. T-box genes related to brachyury have also been implicated in hindgut patterning, and the results presented here support models for an evolutionarily ancient role for these genes in hindgut formation (Woollard, 2000).

Based on this conservation of primitive function it is proposed that mab-9 may be regarded as the C. elegans Brachyury ortholog. Consequently, it becomes useful to consider the phylogenetic relationship among nematodes, arthropods, and chordates. The classical phylogeny based on morphological criteria places nematodes as an outgroup taxon to both arthropods and chordates. This would fit well with the observation that mab-9 is expressed and functions in the posterior gut but not in mesoderm. Some cladistic and molecular phylogenetic analyses, however, challenge the view that nematodes branched off before the arthropod-vertebrate split, and this controversy remains unresolved. This study shows that mab-9 is expressed in two posterior hindgut cells, B and F, to distinguish these two cells from their anterior neighbors Y and U, respectively. It is noteworthy that B and Y straddle the rectum in a similar position just ventral to F and U. mab-9 therefore functions by distinguishing the posterior members of two distinct but neighboring cell pairs. Two other genes have been identified recently that are required for distinguishing fates within these four hindgut cells. These two genes, egl-38 and lin-48, are also required to make certain cells different from others: egl-38, a Pax type transcription factor, distinguishes F and U from B and Y, whereas lin-48 distinguishes U from B. At least two of these genes, mab-9 and egl-38, are transcription factors. The next challenge will be to identify targets of these fate-determining transcriptional controls to establish exactly how these genes might act in combination with each other to pattern the developing hindgut (Woollard, 2000).

Chordates are thought to have emerged from some common ancestor of deuterostomes by organizing shared anatomical and embryological features including a notochord, a dorsal nerve cord and pharyngeal gill slits. Because the notochord is the most prominent feature of chordates and because the Brachyury (T) gene is essential for notochord formation, the T gene is a key molecular probe with which to explore the origin and evolution of chordates. The sea urchin (echinoderm) T (HpTa) gene contains a long open reading frame that encodes a polypeptide of 434 amino acids. Although the overall degree of amino acid identity was not very high (52%, sea urchin/mouse), in the T domain of the N terminus the amino acid identity was 73% (sea urchin/mouse). The HpTa gene is present as a single copy per haploid genome. As with the chordate T gene, the expression of HpTa is transient, being first detected in the swimming blastula, maximally transcribed in the gastrula, decreasing at the prism larval stage and barely detectable at the pluteus larval stage. HpTa transcripts were found in the secondary mesenchyme founder cells, vegetal plate of the mesenchyme blastula, extending tip of the invaginating archenteron and, finally, the secondary mesenchyme cells at the late-gastrula stage (Harada, 1995).

Echinoderms, hemichordates and chordates are deuterostomes and share a number of developmental features. The Brachyury gene is responsible for formation of the notochord, the feature that most defines the chordate phylum and thus may be a key to understanding chordate origin and evolution. Previous studies have shown that the ascidian Brachyury (As-T and Ci-Bra) is expressed in the notochord and that a sea urchin Brachyury (HpTa) is expressed in the secondary mesenchyme founder cells. A hemichordate Brachyury (PfBra) is expressed in a novel pattern in an archenteron invagination region and a stomodeum invagination region in the gastrula. The present study demonstrates that the expression pattern of Brachyury (ApBra) of starfish embryos resembles that of PfBra in hemichordate embryos but not of HpTa in sea urchin embryos: namely, that ApBra is expressed in an archenteron invagination region and a stomodaeum invagination region (Shoguchi, 1999).

This work concerns the expression of two transcription factors during the development of the sea urchin Strongylocentrotus purpuratus: SpNot, the ortholog of the vertebrate Not gene, and SpBra, the ortholog of the vertebrate Brachyury gene are both expressed in the chordate notochord. It is of interest to see whether they are both expressed in a common structure in echinoderms. SpNot is a Hox gene distantly related to Drosophila empty spiracles, but more closely related to another Drosophila Hox gene, 90Bre, also termed E103, for which the literature is incomplete. SpNot transcripts are detected by in situ hybridization in the vegetal plate at the mesenchyme-blastula stage. Later the gene is expressed in the secondary mesenchyme, but expression is no longer detectable after gastrulation. SpNot is upregulated during larval development, in the invaginating vestibule of the adult rudiment. Transcripts are also found in several larva-specific tissues, including the epaulets, blastocoelar cells, and pigment cells. SpBra also displays a discontinuous pattern of expression. Much like SpNot, this gene is expressed during embryogenesis in the embryonic vegetal plate and secondary mesenchyme founder cells, and expression is then extinguished. The gene is upregulated over a week later in the feeding larva, in the vestibule of the adult rudiment. In contrast to SpNot, SpBra is also expressed in the mesoderm of both left and right hydrocoels, and it is not expressed in any larva-specific tissues (Peterson, 1999b).

The spatial expression profile determined in this study is compared with that of the orthologous Brachyury gene in an indirectly developing enteropneust hemichordate, a representative of the sister group to the echinoderms within the deuterostomes. Both SpNot and SpBra are utilized discontinuously, displaying unconnected embryonic and larval phases of expression. Moreover, the expression of these two genes demonstrated in the vertebrate dorsal mesoderm is not found in the development of the echinoderm adult body plan. Thus, SpNot is not expressed at all in the coelomic mesoderm, but only in the invaginating vestibule of the rudiment. It is also expressed in various mesenchymal cells and larva-specific ectodermal derivatives. Expression of SpBra in larval stages occurs only in the mesodermal hydrocoel and the vestibule, i.e., in the major anlagen from which the echinoid body plan develops. Because Brachyury and Not are both expressed in the chordate notochord, precursors of the notochord were sought in the sea urchin, but none were found and it was concluded they do not exist in this species. The notochord is best considered a structure unique to chordates, and it would then follow that in the early evolution of chordates, these two genes were recruited for the specification of the notochord. The only overlapping expression domains of SpBra and SpNot in the larva are in the vestibule, a nonmesodermal structure exclusive to the euechinoids (Peterson, 1999b).

These observations illuminate the genetic basis underlying the process of maximal indirect development in basal deuterostomes. Maximal indirect development requires the construction of two very different organisms, a relatively simple embryo/larva and a complex adult, which are formed by quantitively different modes of development. The embryo/larva is formulated by specification processes that proceed immediately to the institution of differentiation programs and the generation of cell types, beginning even in cleavage. Brachyury appears to be an excellent marker for the progeny of the set-aside cells of the sea urchin embryo. Patches of embryonic cells are set aside from the immediate specification-to-differentiation process of the embryo. Postembryonic development of the adult body plan can be considered to be initiated by genes encoding such pattern-forming transcription factors at the regulatory level, activated within the larval progeny of the set-aside cells (Peterson, 1999b).

The deuterostomes comprise three monophyletic phyla: the echinoderms, the chordates and a third, lesser-known group: the hemichordates. Hemichordates consist of two subgroups: pterobrances and acorn worms or enteropneusts. Echinoderms and hemichordates are considered to be each other's closest relatives (i.e., they are sister taxa). This work concerns the formation of mesoderm in the development of an enteropneust hemichordate, Ptychodera flava, and the expression of the Brachyury gene during this process. Both enteropneusts and echinoderms undergo what can be termed 'maximal indirect development', whereby an elegant but simple larval stage precedes the development of the adult body plan. The adult body plan of maximal indirect developers arises from groups of cells that are 'set-aside' from participation in the construction of the embryo/larva. In enteropneusts and echinoderms, endomesodermal set-aside cells produce the tripartite coeloms from which the mesodermal components of the adult body plan arise. Brachyury expression occurs in two distinct phases. In the embryo, Brachyury is transcribed during gastrulation in the future oral and anal regions of the gut, but transcripts are no longer detected by 2 weeks of development. Brachyury expression is not detected during the 5 months of larval planktonic existence. During this time, the adult coeloms begin to develop, originating as coalescences of cells that appear to delaminate from the wall of the gut. Brachyury expression cannot be detected again until metamorphosis, when transcripts appear in the mesoderm of the adult proboscis, collar and the very posterior region of the trunk. It is also expressed in the posterior end of the gut. At no time is Brachyury expressed in the stomochord, the putative homolog of the chordate notochord. These observations illuminate the process of maximal indirect development in Ptychodera and, by comparison with patterns of Brachyury expression in the indirect development of echinoderms, their sister group, they reveal the evolutionary history of Brachyury utilization in deuterostomes (Peterson, 1999a).

Brachyury is not expressed in the mesoderm of any arthropod so far examned. However Brachyury is evidently recruited for mesoderm specification at the base of the deuterostomes. It is hypothesized that the expression seen in the trunk mesoderm of developing vertebrate embryos is homologous to the expression described here in the posterior metacoel (trunk mesoderm) of enteropneusts. The coincident expression in gut and mesoderm suggests an inductive relationship between the two. Hence, the link between expression in posterior mesoderm and gut may be a phylogenetically old but intimate one. Within the deuterostomes each major clad recuited Brachyury for the specification of novel mesodermal structures: Chordates recruited Brachyury for the specification of the notochord; the latest common ancestor of echinoderms and enteropneusis recruited Brachyury for specificaion of the mesoderm of the middle coelom or mesocoel (the hydrocoel in echinoderms). In echinoderms Brachyury is expressed in secondary embryonic mesenchyme. Euechinoids express Brachyury in a structure that is unique with respect to other echinoderms, viz. the larval vestibule, which provides the ectodermal component of the adult rudiment, including the anlagen for the adult body wall epithelium and the central nervous system. Enteropneusts display a unique pattern of expression in the oral region of the embryonic gut and in the mesoderm of the adult protosome. The protosome of hemichordates is very muscular and is the primary propulsive organ of the adult. On the contrary, the homolog in echinoderms plays no role in locomotion and functions solely as an excretory organ (Peterson, 1999a).

Studies have sought to identify and characterize genes involved in the gastrulation and morphogenetic movements that occur during sea urchin embryogenesis. An ortholog of the T-box family transcription factor, Brachyury, was cloned through a candidate gene approach. Brachyury (T) is the founding member of this T-box transcription factor family and has been implicated in gastrulation movements in Xenopus, zebrafish, and mouse embryogenesis. Polyclonal serum was generated to LvBrac in order to characterize protein expression. LvBrac initially appears at mesenchyme blastula stage in two distinct regions with embryonic expression perduring until pluteus stage. Vegetally, LvBrac expression is in endoderm and lies circumferentially around the blastopore. This torus-shaped area of LvBrac expression remains constant in size as endoderm cells express LvBrac upon moving into that circumference and cease LvBrac expression as they leave the circumference. Vegetal expression remains around the anus through pluteus stage. The second domain of LvBrac expression first appears broadly in the oral ectoderm at mesenchyme blastula stage and at later, at embryonic stages, it is refined to just the stomodael opening. Vegetal LvBrac expression depends on autonomous ß-catenin signaling in macromeres and does not require micromere or veg2-inductive signals. LvBrac is necessary for the morphogenetic movements occurring in both expression regions. A dominant-interfering construct was generated by fusing the DNA binding domain of LvBrac to the transcriptional repression module of the Drosophila Engrailed gene in order to perturb gene function. Microinjection of mRNA encoding this LvBrac-EN construct resulted in a block in gastrulation movements but not expression of endoderm and mesoderm marker genes. Furthermore, injection of LvBrac-EN into one of two blastomeres results in normal gastrulation movements of tissues derived from the injected blastomere, indicating that LvBrac downstream function may be nonautonomous during sea urchin gastrulation (Gross, 2001).

A Brachyury ortholog (PvuBra) in the marine gastropod Patella vulgata is reported. In this mollusc, the embryo displays an equal cleavage pattern until the 32-cell stage. There, an inductive event takes place that sets up the bilateral symmetry, by specifying one of the four initially equipotent vegetal macromeres as the posterior pole of all subsequent morphogenesis. This macromere, usually designated as 3D, will subsequently act as an organizer. The 3D macromere expresses PvuBra as soon as its fate is determined. 3D determination and activity also involve the activation of the MAP kinase ERK, and PvuBra expression in 3D requires ERK activity. PvuBra expression then rapidly spreads to neighboring cells that cleave in a bilateral fashion and whose progeny will constitute the posterior edge of the blastopore during gastrulation, suggesting a role for PvuBra in regulating cell movements and cleavage morphology in Patella. Until the completion of gastrulation, PvuBra expression is maintained at the posterior pole, and along the developing anterior-posterior axis. Comparing this expression pattern with what is known in other bilateria, it is suggested that Brachyury might have a conserved role in the regulation of anterior-posterior patterning among Bilateria, through the maintenance of a posterior growth zone, suggesting that a teloblastic mode of axis formation might be ancestral to the Bilateria (Lartillot, 2002).

A striking aspect of the PvuBra expression profile is that it illuminates the progressive growth of the AP axis during gastrulation in Patella. PvuBra is expressed in 3D, and then in 2d2 which, according to the prevailing interpretation, behaves like a stem cell, budding off the cells of the ventral midline. This interpretation is mostly based on the assumption of cellular continuity of PvuBra expression, and awaits further experimental confirmation. More generally, posterior stem cells, or teloblasts, are seen in other spiralians, such as annelids, where they give rise to most of the ectoderm and the mesoderm of the trunk. Significantly, these teloblasts are derived from the D quadrant exclusively. Thus, in spiralians, the D quadrant seems to play a conserved role in AP axis formation, in that it gives birth to the posterior growth zone. The results obtained in Patella underline the teloblastic mode of AP axis formation that also prevails in molluscs and, more importantly, suggest that Brachyury might be involved in the developmental activity of the posterior pole (Lartillot, 2002).

On a broader scale, it appears that the progressive growth of the AP axis, in an anterior to posterior sequence, and through the activity of a posterior growth zone, is observed in diverse phyla across Bilateria as, for instance, in short-germ insects, polychaetes and chordates. In vertebrates, the posterior pole of the developing axis is the organizer, in the form of the superior lip of the blastopore of fish and frog, or the node of amniotes, and later on, the chordo-neural hinge of the tail bud. Brachyury is expressed in the vertebrate organizer during the major part of embryonic development, and genetic studies in mouse and fish show that this expression is necessary for AP axis formation. Similarly, in Patella, as soon as the four-fold symmetry has been broken, PvuBra is a expressed in the D quadrant, and remains a marker of the posterior pole of the AP axis up to the end of larval development, until the whole axis is laid down. This striking similarity is in favour of the hypothesis that the progressive growth of the AP axis represents a conserved developmental process which, already in Urbilateria (the last common ancestor of insects and vertebrates), would have taken the form of an organizing posterior growth zone, controlled by a genetic system involving Brachyury, among others. This posterior pole would have evolved into the organizer of vertebrates, and into the set of teloblasts in Spiralia (Lartillot, 2002).

Brachyury is a transcription factor that functions in gastrulation and endoderm development throughout the Bilateria. Genes that are expressed downstream of brachyury during gastrulation of the sea urchin embryo are identified in this study. Screens with two different complex probes generated by subtractive hybridization were carried out on high-density arrays of embryonic cDNA libraries. An mRNA sequence population from embryos expressing brachyury at its peak stage of expression was subtracted with message sequence from embryos in which Brachyury function had been 'knocked-out' by injection of a morpholine-substituted antisense oligonucleotide was used to generate a differential probe for brachyury target genes. Another probe was made by using an mRNA population from embryos that mis-express brachyury at a stage just prior to the normal onset of expression, subtracted with message sequence taken from normal embryos at this stage. Screens carried out with these probes target overlapping but distinct sets of downstream genes. After partial sequence characterization, promising genes were independently analyzed by quantitative real-time PCR and by in situ hybridization. Two major classes of genes emerge in this study: genes expressed in the subset of the secondary mesenchyme cells (SMC) that will become pigment cells, and genes that are expressed in portions of the endoderm coincident with brachyury expression. The latter genes are candidates for direct transcriptional targets of Brachyury. Some of the endodermal genes that respond to Brachyury are cytoskeletal modulators that may play a role in gut morphogenesis. This finding is consistent with the block in gastrulation induced by interfering with Brachyury function in sea urchins, and with known or suggested Brachyury function in other species. Other endodermal target genes are expressed in the archenteron and might be terminal differentiation enzymes of the gut. Brachyury expression patterns for Strongylocentrotus purpuratus reported in this paper are entirely consistent with data from other echinoderm species. Brachyury expression in the vegetal plate is confined to the presumptive endodermal cells. Therefore, the SMC genes are likely to be indirect targets of Brachyury-induced signaling from the surrounding endoderm to the central mesoderm, or the effects on these genes may be indirect consequences of gross disruption of the vegetal plate. These results and other data suggest that the brachyury gene transduces information about the state of endodermal specification to genes that modulate morphogenesis and genes that perform terminal functions in the gut (Rast, 2002).

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

The mesoderm-forming gene brachyury regulates ectoderm-endoderm demarcation in the coral Acropora digitifera

Blastoporal expression of the T-box transcription factor gene brachyury (see Drosophila Brachyenteron) is conserved in most metazoans. Cnidarians are basal metazoans that are important for understanding evolution of metazoan body plans. Because they lack mesoderm, they have been used to investigate the evolutionary origins of mesoderm. This study focused on corals, a primitive clade of cnidarians that diverged from sea anemones approximately 500 mya. A microinjection method for coral eggs to was developed to examine Brachyury functions during embryogenesis of the scleractinian coral, Acropora digitifera. Because Acropora embryos undergo pharynx formation after the blastopore closes completely, they are useful to understand Brachyury functions in gastrulation movement and pharynx formation. Blastoporal expression of brachyury is directly activated by Wnt/β-catenin signaling (see Drosophila Wingless pathway) in the ectoderm of coral embryos, indicating that the regulatory axis from Wnt/β-catenin signaling to brachyury is highly conserved among eumetazoans. Loss-of-function analysis demonstrated that Brachyury is required for pharynx formation but not for gastrulation movement. Genome-wide transcriptome analysis demonstrated that genes positively regulated by Brachyury are expressed in the ectoderm of Acropora gastrulae, while negatively regulated genes are in endoderm. Therefore, germ layer demarcation around the blastopore appears to be the evolutionarily conserved role of Brachyury during gastrulation. Compared with Brachyury functions in vertebrate mesoderm-ectoderm and mesoderm-endoderm demarcation, the results suggest that the vertebrate-type mesoderm may have originated from brachyury-expressing ectoderm adjacent to endoderm (Yasuoka, 2016).

Evolutionary conservation of Brachyury structure and sequence

Orthologs of Brachyury, a subfamily of T-box transcription factors, specify distinct cell types in different metazoan phyla, suggesting that the function of these genes has changed through the course of evolution. To investigate this evolutionary process, the activities of Brachyury orthologues from all major phyla have been compared in a single cellular context, the pluripotent Xenopus laevis animal cap. In this assay, an ancestral function is revealed: most orthologs, including the Hydra protein, mimic the action of endogenous Xenopus Brachyury, in that they induce mesoderm but not endoderm. Orthologs from Drosophila and ascidians, however, display an additional derived property, represented in this assay by the induction of endoderm. Misexpression of chimeric versions of Brachyury reveal that the C-terminal half of the protein is important for the strength of the induced response but not for its specificity. In contrast, amino acids located within the T-domain and in a short N-terminal peptide are involved in restricting the activity of Brachyury proteins to induction of mesoderm and not endoderm. Possession of this N-terminal motif correlates with early circumblastoporal expression of Brachyury orthologs. It is proposed that restriction of Brachyury activity by this motif plays a conserved role in the control of Bilaterian gastrulation (Marcellini, 2003).

Alignment of the short upstream N-terminal domain of Brachyury orthologs reveals a motif (KxxQxxxxHLLxAVxxEMxxGSEKGDPTER) that is conserved in most deuterostomes, lophotrochozoans, and the phylogenetically enigmatic chaetognaths, suggesting that these amino acids were present in Urbilateria, the common protostome deuterostome ancestor. Loss of this region correlates with the ability of Brachyury to induce endoderm in the animal cap assay: it is present neither in the outgroup member VegT, nor in ascidian or Drosophila Brachyury. Only HyBra1, which lacks this motif, yet is unable to induce endoderm, represents an exception to this observation (Marcellini, 2003).

To investigate the significance of this motif, three chimeras were constructed in which this small N-terminal domain was swapped between Xbra and As-T, thereby creating the constructs XAX, AXX, and XAA. All three constructs induced endoderm in Xenopus animal caps, revealing that the short N-terminal domain of As-T (AXX chimera) and its T-domain (XAX chimera) are both sufficient to confer endoderm-inducing activity to Xbra. These two domains thus harbor key determinants of the inducing specificity of Brachyury proteins. The failure of HyBra1 to induce endoderm in the absence of the N-terminal domain may be due to the characteristics of its T-domain (Marcellini, 2003).

Insects and tunicates have a derived mode of gastrulation. The formation of their blastopores is in large part independent of Brachyury: this gene is expressed in only a minority of blastoporal cells. This may have led to a reduction of the selective pressure on part of the activity of Brachyury and allowed loss of the N-terminal domain by the accumulation of independent mutations. The nature of these mutations is unlikely to have been dictated by strong constraints since the N termini of related species, such as Ciona intestinalis and Ciona savignyi, show very poor sequence similarity. Interestingly, proteins which possess the conserved N-terminal motif and the ability only to induce mesoderm in the Xenopus assay are able to mimic, in ascidians and Drosophila, the activity of endogenous Brachyury. Thus, loss of the N terminus probably reflects a loss of selective pressure on this motif rather than an obligate requirement for its disappearance (Marcellini, 2003).

Finally, the very similar target DNA sequence preferences of Xenopus VegT and Brachyury, coupled with the absence of amino acid changes at positions contacting DNA in endoderm-inducing proteins, suggests that the intrinsic DNA binding specificity of Brachyury orthologs is not a key determinant of their activity. Rather, it is more likely that most Bilaterian Brachyury proteins interact with cofactors via their N-terminal and T-domains which, directly or indirectly, confer inductive specificity in the animal cap assay. A similar situation has recently been shown for Tbx5, which interacts via its N terminus and T-domain with the homeodomain protein Nkx2.5 during vertebrate heart development. It is predicted that the cofactors of Brachyury will be evolutionarily conserved and that their identification will provide important insights into the core mechanisms of Bilaterian gastrulation (Marcellini, 2003).

In ascidian embryos, Brachyury is expressed exclusively in blastomeres of the notochord lineage and plays an essential role in the notochord cell differentiation. The genetic cascade leading to the transcriptional activation of Brachyury in A-line notochord cells of Ciona embryos begins with maternally provided ß-catenin, which is essential for endodermal cell specification. ß-catenin directly activates zygotic expression of a forkhead transcription factor gene, FoxD, at the 16-cell stage, which in turn somehow activates a zinc finger transcription factor gene, ZicL (Drosophila homolog: Odd-paired), at the 32-cell stage, and then Brachyury at the 64-cell stage. One of the key questions to be answered is whether ZicL functions as a direct activator of Brachyury transcription, and this was addressed in the present study. A fusion protein was constructed in which a zinc finger domain of Ciona ZicL was connected to the C-terminus of GST. Extensive series of PCR-assisted binding site selection assays and electrophoretic mobility shift assays demonstrated that the most plausible recognition sequence of Ciona ZicL was CCCGCTGTG. The elements CACAGCTGG (complementary sequence: CCAGCTGTG) at -123 and CCAGCTGTG at -168 bp upstream of the putative transcription start site of Ci-Bra are found in a previously identified basal enhancer of this gene. In vitro binding assays indicate that the ZicL fusion protein binds to these elements efficiently. A fusion gene construct in which lacZ was fused with the upstream sequence of Ci-Bra showed the reporter gene expression exclusively in notochord cells when the construct was introduced into fertilized eggs. In contrast, fusion constructs with mutated ZicL-binding-elements failed to show the reporter expression. In addition, suppression of Ci-ZicL abolished the reporter gene expression, while ectopic and/or overexpression of Ci-ZicL resulted in ectopic reporter expression in non-notochord cells. These results provide evidence that ZicL directly activates Brachyury, leading to specification and subsequent differentiation of notochord cells (Yagi, 2004).

The ability to associate with histone-modifying complexes is a conserved function for the T-box family

During cellular differentiation, both permissive and repressive epigenetic modifications must be negotiated to create cell-type-specific gene expression patterns. The T-box transcription factor family is important in numerous developmental systems ranging from embryogenesis to the differentiation of adult tissues. By analyzing point mutations in conserved sequences in the T-box DNA-binding domain, it was found that two overlapping, but physically separable regions are required for the physical and functional interaction with H3K27-demethylase and H3K4-methyltransferase activities. Importantly, the ability to associate with these histone-modifying complexes is a conserved function for the T-box family. These novel mechanisms for T-box-mediated epigenetic regulation are essential, because point mutations that disrupt these interactions are found in a diverse array of human developmental genetic diseases (Miller, 2009).

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

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

Table of contents

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

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