Gene name - torso
Cytological map position - 43C5-E7
Function - terminal gene activation
Keywords - terminal group
Symbol - tor
Genetic map position - 2-
Classification - receptor tyrosine kinase
Cellular location - surface membrane
|Konogami, T., Yang, Y., Ogihara, M. H., Hikiba, J., Kataoka, H. and Saito, K. (2016). Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region. Sci Rep 6: 22437. PubMed ID: 26928300
The insect membrane-protein, Torso, is a member of the receptor-tyrosine-kinase family, and is activated by its ligand, prothoracicotropic hormone (PTTH). Although PTTH is one of the most important regulators of insect development, the mechanism of Torso activation by the hormone has remained elusive. In this study, using heterologous expression in cultured Drosophila S2 cells, ligand-independent dimerization of silkworm Torso was observed, and the receptor molecules in the dimer were found to be linked by intermolecular disulfide bridges. By examining the oligomerization states of several truncation and substitution mutants of Torso, atypical cysteine residues in the transmembrane region were identified as being responsible for the intermolecular linkage in the dimer. The replacement of all of the cysteines in the region with phenylalanines abolished the disulfide-bond-mediated dimerization; however, non-covalent dimerization of the mutant was detected using a cross-linking reagent, both with and without ligand stimulation. This non-covalent dimerization caused apparent receptor autophosphorylation independently of the ligand stimulation, but did not promote the ERK phosphorylation in the downstream signaling pathway. The unique Torso structure with the intermolecular disulfide bridges in the transmembrane region is necessary to maintain the ligand-dependent receptor functions of autophosphorylation and downstream activation.
|Amarnath, S., Stevens, L. M. and Stein, D. S. (2017). Reconstitution of Torso signaling in cultured cells suggests a role for both Trunk and Torsolike in receptor activation. Development [Epub ahead of print]. PubMed ID: 28087630
Formation of the Drosophila embryonic termini is controlled by the localized activation of the receptor tyrosine kinase, Torso. Both Torso and Torso's presumed ligand, Trunk, are expressed uniformly in the early embryo. Polar activation of Torso requires Torsolike, which is expressed by follicle cells adjacent to the ends of the developing oocyte. This study found that Torso expressed at high levels in cultured Drosophila cells is activated by individual application of Trunk, Torsolike or another known Torso ligand, Prothoracicotropic Hormone. In addition to assays of downstream signaling activity, Torso dimerization was detected using bimolecular fluorescence complementation. Trunk and Torsolike were active when co-transfected with Torso and when presented to Torso-expressing cells in conditioned medium. Trunk and Torsolike were also taken up from conditioned medium specifically by cells expressing Torso. At low levels of Torso, similar to those present in the embryo, Trunk and Torsolike alone were ineffective but acted synergistically to stimulate Torso signaling. These results suggest that Torso interacts with both Trunk and Torsolike, which cooperate to mediate dimerization and activation of Torso at the ends of the Drosophila embryo.
|Pae, J., Cinalli, R. M., Marzio, A., Pagano, M. and Lehmann, R. (2017). GCL and CUL3 control the switch between cell lineages by mediating localized degradation of an RTK. Dev Cell 42(2): 130-142.e137. PubMed ID: 28743001
The separation of germline from somatic lineages is fundamental to reproduction and species preservation. This study shows that Drosophila Germ cell-less (GCL) is a critical component in this process by acting as a switch that turns off a somatic lineage pathway. GCL, a conserved BTB (Broad-complex, Tramtrack, and Bric-a-brac) protein, is a substrate-specific adaptor for Cullin3-RING ubiquitin ligase complex (CRL3GCL). CRL3GCL promotes PGC fate by mediating degradation of Torso, a receptor tyrosine kinase (RTK) and major determinant of somatic cell fate. This mode of RTK degradation does not depend upon receptor activation but is prompted by release of GCL from the nuclear envelope during mitosis. The cell-cycle-dependent change in GCL localization provides spatiotemporal specificity for RTK degradation and sequesters CRL3GCL to prevent it from participating in excessive activities. This precisely orchestrated mechanism of CRL3GCL function and regulation defines cell fate at the single-cell level.
|Mineo, A., Fuentes, E., Furriols, M. and Casanova, J. (2018). Holes in the Plasma Membrane Mimic Torso-Like Perforin in Torso Tyrosine Kinase Receptor Activation in the Drosophila Embryo. Genetics. PubMed ID: 30049783
Receptor tyrosine kinase (RTK) pathways play central roles in development, and when abnormally activated they can lead to pathological conditions, including oncogenesis. Thus, RTK activation, mediated by ligand binding, is under tight control, a critical step being the conversion of an inactive precursor into the active form of the ligand. A variety of mechanisms have been shown to be involved in this conversion; however, little attention has been paid to how mechanical phenomena may impinge on this process. This issue was addressed by studying Torso, an RTK activated at both poles of the Drosophila embryo at blastoderm stage. Torso activation is induced by a cleaved form of Trunk, a growth factor-like protein, but it also requires the accumulation of the Torso-like (Tsl) protein at both ends of the blastoderm. Tsl is the only known protein in Drosophila bearing a Membrane Attack Complex/Perforin (MACPF) domain, a motif present in proteins involved in pore formation at cell membranes. However, while different hypotheses have been put forward to account for the function of Tsl in Torso receptor activation, little is known about its molecular role and whether it indeed contributes to membrane pore formation. This study shows that mechanically induced holes in the Drosophila embryo can substitute for Tsl function. These results suggest that Tsl is required for an exchange between the interior of the Drosophila embryo and its surrounding milieu and that mechanically induced cell injuries may contribute to abnormal RTK activation.
All the genes involved in the activation of Torso and its targets make up the so-called terminal group. The synergistic network of interactions and influences involving these genes is known as the terminal pathway. The existence of a terminal pathway was discovered by analysis of mutations that delete both anterior and posterior embryonic structures. This pathway activates genes at both the anterior and posterior axis of the embryo.
Activation of the downstream targets of Torso involves a phosphorylation cascade. Upstream, a protein molecule (ligand), probably Trunk, triggers Torso signaling locally.
torso is the membrane receptor responsible for gene activation in the anterior and posterior ends of the embryo. Since the receptor Torso is uniformly distributed throughout the embryo, its activation must by necessity be local. The targets of Torso are members of the Ras pathway. The first target is the kinase D-raf, which in turn acts upon Ras1. Ras-1 is a docking protein that attaches itself to the cytoplasmic tails of receptors, which in their turn activate downstream targets. The resultant phosphorylation cascade activates tailless and huckebein, two targets of the terminal system. tailless is held in a state of repression by grainyhead/NTF-1 until Torso pathway signals inactivate the repression (Liaw, 1995).
Activation of Torso at the poles of the embryo triggers expression of the terminal zygotic gap genes tailless (tll) and huckebein (hkb). Tailless acts as a repressor of Kruppel and knirps in the central domain of the recently fertilized embryo. Groucho acts throughout the embryo to repress the repressor of Kruppel and knirps, allowing the expression of these gap genes in the central domain of the embryo. Patterning of the non-segmental termini of the Drosophila embryo depends on signaling via the Torso receptor tyrosine kinase. The Gro corepressor acts in this process to confine terminal gap gene expression to the embryonic termini. Embryos lacking maternal gro activity display ectopic tll and hkb transcription; in turn, tll then leads to lack of abdominal expression of the Kruppel and knirps gap genes. torso signaling permits terminal gap gene expression by antagonizing Gro-mediated repression. Groucho-mediated repression of tailless is relieved by the torso pathway suggesting that Groucho is the nuclear target for MAP kinase signaling. It is suggested that Groucho functions as a corepressor along with an unknown protein unrelated to Hairy, since Groucho mediated repression takes place in the absence of known Hairy-related bHLH proteins (Paroush, 1997).
The Torso pathway interacts with Dorsal. DL is phosphorylated through the Torso pathway and thus converted to a repressor (Ronchi, 1993). Torso pathway signals, possibly acting directly on target genes, mask the ability of DL to repress gene expression (Rusch, 1994). The Torso pathway also acts through homeotic genes to regulate proliferating cell nuclear antigen, thus regulating the cell cycle (Yamaguchi, 1995). A possible target of Torso pathway regulated transcription may be Raf (Sprenger, 1993).
The most likely candidate for Torso's ligand is Trunk. Trunk is distributed uniformly through the embryo and is presumably secreted and activated locally. Torso-like has been considered a potential ligand. It is made by follicle cells at either end of the egg. Perhaps Torso-like is part of a cascade for local activation of the Torso ligand (Martin, 1994 and Casanova, 1995).
Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. This study demonstrates that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila, is the PTTH receptor. Trunk, the embryonic Torso ligand, is related to PTTH, and ectopic expression of PTTH in the embryo partially rescues trunk mutants. In larvae, torso is expressed specifically in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates ERK phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. It is concluded that PTTH initiates metamorphosis by activation of the Torso/ERK pathway (Rewitz, 2009).
Many organisms undergo distinct temporal transitions in morphology as a part of their normal life process. In humans, for example, passage through puberty is accompanied by changes in body mass and the acquisition of sexual maturity. Likewise, in all holometabolous insects, metamorphosis transforms the immature larva into a completely new body form that is capable of reproductive activity. In both cases, neuropeptide signaling in response to environmental and nutritional cues triggers the transition process. In insects, the process is initiated by the neuropeptide known as prothoracicotropic hormone (PTTH). PTTH signals to the prothoracic gland (PG), the primary insect endocrine organ, which triggers the production and release of ecdysone, the precursor of the active steroid molting hormone 20-hydroxyecdysone (20E). The increased level of 20E provides a systemic signal that ends the larval growth period and initiates metamorphosis (Rewitz, 2009).
PTTH has been proposed to be structurally similar to certain mammalian growth factors that are ligands for receptor tyrosine kinases (RTKs). Previous studies have also indicated that PTTH signaling results in the phosphorylation of cellular signaling molecules that are linked to the mitogen-activated protein kinase (MAPK) pathway in the PG. In light of the potential involvement of MAPK pathway components in PTTH signaling, the expression of all Drosophila RTKs was examined in the PG to determine whether any showed a tissue-specific expression profile that was consistent with a possible role as a PTTH receptor. It was found that after early embryogenesis, the RTK encoded by torso is expressed specifically in the PG (Rewitz, 2009).
The gene torso belongs to the so-called terminal group of genes that are required for the correct patterning of anterior and posterior structures during early embryogenesis. The presumed ligand for Torso during terminal patterning is Trunk (Trk), which contains a cysteine knot-type motif in the C-terminal region similar to the motif in PTTH. Also like PTTH, Trk is thought to be proteolytically processed from a precursor molecule to generate an active C-terminal fragment that is comparable in length to that of PTTH. Alignment of the protein sequences of Trk and PTTH reveals that they share some conserved structures in the C-terminal region that compose the mature peptide, including all of the six cysteines that are important for intramonomeric bonds of the PTTH homodimeric molecule. Previously, it was noted that Trk is related to Spatzle, but a phylogenetic analysis of different insect cysteine knot-type proteins shows that Trk and PTTH form a separate cluster and that PTTH is the closest paralog of Trk. These results raise the possibility that Trk and PTTH share a conserved three-dimensional structure enabling both to activate Torso despite the modest conservation of primary sequence. Expression of trk is not detected in the wandering third-instar larval (L3) stage using real-time polymerase chain reaction (PCR) (no product after 30 PCR cycles) or by in situ hybridization to the brain-PG complex, supporting the idea that PTTH, and not Trk, is a ligand for Torso in post-blastoderm stages (Rewitz, 2009).
To investigate possible post-embryonic roles of Torso in Drosophila development, RNA interference (RNAi) was used to knock down torso specifically in the PG. The PG-specific phantom (phm)-Gal4 line (phm>) was used to drive expression of RNAi constructs under control of upstream activator sequences (UASs) in the PG. This expression of a torso RNAi construct produced a phenotype that was almost identical to the one created by the loss of PTTH-expressing neurons. Reduction of torso expression in the PG of phm>torso-RNAi larvae delays the onset of pupariation by 5.8 days as compared with the phm> + control animals, similar to the 5.4-day delay of pupariation in animals lacking PTTH. As with the loss of the PTTH-producing neurons, torso silencing in the PG also leads to excessive growth during the prolonged L3 stage, resulting in increased pupal size. To test the specificity of the RNAi, it was confirmed that torso mRNA levels are reduced in phm>torso-RNAi larvae and that the PG cells are morphologically normal, although slightly smaller (Rewitz, 2009).
Because torso is a maternal-effect gene, homozygous mutants derived from heterozygous parents are viable. Therefore, the developmental profile and adult size were examined of animals homozygous and transheterozygous for three different torso mutations. Larvae with mutations in torso exhibited substantial developmental delays, although not as long as those seen by RNAi knockdown, in the time to pupariation as compared with heterozygous controls, and the mutants produced larger adults. The difference in time delay may result from residual maternally loaded torso mRNA. In contrast, trk mutants developed on a normal time scale, and adults were similar in size to heterozygous control adults, demonstrating that the phenotype of torso mutants is independent of early embryonic signaling (Rewitz, 2009).
In animals lacking PTTH-producing neurons, it is the low level of the active molting hormone 20E that causes the developmental delay and tissue overgrowth. To investigate whether the torso loss-of-function phenotype is also caused by low 20E levels, 20E was fed to phm>torso-RNAi larvae. Similar to what was found when the PTTH-producing neurons were removed, feeding these larvae with 20E completely rescued the developmental delay and overgrowth. Taken together, these results demonstrate that reducing Torso signaling in the PG alone phenocopies the loss of PTTH, which is consistent with the notion that Torso mediates PTTH signaling in the PG. If this is the case, it would be expected that the constitutively active torsoRL3 allele might produce precocious pupation, as would overexpression of PTTH. Consistent with this conjecture, it was found, using the daughterless (da)-Gal4 driver (da>), that ubiquitous overexpression of PTTH advances the onset of pupariation by 11.5 hours as compared with (da> +) balancer controls and produces smaller adults. At 25°C, torsoRL3 is activated, and heterozygous torsoRL3/+ animals pupariate 9.2 hours before controls and form smaller adult males (Rewitz, 2009).
To establish whether PTTH can activate Torso in vivo, it was reasoned that if PTTH is a ligand for Torso, then ectopic expression of PTTH in the embryo might elicit partial rescue of trk mutants. To examine this, the maternal nanos (nos)-Gal4 line (nos>) was used to drive ubiquitous early embryonic expression of a UAS-PTTH-hemagglutinin (HA)-tagged transgene in trk mutant embryos. In the blastoderm-stage embryo, activation of Torso by Trk induces expression of the downstream target gene tailless (tll) in the anterior and posterior regions. The inability to activate this target gene in trk or torso mutants leads to the loss of structures posterior to the seventh abdominal segment. Early embryonic expression of PTTH was observed in 13% of blastoderm-stage embryos derived from trk1/trk1; nos>PTTH females. Ectopic expression of PTTH in these embryos was sufficient to activate tll in the posterior part of the embryos. Although PTTH expression did not fully restore wild-type tll expression, the partial rescue elicited by PTTH was sufficient to restore posterior structures, such as the Filzkörper, in several trk mutant embryos. These results provide genetic evidence that PTTH functions as a ligand for Torso in vivo (Rewitz, 2009).
In the embryo, Torso signaling is transduced through the canonical MAPK pathway that includes the Drosophila homologs of Ras (Ras85D), Raf (Draf), MAPK kinase (MEK), and extracellular signal-regulated kinase (ERK). If Torso is indeed the PTTH receptor, it would be expected that disrupting MAPK signaling in the PG would result in a phenotype similar to that resulting from loss of the PTTH-producing neurons and Torso signaling. So far, the role of the MAPK pathway in transduction of the PTTH signal has been determined only by in vitro studies of lepidopteran PG. In Drosophila, the expression of dominant negative forms of Ras and Raf is known to delay development. To further examine the importance of the MAPK pathway in mediating PTTH/Torso signaling, RNAi was used to reduce the expression of several core components of this pathway, including Ras, Raf, and ERK, in the PG. Loss of either Ras, Raf, or ERK delayed pupariation by 4.3, 2.7, and 6.1 days, respectively. ERK silencing in the PG delays pupariation as severely as the reduction of Torso signaling or the complete loss of the PTTH-producing neurons does. The increase in size of phm>ERK-RNAi pupae and adults was also similar to the increase caused by the loss of PTTH or loss of Torso. The developmental delay, as well as the size increase caused by ERK silencing, were negated by 20E feeding. The less-severe phenotypes produced by the loss of Raf and Ras may result from less-efficient knockdown or, in the case of Ras, may reflect partial redundancy with Rap1. Consistent with Ras being downstream of torso, it was also found that expression of constitutively active Ras in the PG completely rescued the torso-RNAi-induced delay and overgrowth phenotype. Taken together, these results indicate that, as during embryonic terminal patterning, Torso regulation of ecdysone production in the PG is primarily mediated by the MAPK pathway, resulting in the activation of ERK (Rewitz, 2009).
To test directly whether stimulation of Torso by PTTH could lead to ERK phosphorylation, a cell culture-based signaling assay was developed. Because active Drosophila PTTH has not been produced in tissue culture, the silkworm Bombyx mori full-length Bombyx torso cDNA was cloned. As in Drosophila, the Bombyx torso ortholog is expressed predominantly in the PG of the final (fifth)-instar larvae. Stimulation of Drosophila S2 cells transfected with Bombyx torso and Drosophila ERK with 10-9 M PTTH led to robust phosphorylation of ERK. PTTH stimulation of ERK phosphorylation was not detected in control S2 cells, either incubated in the absence of PTTH or those stimulated with PTTH but not expressing Bombyx torso. Bombyx PTTH did not stimulate activation of ERK through Drosophila Torso or through the insulin receptor, demonstrating that ERK stimulation by Bombyx PTTH is specific to Bombyx Torso. These results demonstrate that Torso is a functional PTTH receptor that is able to mediate PTTH signaling through the activation of the ERK pathway (Rewitz, 2009).
These observations define another role for the terminal system, which is the initiation of metamorphosis at the end of larval growth. Therefore, insects apparently use the same core system for two developmentally distinct processes: the establishment of terminal cell fate in the embryo and the termination of larval growth at the correct time to ensure an appropriate final adult body size. This identification of the PTTH receptor will facilitate further characterization of the system that determines body size in insects. It will be of interest to ascertain just how similar this system is in overall design to the hypothalamus-pituitary-gonadal axis, which controls the timing of puberty in mammals (Rewitz, 2009).
Exons - at least 13
Torso has structural similarities to growth-factor receptor tyrosine kinases except that the extracellular domain does not resemble that of other known receptor tyrosine kinases. Torso has 12 potential extracellular glycosylation sites, a central transmembrane domain and two putative intracellular kinase domains (Sprenger, 1989).
Insect axis formation is best understood in Drosophila, where rapid anteroposterior patterning of zygotic determinants is directed by maternal gene products. The earliest zygotic control is by gap genes, which determine regions of several contiguous segments and are largely conserved in insects. Isolation of mutations has been used to approach a genetic question: do early zygotic patterning genes control similar anteroposterior domains in the parasitoid wasp Nasonia vitripennis as in Drosophila? Nasonia is advantageous for identifying and studying recessive zygotic lethal mutations because unfertilized eggs develop as males while fertilized eggs develop as females. On first consideration, the Hymenopteran Nasonia and the Dipteran Drosophila appear very similar in their embryonic development, though the Hymenoptera diverged from the Diptera >200 million years ago. Embryos of both species produce larvae in about 1 day at 25°C. In Nasonia, the fertilized egg gives rise to an embryo that undergoes syncytial and cellular blastoderm stages morphologically similar to those of Drosophila. Both Nasonia and Drosophila undergo the long germband mode of embryonic development. Despite these similarities, two observations suggest that the relative importance of maternal versus zygotic patterning functions may differ in the two insects. (1) Although postgastrulation events proceed with very similar timing, the time for early development differs substantially - at 25°C: the events preceding gastrulation take only about 3 hours in Drosophila but almost 10 hours in Nasonia. This difference in timing may allow for greater zygotic control of patterning in Nasonia than in Drosophila. (2) Among the relatives of Nasonia, a polyembryonic mode of development has evolved in which a single fertilized egg gives rise to hundreds or thousands of progeny. Polyembryonic development is likely to rely heavily on zygotic control of patterning. Polyembryony has arisen several times in the Hymenoptera, and the polyembryonic Copidosoma floridanum is in the same superfamily as Nasonia. These considerations pose the following question -- is early development substantially controlled by the zygotic genome in Hymenopterans? This question may be approached genetically, by isolating zygotic mutations that disrupt early anteroposterior patterning in Nasonia. Recessive zygotic mutations have identified three Nasonia genes: head only mutant embryos have posterior defects, resembling loss of both maternal and zygotic Drosophila caudal function; headless mutant embryos have anterior and posterior gap defects, resembling loss of both maternal and zygotic Drosophila hunchback function, and squiggy mutant embryos develop only four full trunk segments, a phenotype more severe than those caused by lack of Drosophila maternal or zygotic terminal gene functions. head only mutant embryos lack all segmentation posterior to the head, in the strongest manifestation of the phenotype, and have only a narrow domain of Ubx-Abd-A expression. head only differs from Drosophila gap genes with respect to the extent of pattern deleted and effects on Ubx-Abd-A. In Drosophila, neither Krüppel nor knirps affects a domain as large as that of head only. Moreover, the wild-type functions of Krüppel and knirps are not required for the positive regulation of Ubx or abd-A in Drosophila (Pultz, 1999).
squiggy mutant embryos have severe defects both anteriorly and posteriorly, leaving only four consistently developed trunk segments. This cuticular phenotype differs substantially from the phenotypes of maternal terminal group genes in Drosophila, such as torso, in which loss-of-function maternal-effect mutations delete pattern elements from both ends of the embryo. The terminal structures deleted in torso embryos are anterior to the gnathal segments and posterior to the seventh abdominal segment, and are thus limited compared to those of the zygotic squiggy mutant embryos. The extensive zygotic control of terminal development by squiggy appears to be a departure from Drosophila developmental mechanisms. The Drosophila maternal terminal gene patterning system is not known to be widely conserved, and the follicle cell types that express torsolike do not appear to be conserved even in the lower Diptera. Terminal patterning in insects may therefore be subject to considerable evolutionary flexibility. Zygotic control of early patterning in head only, headless and squiggy mutants share a common theme: the zygotic Nasonia phenotypes are more extreme than those of Drosophila gap genes and all three genes appear to control processes zygotically that are partially or fully subject to maternal control in the fly. These results indicate greater dependence on the zygotic genome to control early patterning in Nasonia than in the fly (Pultz, 1999).
In the long germ insect Drosophila, all body segments are determined almost simultaneously at the blastoderm stage under the control of the anterior, the posterior, and the terminal genetic system. Most other arthropods (and similarly also vertebrates) develop more slowly as short germ embryos, where only the anterior body segments are specified early in embryogenesis. The body axis extends later by the sequential addition of new segments from the growth zone or the tail bud. The mechanisms that initiate or maintain the elongation of the body axis (axial growth) are poorly understood. The terminal system in the short germ insect Tribolium was functionally analyzed. Unexpectedly, Torso signaling is required for setting up or maintaining a functional growth zone and at the anterior for the extraembryonic serosa. Thus, as in Drosophila, fates at both poles of the blastoderm embryo depend on terminal genes, but different tissues are patterned in Tribolium. Short germ development as seen in Tribolium likely represents the ancestral mode of how the primary body axis is set up during embryogenesis. It is therefore concluded that the ancient function of the terminal system mainly was to define a growth zone and that in phylogenetically derived insects like Drosophila, Torso signaling became restricted to the determination of terminal body structures (Schoppmeier, 2005).
In Drosophila, the anterior- and posterior-most terminal body regions of the embryo depend on the maternal terminal-group genes. One of them, the torso-like (tsl) gene is expressed in somatic follicle cells located at the anterior and posterior pole of the oocyte. In the embryo, tsl contributes to the local activation of the receptor tyrosine kinase Torso at the egg poles. The signal is transduced to the nucleus via a Ras-Raf-MAP-K/Erk phosphorylation cascade, and leads to the expression of the zygotic target genes tailless (tll) and huckebein (hkb) at the posterior terminus of the embryo. Failure to activate Torso signaling results in defects in the head skeleton and loss of all segments posterior to abdominal segment 7, in addition to loss of the hindgut and posterior midgut anlagen (Schoppmeier, 2005 and references therein).
Whether an anteriorly acting terminal system is a general feature of all insects has been challenged because under certain conditions, Torso function at the anterior is dispensable for head development in Drosophila. This hypothesis is supported by the expression of the Tribolium ortholog of tll at the posterior, but not at the anterior pole of blastoderm stage embryos. Thus, in Tribolium, posterior terminal cells appear to be determined before the onset of abdomen formation. It is unknown, however, whether these cells specify posterior fate after axis elongation and abdomen formation is completed or whether they also contribute to earlier steps of segmentation (Schoppmeier, 2005).
The orthologs of the key components of the Torso pathway have been isolated in the short germ beetle Tribolium torso (Tc-tor) and torso-like (Tc-tsl). As in Drosophila, Tc-torso mRNA is maternally inherited by the embryo and expressed ubiquitously in freshly laid eggs, and Tc-tsl is expressed during oogenesis anteriorly and posteriorly in the follicle cells of the oocyte (Schoppmeier, 2005).
Knocking down the function of Tc-torso or Tc-tsl using parental RNA interference leads to identical embryonic phenotypes. Whereas the head and the anterior thorax are unaffected, unexpectedly the most extreme Tc-torsoRNAi and Tc-tslRNAi embryos lack all structures that develop during postblastodermal abdominal growth. Thus, the head and thoracic segments that form in torso or tsl RNAi embryos likely represent the structures, which are determined already during the Tribolium blastoderm stage. Less strongly affected embryos fail to form the full number of abdominal segments (Schoppmeier, 2005).
To determine whether the Tc-torso RNAi phenotype does not reflect a late function of maintaining abdominal fate prior to cuticularization, the expression of Engrailed protein was examined in Tc-torsoRNAi embryos at a stage when abdominal segments should already have developed. Indeed, in strongly affected embryos, Engrailed stripes corresponding to the head and thorax, but not to abdominal segments, are present (Schoppmeier, 2005).
The emergence of segments was visualized in embryos with impaired Torso signaling by analyzing the Tc-even-skipped (Tc-eve) expression pattern. In wild-type embryos, Tc-eve is initially expressed in a double segmental pattern that later resolves into secondary segmental stripes. Tc-tsl RNAi does not interfere with the formation of the first two primary Tc-eve stripes that give rise to the gnathal and the first thoracic (T1) segments. However, although the third primary Tc-eve expression domain (Tc-eve stripe 3) forms normally, this domain does not resolve into segmental stripes, and no additional primary eve-stripes form. In the wild-type, Tc-eve stripe 3 covers the region where the second (T2) and third thoracic (T3) segment will develop. Although Tc-eve stripe 3 does not split in Tc-tsl RNAi embryos, this domain gives rise to the second thoracic segment. Thus, Torso signaling is required for the initiation of axial growth or maintaining the segmentation process (Schoppmeier, 2005).
As revealed by DAPI staining and by morphology, posterior invagination of cells is abolished in both Torso- and tsl RNAi embryos, and as a consequence, no posterior pit forms. To understand how Torso signaling is propagated at the posterior pole and to test whether downstream gene activity is affected in the growth zone in Tc-torsoRNAi embryos, the activity of the Map-kinase and the expression of Tc-wingless (Tc-wg), Tc-tailless (Tc-tll), Tc-caudal (Tc-cad), and Tc-forkhead (Tc-fkh) RNA was analyzed in early embryos (Schoppmeier, 2005).
The active state of the Torso receptor is transduced to the nucleus via the Ras-Raf signal transduction pathway and leads to the activation of zygotic target genes. The activity of this pathway can be visualized with an antibody that recognizes ErkPP but does not discriminate between the different pathways that involve ErkPP signaling. In nontreated embryos, ErkPP can be detected in a subpopulation of the serosa, a single row of cells at the border of the serosa and the embryonic anlage; at the rims of the mesoderm; and at the posterior pole. In torsoRNAi embryos posterior ErkPP expression is lost, further indicating that ErkPP is involved in propagating terminal signaling. ErkPP expression in the serosa is mildly affected whereas the other sites where ErkPP activity is detected in the wild-type are normal. ErkPP activity in the amnion appears not to be reduced; however, the amnion itself does also not form properly. Whether this is a direct or indirect consequence of Torso reduction is unclear (Schoppmeier, 2005).
In addition to segmental stripes, a terminal wingless (wg) expression domain first seen at the blastoderm stage is present throughout the phase of body elongation in the growth zone of the wild-type. In Tc-torsoRNAi embryos, the posterior terminal Tc-wg domain is missing at the blastoderm stage, as well as in older embryos corresponding in age to wild-type embryos undergoing body axis extension. Drosophila-torso mutant embryos also lack the posterior terminal wg expression domain, indicating, that the dependence of wg on torso is conserved. The segmental wg stripes that were built prior to the growth process, form close to the posterior end. This shows that a presegmented region (PSR) normally separating the last segment formed at the posterior end of the embryo is strongly reduced or absent in torsoRNAi embryos. The absence of Tc-cad and Tc-tll in torsoRNAi embryos establishes these genes as potential targets of terminal signaling also in Tribolium (Schoppmeier, 2005).
As judged from the lack of the posterior Tc-forkhead (Tc-fkh) expression domain, Tc-torsoRNAi embryos do not develop a hindgut. Tc-fkh itself, which is also expressed in the growth zone throughout most of the segmentation process, seems not to be required in the axis elongation process because Tc-fkh RNAi leads only to malformation of the hindgut (Schoppmeier, 2005).
The irregular expression of ErkPP in the serosa of embryos deficient for Torso signaling suggests a function for this pathway also in patterning this extraembryonic tissue. Indeed, in Tc-tslRNAi embryos, the serosa is severely reduced in size whereas the presumptive head region appears enlarged and extended toward the anterior. This finding is corroborated by the expanded expression domain of the head marker gene Tc-006A12 in Tc-tslRNAi embryos, which in wild-type embryos is expressed just posterior of the serosa in a wedge-shaped domain. Nevertheless, embryos develop with normal head structures, similarly as in embryos where serosa reduction results from reduction of zen-1 activity (Schoppmeier, 2005).
The anterior phenotype, however, differs among embryos depleted for either Tc-torso or Tc-tsl. Compared with the Tc-tsl RNAi, the serosa is less affected with Tc-torso RNAi, indicating that Tc-tsl has been more sufficiently downregulated via RNAi than Tc-torso (Schoppmeier, 2005).
Thus, in a short germ embryo Torso signaling is—as in Drosophila—required for patterning both the anterior and the posterior region of the embryo. Due to the difference in the anlagenplan of short and long germ embryos, however, different tissues and different processes depend on the Torso pathway (Schoppmeier, 2005).
This study has presented the first functional characterization of the terminal-class genes torso and torso-like outside the dipterans. At the anterior, Torso signaling is involved in the specification of the anterior-most structure in the Tribolium egg, the extraembryonic serosa. At the posterior, Tc-torso-like and Tc-torso are required for terminal fates, including the posterior gut primordial and for body axis growth (Schoppmeier, 2005).
These results show that the terminal system in Tribolium functions to establish the growth zone and to initiate rostrocaudal growth. In the absence of the terminal signal, caudal expression and pair-rule patterning is not maintained during later stages of development. It is not known how the loss of posterior patterning and the loss of posterior growth relate to each other. Since the growth zone specific expression domain of wg depends on torso one could speculate that the posterior domain of Tc-wg is required for both, regulation of cell proliferation and coordination of continued segmental patterning. The involvement of the Wg pathway in the axis elongation process has already been demonstrated for Gryllus (Schoppmeier, 2005).
Insects like Drosophila that develop as long germ embryos have short life cycles and are thus perfectly adapted to quickly changing environments like rotting fruits. During Drosophila embryogenesis, the posterior-most Engrailed stripe corresponding to abdominal segment 9 forms under the influence of Torso slightly later than the other stripes. This could be seen as a rudimentary elongation of the body axis in Drosophila and likely reflects the ancient function of Torso signaling (Schoppmeier, 2005).
In contrast to long germ embryos, the formation of the complete trunk occurs in a secondary growth process in short germ embryos of arthropods and vertebrates. It is proposed that the involvement of Torso signaling in body axis growth likely represents the ancient function of this gene in the development of short germ insects. Thus, during the evolution from short to long germ development, Torso signaling lost its major function in axial growth and was recruited as an additional gradient system to specify the position of posterior abdominal segmentation gene domains during the blastoderm stage. Whether Torso signaling is involved in body axis growth also in other short germ animals remains to be shown (Schoppmeier, 2005).
date revised: 10 March 99
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