transcription factor - bHLH - DV pathway - a switch in the development of muscle - High levels required for somatic myogenesis - this blocks formation of other mesodermal derivatives such as visceral mesoderm and heart
Gene model reviewed during 5.52
Shares 5' exon(s) with upstream non-coding gene; shared promoter (one isoform only of this gene).
Gene model reviewed during 5.56
Efficient DNA binding requires dimerization with another bHLH protein. Homodimer.
Click to get a list of regulatory features (enhancers, TFBS, etc.) and gene disruptions (point mutations, indels, etc.) within or overlapping Dmel\twi using the Feature Mapper tool.
Both twi and sna transcript are first detectable during nuclear cycle 12 as a diffuse band half the width of the presumptive mesoderm. Although the expression patterns of sna and twi transcript and protein are similar early in embryogenesis, there are some subtle differences. At mid-cellularization, the sna transcript and protein expression boundaries sharply delimit the presumptive mesoderm. At the same time, twi transcript and protein is expressed in a gradient that extends past the sna expression zone into the presumptive ectoderm. The twi transcript and protein expression pattern gets sharper later, during gastrulation. twi protein is expressed in the mesoderm throughout germ band extension, whereas the sna protein product disappears from the mesoderm partway through germ band extension, and appears in neurectodermal cells which might be neuroblasts.
twi expression is detected in a limited number of ventral cells in stage 5 embryos. The ventral furrow forms shortly thereafter, and twi transcript accumulates in the invaginated cells of the ventral furrow (presumptive mesodermal cells). At stage 8, twi is expressed in the mesodermal layer of the germ band.
Protein can be detected in myoblasts in the presumptive and larval wing disc.
Both twi and sna transcript are first detectable during nuclear cycle 12 as a diffuse band half the width of the presumptive mesoderm. Although the expression patterns of sna and twi transcript and protein are similar early in embryogenesis, there are some subtle differences. At mid-cellularization, the sna transcript and protein expression boundaries sharply delimit the presumptive mesoderm. At the same time, twi transcript and protein is expressed in a gradient which overlaps and extends past the sna expression zone into the presumptive ectoderm. The twi transcript and protein expression pattern gets sharper later, during gastrulation. twi protein is expressed in the mesoderm throughout germ band extension, whereas the sna protein product disappears from the mesoderm partway through germ band extension, and appears in neurectodermal cells which might be neuroblasts.
During stage 5 of embryogenesis, the twi protein is expressed ventrally, extending past the anterior and posterior poles. During stage 6, twi protein is seen in the presumptive mesodermal cells of the invaginating ventral furrow, in endodermal cells in the anterior and posterior regions of the embryo, and in the ectodermal cells of the anterior midgut primordium. The mesodermal cells forming the ventral furrow, as well as the four adjoining mesectodermal cells express twi protein at stage 7. At stage 7, twi protein is no longer detected in the cells which will form the posterior midgut rudiment, although labeling is still seen in the anterior midgut rudiment. twi protein becomes restricted to the mesoderm starting at stage 9. In stage 11 embryos, only a few mesodermal cells in each segment express twi protein. At stage 12, a subset of the cells of the somatopleura and the splanchnopleura (mesodermally derived cells which will give rise to the somatic and visceral musculature, respectively) are labeled by anti-twi antibody, and by stage 14, only the splanchnopleura labeling is visible.
GBrowse - Visual display of RNA-Seq signalsView Dmel\twi in GBrowse 2
Please Note FlyBase no longer curates genomic clone accessions so this list may not be complete
Please Note This section lists cDNAs and ESTs that fall within the genomic extent of the gene model, which may include cDNAs and ESTs of genes within introns, or of overlapping genes. Please see GBrowse for alignment of the cDNAs and ESTs to the gene model.
For each fully sequenced cDNA the DGRC maintains various forms of the cDNA (e.g tagged or untagged) in several different host vectors for subsequent cloning and expression in Drosophila and Drosophila cell lines.
twi function is required during the late larval stage for splitting of the larval oblique muscles that form the templates of the adult dorsal longitudinal indirect flight muscles.
twi protein directly regulates Mef2 expression in adult somatic muscle precursor cells via a 175bp enhancer located 2245bp upstream of the Mef2 transcriptional start site. Activation of Mef2 transcription via this enhancer by twi protein is essential for normal adult muscle development.
In a sample of 79 genes with multiple introns, 33 showed significant heterogeneity in G+C content among introns of the same gene and significant positive correspondence between the intron and the third codon position G+C content within genes. These results are consistent with selection adding against preferred codons at the start of genes.
The normal location of the ventral furrow in embryos with uniformly expressed fog suggests the existence of a fog-independent pathway determining mesoderm-specific cell behaviours and invagination. Epistasis experiments indicate this pathway requires sna but not twi expression.
tin is a direct transcriptional target for twi and its own gene product in visceral mesodermal cells, supporting the idea that twi and tin function in the subdivision of the mesoderm during embryogenesis.
dl and twi proteins synergistically activate transcription in cell culture from a promoter containing binding sites for both factors. A region outside of the conserved bHLH domain of the twi protein is required for the synergy. Protein-protein interaction assays show that dl and twi proteins bind to one another in vitro.
Expression of esg in the neuroectoderm is studied, the expression pattern prefigures that of the ASC genes. Dorsoventral pattern of esg expression in the blastoderm is determined by three independent repressive cues (dpp, sna and twi).
High levels of twi product are required for somatic myogenesis and block the formation of other mesodermal derivatives. Ectopic expression of twi can drive ectodermal cells into myogenesis. After an initial role in gastrulation, twi regulates mesodermal differentiation and propels a specific subset of mesodermal cells into somatic myogenesis.
Analysis of the motorneuron structure in twi mutant embryos demonstrates post-synaptic target muscles are not required for the outgrowth of presynaptic motor axons into the periphery, nor are they required to induce the expression of genes coding for the components of active zones or for the assembly of active zones themselves. Synthesis of the active zone appears to be an independent function of the presynaptic motorneuron, which is integrated into the development of the neuromuscular synapse.
At stage 10 two different groups of mesodermal cells can be observed by twi protein expression: group A have weak twi expression and give rise to internal mesodermal structures (visceral mesoderm) and group A have strong twi expression and give rise to external mesodermal structures (heart and somatic mesoderm).
sna is sufficient to induce the formation of an attenuated ventral furrow in the absence of twi+ gene activity. Expression of sna in the absence of twi uncouples ventral furrow formation and mesoderm differentiation, invaginating cells fail to express various mesoderm marker genes.
The coding region and regulatory regions of twi and Dvir\twi are compared. Within the coding region the basic helix-loop-helix DNA binding and dimerisation domain are highly conserved. A high degree of conservation is seen between the more distal of the two ventral activator elements in the 5' regulatory regions of twi and Dvir\twi. The more proximal ventral activator is missing in the equivalent position in Dvir\twi, instead a region in the second intron resembles the proximal element. Transformation experiments reveal the evolved enhancer elements are similar in structure and functional properties.
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 twi and sna are required for foregut development.
The prepattern of the adult musculature, revealed in persistent twi-expressing cells in the embryo, is not dependent on the embryonic peripheral motor nerves.
twist-expressing cells associate with the segmental nerves in the thoracic as well as the abdominal segments of the third larval instar.
Promoter fusions using elements of the twi, ve, da and sna promoters indicate that low affinity dl-binding sites restrict target gene expression to the presumptive mesoderm, where there are peak levels of dl expression. The twi promoter includes a proximal and a distal element, and the proximal element contains two low affinity dl binding sites. The proximal element alone directs twi expression in the ventralmost 12 to 14 nuclei while the complete promoter with both proximal and distal elements directs twi expression in the ventralmost 18 to 20 cells. Promoters containing the Et (veinlet) or Eds (dl and snail) E boxes display opposite behaviour in da and twi mutants, suggesting they are regulated by different basic helix loop helix proteins.
The BRE region of Ubx includes binding sites for hb, ftz, tll, en and twi. The binding of their products and the interplay between them is responsible for generating the expression pattern directed by the BRE.
In mutant embryos lacking the entire mesoderm or failing to differentiate the visceral mesoderm, the anterior and posterior midgut primordia form but do not migrate properly.
twi mutants fail to differentiate ventrally derived mesoderm.
Expression of twi assayed in embryos from females expressing wild type dl and dl-lacZ fusion proteins. Reduction in intensity of twi expression at gastrulation correlates well with degree of dorsalization of embryos, suggesting effect of dl mediated through its downstream target genes.
A twi binding site has been identified in the neural ectoderm expression region (NEE) of ve promoter: twi activates ve expression. The twi gene is a target gene of dl.
dl binding site domain exchange experiments, using Ecol\lacZ reporter gene constructs, between the zen and twi promoters demonstrate that dl is intrinsically an activator and that repression requires additional factors present in the distal region of the zen promoter, the VR.
Deletion analysis of the twi promoter using Ecol\lacZ reporter gene constructs demonstrates that the repressor/activator activity of the dl binding sites depends on its context, not an intrinsic property.
The zygotically acting DV genes repress ac expression within specific DV domains.
twi expression marks embryonic precursors of the cells that make the adult muscles.
twi-expressing lineages of cells have been ablated in the larva, and the impact on muscle pattern in the adult studied.
A combination of promoter fusion-P-element transformation assays (1.2kb twi promoter fragment is sufficient to generate normal twi pattern of Ecol\lacZ expression) and in vitro DNA binding assays coupled with site directed mutagenesis (revealing four dl-binding sites in the twi promoter) have been used to establish a link between the dl-binding sites and twi expression in early embryos. The dorsal ventral limits of twi expression depend on the number and affinity of dl binding sites present in the twi promoter.
Establishment of the mesoderm neuroectoderm boundary involves the interaction of twi, sna and dl proteins.
Deletion analysis of the twi promoter region using Ecol\lacZ reporter gene construct demonstrate that the region extending from -68 to -269 relative to the transcription start site contains an element that is required for ventral activation of twi in the embryo. This element "z" lies between -55 and -205 and it influences overall promoter strength. A second functional element lies between -537 and -269 and constitutes a ventral activator region. In vitro transcription reactions and S1 nuclease analysis demonstrate the presence of z binding sites within 120bp region of twi promoter proximal to the transcription start site.
Mutations in zygotic dorsal class gene twi interact with RpII140wimp.
Lateralized embryos have twi expression at the poles. Dorsalized embryos show twi expression during late stages of embryogenesis. Polar expression of twi requires genes of the terminal group. twi is a primary patterning gene for dorsal mesoderm as expression is unaffected by mutations in zygotic dorsal-ventral genes.
Zygotically active locus involved in the terminal developmental program in the embryo.
Phenotypic rescue and twi-lacZ expression studies demonstrate that 5' sequences -4.0kb up to +6.2kb 3' sequences are required for normal twi expression. Recurrent deletions in the 3kb twi promoter region have found dl responsive elements (DREs). dl acts as a sequence specific trans-activator of the twi promoter. Gel retardation assays have been used to investigate binding of dl protein to synthetic oligonucleotides corresponding to the proximal and distal activator region of the twi promoter.
Involved in the regulatory hierarchy responsible for the asymmetric distribution and function of zygotic regulatory gene products along the DV axis of early embryos.
Molecular analysis of the twi gene and its product reveals that it encodes a nuclear protein expressed in the endoderm and mesoderm anlagen and derived tissues.
In homozygous embryos invagination of the ventral presumptive mesodermal cells fails to occur and the resulting embryos are devoid of internal organs.
Mutations in dl, pll, ea, or Tl abolish the expression of twi. At least one dose of dl+ in females is necessary for transcription of twi (Thisse, Stoetzel, El Messal and Perrin-Schmitt, 1987). twi shows extensive identity to a pair of myc-related polypeptides whose dimerized products bind to a sequence in the immunoglobulin kappa chain enhancer; the identical regions have the potential to form two amphipathic helices separated by an intervening loop (Murre, McCaw and Baltimore, 1989).
The wild-type allele of twi is involved in the establishment of germ layers. Mutants are embryonic lethals (zygotic), partially dorsalized and without mesodermal differentiation. A normal blastoderm is formed; at gastrulation, no ventral furrow is visible, but the endoderm invaginates, a cephalic furrow is formed and the germband elongated. The embryo is twisted or coiled in the egg case, often with posterior side up. There are few mesodermally derived internal tissues. Some embryos fail to make a properly differentiated cuticle, although 40-100% make normal ectodermal derivatives (Simpson, 1983). There is no maternal effect in germ-line chimeras. twi/+ heterozygous embryos have delayed ventral furrow formation. The TSP of the twi gene is around gastrulation (Thisse, Stoetzel, El Messal and Perrin-Schmitt, 1987).