Interactive Fly, Drosophila

There is no obvious TATA box, although there is an AT rich region between positions -40 to -28 (Thisse, 1988). A comparison of the transcriptional regulatory regions reveals a high degree of conservation in the more distal of the two ventral activator regions that have been mapped in the twist 5' flanking region. However, the more proximal VAR is absent at the corresponding position in the D. virilis twist gene. Instead, there is a region in the second intron of the D. virilis gene that resembles the proximal element of the D. melanogaster gene, in that it consists of little more than a series of whole and half binding sites for the Dorsal morphogen. In transformation experiments, the intronic D. virilis element directs an expression pattern that is indistinguishable from that directed by the D. melanogaster proximal VAR. Thus, the twi genes from these two species appear to have evolved enhancer elements with very similar structural and functional properties (Pan, 1994).

Transcriptional Regulation

Four zygotic patterning genes, decapentaplegic (dpp), zerknüllt (zen), twist , and snail are initially expressed either dorsally or ventrally in the segmented region of the embryo, and at the poles. In the segmented region of the embryo, correct expression of these genes depends on cues from the maternal morphogen Dorsal (DL). The DL gradient appears to be interpreted on three levels: dorsal cells express dpp and zen, but not twi and sna; lateral cells lack expression of all four genes; ventral cells express twi and sna, but not dpp and zen. DL appears to activate the expression of twi and sna and repress the expression of dpp and zen. Polar expression of dpp and zen requires the terminal system to override the repression by DL, while that of twi and sna requires the terminal system to augment activation by DL (Ray, 1991).

Dorsal activates twist, and it also functions as a direct transcriptional repressor of a second target gene, zerknüllt. By exchanging DL binding sites between the promoters it can be shown that activator sites from the twi promoter can mediate repression when placed in the context of the zen promoter, and that repressor sites from zen can mediate activation in the context of the twi promoter. This represents the first demonstration that common binding sites for any DNA binding protein can mediate both activation and repression in a developing embryo (Jiang, 1992).

Although CREB-binding protein (CBP: nejire) functions as a co-activator of many transcription factors, relatively little is known about the physiological role of CBP. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haplo-insufficiency disorder characterized by abnormal pattern formation. Drosophila CBP is maternally expressed, suggesting that it plays a role in early embryogenesis. Mesoderm formation is one of the most important events during early embryogenesis. To initiate the differentiation of the mesoderm in Drosophila, multiple zygotic genes such as twist (twi) and snail (sna), which encode a basic-helix-loop-helix and a zinc finger transcription factor, respectively, are required. The transcription of these genes is induced by maternal Dorsal protein, a transcription factor that is homologous to the NF-kappa B family of proteins. Drosophila CBP mutants fail to express twi and generate twisted embryos. This is explained by results showing that dCBP is necessary for Dorsal-mediated activation of the twi promoter (Akimaru, 1997).

Subsets of differentiating muscles in the Drosophila embryo express transcription factors, such as NK1/S59 and vestigial. These genes control the development of specific muscle properties. Myogenesis in embryos mutant for wingless is grossly deranged. Mesodermal expression of S59 is lost, whereas some vestigial-expressing muscles develop. wingless dependence and independence of specific muscle subsets correlates with an early derangement of twist expression in wingless mutants (Bate, 1993).

Huckebein (hkb) sets the anterior and the posterior borders of the ventral furrow, but acts through different modes of regulation. In the posterior part of the blastoderm, HKB represses the expression of sna in the endodermal primordium. In the anterior part, HKB antagonizes the activation of target genes by twi and sna. Here, Bicoid permits the co-expression of hkb, sna and twi, which are all required for the development of the anterior digestive tract. Mesodermal fate is determined where sna and twi but not hkb are expressed. Anteriorly hkb together with sna determines endodermal fate, and hkb together with sna and twi are required for foregut development (Reuter, 1994).

Sloppy paired (Slp) and Even-skipped are involved in cell fate determination and segmentation in the Drosophila mesoderm. The primordia for heart, fat body, and visceral and somatic muscles arise in specific areas of each segment in the Drosophila mesoderm. The primordium of the somatic muscles, which expresses high levels of twist, a crucial factor of somatic muscle determination, is lost in sloppy-paired mutants. The effect of slp on Twist levels is probably partly, but not completely mediated by wg. wg mutant embryos show a premature and ectopic decay of Twist, but not to the same degree as seen in slp embryos. Whereas patches of cells expressing high levels of Twist are initially established in wg mutant embryos, no Twist is seen in the trunk region of slp mutant embryos after stage 11. At the same time that twist expression is lost in slp mutants, the primordium of the visceral muscles is expanded (Riechmann, 1997).

bagpipe and serpent expressing mesodermal domains corresponding to the ectodermal even-skipped domains, alternate with the sloppy-paired expressing high-twist mesodermal domains. Ectodermal even-skipped is thought to act through engrailed and subsequently hedgehog to promote bagpipe expression in cardiac and dorsal muscle and serpent in the fat body (Azpiazu, 1996). Ectodermal Dpp is required for the maintenance of mesodermal tinman, which in turn activates bap expression in the eve domain. The visceral muscle and fat body primordia require even-skipped for their development and the mesoderm is thought to be unsegmented in even-skipped mutants. However, it has been found that even-skipped mutants retain the segmental modulation of the expression of twist. Both the domain of even-skipped function and the level of twist expression are regulated by sloppy-paired, and eve serves reciprocally to regulate the slp domain. sloppy-paired thus controls segmental allocation of mesodermal cells to different fates (Riechmann, 1997).

The Drosophila protein DSP1, an HMG-1/2-like protein, binds DNA in a highly cooperative manner with three members of the Rel family of transcriptional regulators (NF-kappaB, the p50 subunit of NF-kappaB, and the Rel domain of Dorsal). This cooperativity is apparent with DNA molecules bearing consensus Rel-protein-binding sites and is unaffected by the presence of a negative regulatory element, a sequence previously proposed to be important for mediating repression by these Rel proteins. The cooperativity observed in these DNA-binding assays is paralleled by interactions between protein pairs in the absence of DNA. In HeLa cells, as assayed by transient transfection, expression of DSP1 increases activation by Dorsal from the twist promoter and inhibits that activation from the zen promoter, consistent with the previously proposed idea that DSP1 can affect the action of Dorsal in a promoter-specific fashion (Brickman, 1999).

DSP1 has opposite effects on the activity of Dorsal assayed with regulatory sequences excised from the twist and zen promoters. These experiments were performed by transiently transfecting mammalian cells in culture. Thus, reporters containing either a 180-bp fragment from zen (a fragment sufficient to mediate repression in Drosophila) or the entire regulatory region of twist (from -1,438 to +38) were activated by cotransfection with DNA encoding Dorsal. Cotransfection with DNA encoding DSP1 has just the opposite effects on this Dorsal mediated activation of the two promoters: activation from the twist promoter is stimulated 4-fold, whereas that from the zen promoter is inhibited 3-fold. DSP1's stimulation of Dorsal-mediated activation from the twist promoter can be mapped to the defined enhancer elements or VARs. Thus, DSP1 also stimulates Dorsal-mediated activation if the template bears, instead of the intact twist promoter, a cassette that contains the two VARs that drive ventral-specific expression of the twist gene in the Drosophila embryo. The two VARs together constitute approximately 300 bp and contain multiple Rel-protein-binding sites (Brickman, 1999).

It is not known what DNA sequences in the zen and twist promoters determine the opposite effects of DSP1 on dorsal-mediated activation. The finding that a negative regulatory element (NRE) has no effect on cooperative binding to DNA of DSP1 and various Rel proteins prompted a reexamination of the earlier claims that DSP1 converts Dorsal, the p50 homodimer, and the NF-kappaB heterodimer into repressors and that this effect requires the NRE. In each case, DSP1 inhibits Rel-protein-dependent activation both in the presence and absence of an NRE. In no case was NRE-dependent conversion of the Rel protein to a repressor by cotransfection with DSP1 observed. It is not understood why the current results differ from those reported previously (Brickman, 1999 and references therein).

Sites of the described protein-protein interactions are found in the conserved Rel domains and in the fragment of DSP1 that bears both HMG domains. The Rel domains of p65 and of Dif differ from those of Dorsal and of p50 in that they lack the HMG-domain-interaction site. The HMG domain of DSP1 also interacts with the TATA-binding protein. Similar interactions have been reported for HMG-1 and HMG-2 with the steroid hormone receptors, for HMG-1 with p53, for HMG-1 with HOXD9, and for HMG-2 with Oct2. Thus, the HMG domain may contain a common structural motif for cooperative DNA binding and interaction with other transcription factors. The interaction between TATA-binding protein and DSP1 also seems to be influenced by the glutamine-rich amino-terminal domain in that the full-length DSP1 interacts more avidly with TATA-binding protein than does the HMG-1 domain. These experiments suggest that the amino-terminal glutamine-rich domain may also potentiate the DSP1-Rel protein interaction as well, because all DSP1-Rel interactions seem stronger with full-length DSP1, particularly the weak interactions seen between DSP1 and p65 or Dif, which are observed only with GST-DSP1 and not with GST-DSP1 (178-393) (Brickman, 1999).

Drosophila thoracic muscles are comprised of both direct flight muscles (DFMs) and indirect flight muscles (IFMs). The IFMs can be further subdivided into dorsolongitudinal muscles (DLMs) and dorsoventral muscles (DVMs). The correct patterning of each category of muscles requires the coordination of specific executive regulatory programs. DFM development requires key regulatory genes such as cut (ct) and apterous (ap), whereas IFM development requires vestigial (vg). Using a new vg^null mutant, a total absence of vg is shown to lead to DLM degeneration through an apoptotic process and to a total absence of DVMs in the adult. vg and scalloped (sd), the only known Vg transcriptional coactivator, are coexpressed during IFM development. Moreover, an ectopic expression of ct and ap, two markers of DFM development, is observed in developing IFMs of vg^null pupae. In addition, in vg^null adult flies, degenerating DLMs express twist (twi) ectopically. Evidence is provided that ap ectopic expression can induce per se ectopic twi expression and muscle degeneration. All these data seem to indicate that, in the absence of vg, the IFM developmental program switches into the DFM developmental program. Moreover, the muscle phenotype of vg^null flies can be rescued by using the activity of ap promoter to drive Vg expression. Thus, vg appears to be a key regulatory gene of IFM development (Bernard, 2003).

Thus the absence of Vg leads to IFM degeneration. Some IFM phenotypes have been reported for the vg^83b27R allele, a strong allele of vg. In these flies, the DVMs are absent and some DLMs are missing. It has been shown that this phenotype is fully penetrant in vg^null flies and that apoptosis is involved in loss of IFMs. Since muscle attachment sites are normal in vg^null flies, the process of degeneration is different from that described in ap mutants. Phenotypic analysis shows that degeneration occurs during late metamorphosis (after 48 h APF) (Bernard, 2003).

Thus DLMs degenerate by apoptosis in homozygous vg^null flies. This degeneration could be due to a misprogramming of myoblasts surrounding DLMs during development. The process that leads to apoptosis in these muscles remains to be determined. DLM degeneration is associated with an ectopic expression of Twi transcription factor. During flight muscle development, Twi expression is restricted to myoblasts and that persistent expression in developing muscles leads to muscle degeneration. Thus, Twi expression in vg^null mutants could be responsible for DLM degeneration. Finally, it has been shown that ectopic ap expression induces Twi expression in DLMs. Since AP and twi are known to be, respectively, activator and target of the N pathway, it can be hypothesized that AP activates Twi ectopically in vg^null DLMs through the N pathway. If this hypothesis is confirmed, it can be asked why Ap does not activate Twi during normal DFM development. It is likely that numerous genes, other than vg and ap, are differentially activated during DFM and IFM development. Twi activation by AP could be repressed by one of these genes during DFM development (Bernard, 2003).

Morphogenetic movements are closely regulated by the expression of developmental genes. This study examines whether developmental gene expression can in turn be mechanically regulated by morphogenetic movements. The effects of mechanical stress were examined on the expression of Twist, which is normally expressed only in the most ventral cells of the cellular blastoderm embryo under the control of the Dorsal morphogen gradient. At embryogenesis gastrulation (stage 7), Twist is also expressed in the anterior foregut and stomodeal primordia. Submitting the early Drosophila embryo to a transient 10% uniaxial lateral deformation induces the ectopic expression of Twist around the entire dorsal-ventral axis and results in the ventralization of the embryo. This induction is independent of the Dorsal gradient and is triggered by mechanically induced Armadillo nuclear translocation. Twist is not expressed in the anterior foregut and stomodeal primordia at stage 7 in mutants that block the morphogenetic movement of germ-band extension. The mutants can be rescued with gentle compression of these cells, the stomodeal-cell compression normally caused by the germ-band extension is interpretated as inducing the expression of Twist. Correspondingly, laser ablation of dorsal cells in wild-type embryos relaxes stomodeal cell compression and reduces Twist expression in the stomodeal primordium. The induction of Twist in these cells depends on the nuclear translocation of Armadillo. It is proposed that anterior-gut formation is mechanically induced by the movement of germ-band extension through the induction of Twist expression in stomodeal cells (Farge, 2003).

Therefore, lateral compression of the early embryo induces the ectopic expression of Twist around the entire dorsal-ventral axis and results in the ventralization of the embryo. Despite the probable variations in the direction and amplitude of the deformation of each cell as a function of its location in the embryo, all cells respond to this stress. This suggests that their transcriptional response is triggered by deformation per se and does not depend on the exact geometry and amplitude of the mechanical strain applied to each cell. However, it is unclear how the forces required to artificially deform the embryo lead to embryonic epithelium stresses and strains that are related to endogenous forces and deformations present in the embryo during development (Farge, 2003).

Importantly, the mechanical induction of Twist is independent of the maternal determinants of dorsal-ventral polarity. Instead, this induction depends on the nuclear translocation of Armadillo and its ability to activate transcription. The mechanism that triggers the nuclear translocation of Armadillo in response to mechanical stress is unknown. One possibility is that mechanical strain activates a noncanonical Wingless transduction pathway, which releases the cytoplasmic pool of Armadillo from Axin and allows it to enter the nucleus. Alternatively, mechanical strain might trigger the release and nuclear localization of the pool of Armadillo that is associated with Cadherin at the zonula-adherens. Indeed, this might provide a reason for dual function of Armadillo as an essential component of Cadherin adhesion complex and as a transcription factor (Farge, 2003).

It is interesting to note that the Armadillo homolog, beta-catenin, translocates into the nuclei at the dorsal pole of early frog and fish embryos, where it plays a role in determining dorsal-ventral polarity. Furthermore, the ectopic nuclear localization of beta-catenin induces the dorsalization of vertebrate embryos. Because the dorsal-ventral axis of invertebrates is inverted with respect to that of vertebrates, this corresponds well with the ventralization observed in Drosophila embryos upon the mechanical induction of Armadillo nuclear localization. Thus, mechanical compression may reactivate a conserved and ancient pathway for dorsal-ventral axis formation (Farge, 2003).

The results presented in this study suggest that the expression of Twist in foregut and stomodeal-primordia cells at the onset of gastrulation is mechanically induced by the compression caused by germ-band extension and that this is also mediated by the nuclear translocation of Armadillo. twist is involved in the differentiation and the formation of both the foregut and the anterior midgut. Interestingly, neither the anterior midgut nor the stomodeum invaginate in embryos that lack the mechanical compression and do not express Twist when epithelial dorsal cells have been photo-ablated. It is proposed that, through mechanical induction of twist, the anterior-gut formation is induced by stomodeal cell compression in response to germ-band extension (Farge, 2003).

In addition to its role in dorso-ventral axis formation, Armadillo is thought to induce the differentiation and invagination of the meso-endoderm cells that give rise to the gut in other vertebrate and nonvertebrate embryos. Although the maternal signals that induce the nuclear translocation of beta-catenin in zebrafish and Sea Urchins are not known, they have been shown to be independent of the classical determinant Wingless. These results in Drosophila raise the possibility that the nuclear translocation of Armadillo/beta-catenin in the gut primordia of these embryos might be mechanically induced by morphogenetic movements that are homologous to germ-band extension. Indeed, the nuclear translocation of beta-catenin and the formation of the meso-endodermal gut invagination/involution are concomitant with convergent extension, which tends to compress the meso-endoderm cells (Farge, 2003).

These parallels led to the speculation that mechanical induction may be an ancient mechanism for inducing gut formation. This could have evolved from a primitive reflex response to mechanical deformation. Such a response might have been the phagocytosis of particles in response to physical contact, which has been proposed to be the 'feeding-response' of the earliest organisms. The generation of a permanent gut might have then been stabilized by the Armadillo-induced expression of meso-endodermal genes in response to genetically controlled endogenous morphogenetic movements, such as cell intercalation generating convergent extension. These experiments may have reactivated the genetic pathway of such 'fossil sensorial behavior' in early Drosophila embryos (Farge, 2003).

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