Gene name - brachyenteron
Synonyms - T-related gene, TRG, aproctous
Cytological map position - 68D/E
Function - T-box transcription factor
Keyword(s) - selector, gut ectoderm, hindgut
Symbol - byn
Genetic map position - 3-
Classification - Brachyury (T) homolog
Cellular location - nuclear
Trying to make evolutionary sense of the gastrulation process [Images], looking for a unity between insects and vertebrates with regard to the genes involved and the movements of tissues, simply does not work. Vertebrate Brachyury has a major role in differentiation of the notochord and in the formation of the mesendoderm. A similar role for Brachyenteron, or T-related gene, a Drosophila Brachyury homolog, is not found in insects. Brachyenteron is required for specification of hindgut and anal pads. A second Brachyury homolog, Optomotor blind shows anterior activity in the brain and central nervous system, again not functionally identical to Brachyury. Thus the fly has specialized Brachyury homologs for the anterior and posterior domains serving different functions from Brachyury in chordates (Kispert, 1994a and Murakami, 1995).
The formation of mesoderm and endoderm requires one gastrulation process in frogs and fish, while insects use separate processes for each type of tissue formed. In zebrafish, cells of the future mesoderm and endoderm involute beneath the margin of neighboring blastomeres, with separate fates for different involuting cells (Schulle-Merkes, 1994). In Xenopus there is mass movement through a single blastpore (Keller, 1992). In Drosophila however, mesoderm is formed by ventral invagination while endoderm is formed by invagination from separate anterior and posterior primordia.
The apparent unity of the gastrulation process in fish and frogs belies its complexity, and the complex origins of mesoderm and endoderm. Regardless of the divergent morphogeneic movements when comparing insects and vertebrates, the level of complexity remains high in either case. For example, in spite of the seemingly unitary origin of mesoderm, an examination of zebrafish goosecoid, coding for a homeodomain protein and no tail, the fish Brachyury homolog, reveals a complex origin: initially expressions overlap, but with time, these genes take on separate roles. goosecoid is expressed in a precoidal area while no tail is expressed in the presumptive mesoderm.
Whereas Xenopus and fish Brachyury homologs are involved in induction of mesoderm, it has been thought neither of the Drosophila Brachyury homologs has anything to do with this process (Schulte-Meeker, 1994). optomotor blind is expressed in neuronal and glial cells in the developing nervous systerm (Poeck, 1993), while Trg is expressed in the hindgut. Both mark the future site of involution, and continue to be expressed as the involution process proceeds (Kispert, 1994a and Murakami, 1995). Thus Trg appears to be involved in the development of the proctodeum, having no immediately apparent phylogenetic relationship to the notochord and optomotor blind shows neural expression, again far removed from a mesodermal function. Homologs of Trg are also expressed in the developing hindgut of Tribolium and Locusta embryos (see Tribolium early embryonic development) suggesting a highly conserved function of Trg in insects. This conservation and the high similarity of T and Trg raise the question of a common evolutionary origin of the hindgut of insects and the notochord of chordates (Kispert, 1994b).
This conundrum has largely disappeared in the face of an excellent study by Kusch (1999) on the origin of the caudal visceral mesoderm of Drosophila (CVM). The Drosophila Brachyury homolog brachyenteron (byn) is essential for the development of hindgut, anal pads and Malpighian tubules. byn is activated by the terminal gap gene tailless (tll) in a region of 0%-20% egg length of the syncytium (0% = posterior tip). With completion of cellularization, the byn expression becomes downregulated in the posteriormost cap of the embryo, which will later form the posterior midgut, by the terminal gap gene huckebein (hkb). Thus, the expression of byn is confined to a ring of cells from about 10%-20% egg length. The dorsal and the lateral aspects of that ring correspond to the proctodeum, from which the hindgut, the anal pads and the Malpighian tubules later develop. Intriguingly, hkb also determines the posterior extent of the ventral mesoderm primordium by repressing the mesodermal determinant snail (sna). This suggests that the ventralmost aspect of byn expression might comprise the posterior tip of the mesoderm primordium (Kusch, 1999).
The visceral musculature of the larval midgut of Drosophila has a lattice-type structure and consists of an inner stratum of circular fibers and an outer stratum of longitudinal fibers. The longitudinal fibers originate from the posterior tip of the mesoderm anlage, which has been termed the caudal visceral mesoderm. The CVM migrates in an orderly movement anteriorly and eventually forms an outer layer of longitudinal muscle fibers surrounding the midgut. The progenitors of a second tissue, the inner sheet of circular muscles of the midgut, are recruited from 11 parasegmentally arranged clusters of dorsal mesoderm in the trunk region and are therefore referred to as trunk visceral mesoderm (TVM) (Kusch, 1999).
In this study, the specification of the CVM has been investigated and particularly the role of the Drosophila Brachyury-homolog brachyenteron. Supported by fork head, brachyenteron mediates the early specification of the CVM along with zinc-finger homeodomain protein-1. This is the first function described for brachyenteron or fork head in the mesoderm of Drosophila. The mode of cooperation resembles the interaction of the Xenopus homologs Xbra and Pintallavis. Another function of brachyenteron is to establish the surface properties of the CVM cells; these properties are essential for orderly cell migration along the trunk-derived visceral mesoderm. During this movement, the CVM cells, under the control of brachyenteron, induce the formation of one muscle/pericardial precursor cell in each parasegment. It is here proposed that the functions of brachyenteron in mesodermal development of Drosophila are comparable to the roles of the vertebrate Brachyury genes during gastrulation (Kusch, 1999).
During germband retraction and midgut closure, the progenitors of the the outer, longitudinally oriented fibers of the visceral mesoderm, the CVM, perform an ordered movement that can be subdivided into three phases. The first migratory phase starts at early germband retraction when the cells begin to move anteriorly from their position at the posterior tip of the mesodermal germ layer and split into two tightly packed, bilaterally symmetrical clusters on each side of the posterior midgut primordium. When these clusters have reached the anterior tip of the posterior midgut primordium, the cells detach from each other and disperse anteriorly as two rows along the germband, the second phase of the migration. During this movement, the cells are arranged along the dorsal and ventral edge of the midgut primordia and are in close contact with the band of progenitors of the circular muscle fibers. The band seems to serve as a migration substratum. During the last phase of the migration, which takes place as the midgut encloses the yolk, the progenitors of the longitudinal muscle fibers spread regularly over the underlying circular muscle fibers. The cells acquire a spindle shape, then stretch in an anteroposterior direction and form about 16-20 regularly spaced longitudinal muscle fibers. These fibers reach from the proventriculus to the midgut-hindgut transition where the ureters of the Malpighian tubules insert. The foregut and the hindgut lack any longitudinal muscles and are solely covered by the inner layer of circular muscles (Kusch, 1999).
The specification of the CVM and its fate were monitored by the detection of Byn protein or the expression of CVM-specific markers like croc-lacZ and cpo-lacZ. The initial byn expression at the posterior pole is regulated by tll and hkb. Thus it is likely that the CVM cells are specified under the control of the same genes. In fact, in hkb embryos, the size of the CVM primordium is enlarged and comprises more cells than normal. This corroborates the notion that the CVM primordium constitutes the most posteriorly located mesoderm primordium. tll expression reaches more anteriorly than the hkb domain and encompasses the primordia of the proctodeum and of the CVM. One would therefore expect that the formation of the CVM is entirely dependent on tll. Indeed, this is the case: the CVM is missing in tll mutant embryos. Part of the function of tll seems to be mediated by byn. In byn mutants, a significantly reduced number of CVM cells is seen, and these few cells form clusters that are less compact and migrate significantly slower than in wild type. Later, they fail to contact the TVM and do not distribute along the germband. During stage 11, most of the cells acquire a condensed appearance resembling apoptotic bodies. A high level of apoptosis is detected in the proctodeum of byn embryos as well as in the posteriormost mesoderm. By stage 13, cells with the properties of the CVM are not detectable any longer in the mutants and, as expected from this, the dissected midguts of byn embryos lack the outer, longitudinal muscle fibers (Kusch, 1999).
byn embryos show morphological aberrations at a time before the CVM begins to migrate anteriorly. The severely shortened hindgut causes a significant shift in the spatial relationship of the various primordia at the posterior region of the embryo and thereby might indirectly affect the migration of the CVM. In order to exclude such an indirect influence, byn embryos were generated that expressed byn in the CVM precursors, but not in the hindgut. In such embryos, the CVM survives and disperses virtually the same as in wild type along the TVM, whereas the proctodeum remains rudimentary as in ordinary byn mutants. These results demonstrate that the defective migration and the death of the CVM cannot be attributed to the disordered morphology of the posterior gut structures. It has therefore been concluded that byn in the mesoderm is essential for the adhesive and migratory properties of the CVM precursors. byn cannot be the only gene that mediates the function of tll in the specification and further development of the CVM since the lack of tll causes a far stronger phenotype than the lack of byn. In addition to byn, the gene fkh is known to act downstream of tll in the posterior gut. fkh is expressed in a large domain at the posterior pole that encompasses the byn expression domain including the ventral, mesodermal aspect. In fkh mutants, the CVM specification seems less impaired than in byn mutants: the number of CVM cells is initially quite normal. However, as in byn mutants, the cells fail to migrate along the germband although differentiation of the migration substratum, the TVM, is not affected. By stage 14, most of the CVM cells have been eliminated by apoptosis. On this level of analysis, fkh mutants resemble embryos homozygous for weak byn alleles. However, the phenotype of byn fkh double mutants shows that byn and fkh either have distinct functions in the specification of the CVM or act synergistically. In double mutants, no CVM cells are distinguishable, just as in tll mutants. Therefore, the function of tll in the specification of the CVM appears to be mediated by byn and fkh (Kusch, 1999).
Only the anterior and the posterior mesoderm are competent to be specified by byn as CVM, in conjunction with fkh. Therefore, at least one other gene must exist that confines the competence to form CVM to these two regions. A good candidate for this gene is zinc finger homeodomain protein-1 (zfh-1). At the blastoderm stage, zfh-1 is expressed in high levels in the terminal regions of the mesoderm including the primordium of the CVM. zfh-1 is essential for the migration of the CVM: in zfh-1 mutant embryos, CVM-specific gene expression such as croc-lacZ is deleted. From the restricted effects of ectopic byn /fkh, it has been proposed that the two genes are capable of specifying CVM development only in the region of high zfh-1 expression. zfh-1, byn and fkh act in parallel downstream of tll. High levels of caudal zfh-1, as with byn and fkh, are dependent on tll, and there is no crossregulation between zfh-1, byn and fkh (Kusch, 1999).
The caudal visceral mesoderm is specified independent of twist function. The overlapping expressions of the two zygotic genes twi and snail (sna) are essential for gastrulation and specification of the mesoderm. twi is an activator of mesodermal gene expression, whereas sna mainly functions as a repressor of neuroectodermal gene expression in the mesoderm. Deviating from this rule, mesodermal zfh-1 expression is missing in sna mutants, whereas high zfh-1 expression is unaffected in the termini of the mesoderm in twi mutants. The expression of byn and posterior fkh does not depend on sna or twi function, raising the question whether the CVM might be at least partially specified in twi or sna mutants. In sna embryos, no byn-expressing cells can be detected in a mesoderm-specific position, i.e. between the epidermis and the midgut epithelium. Such embryos lack CVM-specific gene expression, which is in accordance with the findings that zfh-1 depends on sna and CVM development on zfh-1. Strikingly, in twi mutants, byn-expressing cells can be found at an internal, mesoderm-typical position in the tail region. It is not clear how the cells find their way into the twi embryos, which are characterized by the failure to form a ventral furrow. The cells probably immigrate after they have been internalized together with the adjoining posterior gut anlagen. Later the cells begin to express CVM-specific marker genes and undergo the first phase of the normal migration movement: they arrange as two clusters ventrolaterally to the posterior midgut. The cells initiate the second phase of migration as well; they become migratory, disperse and acquire the typical spindle shape of normal CVM cells. However, most likely because of the absence of their putative migration substratum, the TVM, they merely spread over the posterior midgut in twi embryos. Thus, the CVM is not only internalized during gastrulation independent of twi function, but also acquires at least some of its adhesive and migratory properties. This view is consistent with the finding that the expressions of byn, fkh and zfh-1, which are required for the specification of the CVM, are not affected in twi mutants (Kusch, 1999).
The defects in the CVM of a byn mutant suggest that byn is not only involved in the early specification of the CVM, but also plays an essential role in establishing the adhesive and migratory properties of the CVM cells. These do not properly form the two bilaterally symmetrical clusters and later fail to disperse along the TVM in byn mutants. Moreover, the ubiquituous mesodermal byn expression strongly affects the normal migratory behaviour of the CVM. The cells do not exclusively migrate along the TVM towards the anterior, but attach to any other cell in the mesoderm. As a consequence, migration is not restricted dorsoventrally and the cells do not reach the anterior half of the midgut. In these experimental embryos, it is impossible to physically separate splanchopleura and somatopleura, since they are firmly attached to each other. Normally this is not the case. It is concluded from these observations that ectopic byn expression changes the adhesive properties of the mesoderm. The specific effects of ectopic byn on the surface properties of other mesoderm cells also include the rescue of the germ cell migration defect of byn mutants. Normally, the germ cells pass through the posterior midgut epithelium as it becomes mesenchymal, prior to germband retraction. From the basal side of the endoderm, the germ cells migrate to the adjoining mesoderm of the body wall. Later, they migrate anteriorly and intermingle with the somatic gonadal mesoderm. It has been suggested that the CVM plays an important role in directing or facilitating this transition of the germ cells to the mesoderm. During the first phase of migration, the CVM cell clusters are located at a position where the germ cells pass through the epithelium of the posterior midgut before they migrate to the gonadal mesoderm. In byn mutants, the germ cells pass through the midgut epithelium as in wild type, but virtually all cells distribute over the ventral surface of the posterior midgut rather than contacting the mesoderm. byn is neither expressed in the germ cells nor in the gonadal mesoderm and the latter develops normally in byn mutants. Therefore, the observed phenotype is most likely due to defects in a byn-dependent signaling or the adhesive properties of the CVM. This idea is supported by the finding that ubiquitous expression of byn in the mesoderm rescues the defective germ cell migration in byn mutants almost completely: only a few germ cells do not coalesce with the somatic gonadal precursors. Thus, either the few rescued CVM cells of byn embryos at the normal position (close to the migrating germ cells) or the adhesive or signaling properties that ectopic byn confers to other mesodermal cells, are sufficient for the transition of the germ cells from the midgut to the gonadal mesoderm (Kusch, 1999).
A dynamic functional evolutionary diversification has recently been proposed for the Brachyury genes. The Brachyury proteins are a subfamily of the T-domain transcription factors, which are classified by a highly conserved DNA-binding domain. Brachyury relatives have been found in the diploblastic Cnidarian as well as triploblasts like chordates, protochordates, echinoderms, hemichordates and insects, suggesting that evolutionarily conserved mechanisms are regulated by these factors. However, initial comparisons of the expression and function of Brachyury genes between deuterostomes and protostomes indicate diverse rather than common functions. In deuterostomes, Brachyury seemed to play a critical role in mesoderm development, whereas the protostome Brachyury proteins have been shown to be involved in hindgut development. For instance, in vertebrates, Brachyury is transiently expressed in all nascent mesendodermal cells. As development proceeds, the expression becomes restricted to the notochord and tailbud. In cephalochordates and urochordates, the distinct aspects of mesodermal expression have been divided between two paralogs, one expressed in the posterior mesoderm and the other expressed in the notochord, whereas no expression is detectable in the gut. In echinoderms, Brachyury has only been reported to be expressed in the migrating secondary mesenchyme cells. However, it has recently been shown that the hemichordate Brachyury homolog is not only expressed in the mesoderm but also in the oral and anal region of the gut. Since Brachyury is transiently expressed in vertebrates in the developing hindgut epithelium of the tailbud region, it has been proposed that the expression of Brachyury in the posterior gut reflects its original function in development. Intriguingly, the Hydra Brachyury gene is expressed in the cells of the gut opening. In the light of the findings reported in this paper, it is concluded that the role of byn in the mesoderm of Drosophila is reminiscent of and likely to be homologous to the proposed mesodermal functions of vertebrate Brachyury genes (Kusch, 1999).
Base pairs in 5' UTR - 336
Base pairs in 3' UTR - 311
TRG has a stretch of 200 amino acids with extensive homology to chordate T (Brachyury) gene products from mouse, Xenopus, zebrafish and ascidians (phylum Urochordataq). Drosophila Optomotor-blind also contains a T-domain, but OMB T-domain is divergent from that of the other species in that it contains insertions. Functional homology of Drosophila Trg with Brachyury is not apparent. Whereas Brachyury has a major role in differentiation of the notochord and in the formation of the mesendoderm, a similar role is not found in insects (Kispert, 1994a).
The mouse Brachyury (T) gene is the prototype of a growing family of so-called T-box genes that encode transcriptional regulators and have been identified in a variety of invertebrates and vertebrates, including humans. Mutations in Brachyury and other T-box genes result in drastic embryonic phenotypes, indicating that T-box gene products are essential in tissue specification, morphogenesis and organogenesis. The T-box encodes a DNA-binding domain of about 180 amino-acid residues: the T domain. The X-ray structure of the T domain from Xenopus laevis was examined in complex with a 24-nucleotide palindromic DNA duplex. The protein is bound as a dimer, interacting with the major and the minor grooves of the DNA. A new type of specific DNA contact is seen, in which a carboxy-terminal helix is deeply embedded into an enlarged minor groove without bending the DNA. Hydrophobic interactions and an unusual main-chain carbonyl contact to a guanine account for sequence-specific recognition in the minor groove by this helix. Thus the structure of this T domain complex with DNA reveals a new way in which a protein can recognize DNA (Muller, 1997).
date revised: 30 October 99
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