BIOLOGICAL OVERVIEW

Trunk is a secreted protein with growth hormone characteristics. Its sequence resembles Spätzle, the putative ligand for Toll; both are directly involved in dorso-ventral polarity. Spätzle is activated proteolytically by Easter on the ventral side of the embryo.

Trunk is distributed to nurse cells but not follicle cells in oogenesis, and is transferred to the mature oocyte. The distribution in the early embryo is uniform. This raises a question: with its uniform distribution in egg and zygote, why should Trunk be a candidate for the Torso ligand, the major determinant in anterior-posterior polarity? One might reasonably expect a ligand involved in polarity to be asymmetrically distributed. The answer is to be found in its mutant phenotype, known now for over a decade (Schumpbach, 1986). trunk mutants resemble torso mutants in their mutual loss of both anterior and posterior structures. Given the uniform distribution of Trunk, assuming it is the ligand of Torso, how do its effects become localized? Since Torso is an oocyte protein, wouldn't its ligand be expected to be produced by follicle cells and not the oocyte?

A possible second candidate for the Torso receptor is torso-like (tsl). tsl has all the necessary qualities of a Torso ligand save one. TSL is produced by anterior and posterior follicle cells. This puts it in the right place at the right time. Mutants of tsl also produce similar polar deficiencies. However the structure of TSL does not resemble that of ligands for hormone receptors. If in fact Trunk is the Torso ligand, then what is the role for TSL? There is precedent for the requirement of follicular protein in the activation of Spätzle, the Toll ligand. easter and several other genes are transcribed in follicle cells and have roles in the local activation of Spätzle. Perhaps TSL fulfills a similar function in the local activation of Trunk (Casanova, 1995).

It seems likely that Trunk, a ubiquitiously distributed ligand, may become locally activated by locally supplied enzymes. Torso, the putative receptor for Trunk, is uniformly distributed in the egg. Therefore, by local activation, a ubiquitously distributed ligand could function to trigger a receptor, even if both receptor and ligand are carried by the oocyte.

GENE STRUCTURE

cDNA clone length

There appear to be multiple transcripts for trunk that differ in their polyadenylation sites. Two of the termination sites are found within the coding sequence and could yield truncated proteins. cDNAs generated by early termination signals have been isolated (Casanova, 1995).

Bases in 5' UTR - 41

Bases in 3' UTR - 60

PROTEIN STRUCTURE

Amino Acids - 226

Structural Domains

trunk encodes a protein that resembles Spitz in several respects. In particular, the sequence suggests that TRK is a secreted protein and that it contains an internal site for proteolytic cleavage. Furthermore, the carboxy-terminal domain of TRK has an arrangement of cysteines similar to that of Spätzle (Casanova, 1995).

REGULATION

Proteolytic Processing of Trunk

The Torso tyrosine kinase receptor is distributed along the surface of the embryo but it is only activated at the poles by a diffusible extracellular ligand generated at each pole that is trapped by the receptor, thereby impeding further diffusion. Although it is known that this signal depends on the activity of several genes, such as torso-like and trunk, it is still unclear how it is generated. The identification of the signal responsible for the Torso receptor activation is an essential step towards understanding the mechanism that regulates the local restriction of torso signaling (Casali, 2001).

The molecular characterization of the trk gene has shown that it encodes a protein with analogies to extracellular ligands and is likely to be cleaved. More precisely, the Trk protein resembles Spz, the putative ligand for the Toll receptor. The analysis of the trk sequence has revealed a putative cleavage site at position 67 in the predicted protein that would generate a carboxy-terminal fragment of 159 amino acids (Trk^C-159). In addition, the conserved arginine immediately upstream of this putative cleavage site is changed to a glutamine in the trk⁶ loss-of-function mutation. The observation that a mutation in a putative cleavage site eliminates the biological function of the Trk protein suggests that cleavage is an essential step for Trk activity. Further analysis of the Trk sequence has unveiled an additional putative cleavage site at position 119, which would give rise to a carboxy-terminal fragment of 108 amino acids (Trk^C-108). Several reasons suggested that this additional site could also be important for Trk activity. (1) Trk shares some sequence similarity with the prothoracicotropic hormone (PTTH) of Bombyx mori, which is presumed to be cleaved at different sites to yield a final carboxy-terminal fragment of 109 amino acids. (2) Spz is cleaved at a similar position between Arg220 and Val221 The cleavage site of Spz, VSSRVGGS, bears some similarity to the putative cleavage site of Trk: FHDRVGHP. (3) Most of the active forms of the growth factors with a cystine knot motif possess only a small fragment amino terminal to the first cysteine of the knot. In this regard, the Trk carboxy-terminal fragment generated by cleavage at position 119 (Trk^C-108) fits this 'rule' better than the fragment generated by cleavage at position 67. The analysis of the Trk sequence, the presence of putative cleavage sites and the mutation on a cleavage site that abolishes Trk function prompted a study of the biological activity of deleted forms of the trk gene (Casali, 2001).

A fragment containing the carboxy-terminal 108 amino acids of the trunk protein retains Trunk activity and is sufficient to activate Torso signaling. This fragment bypasses the requirements for the other genes involved in the activation of the Torso receptor. These results suggest that a cleaved form of the Trunk protein acts as a signal for the Torso receptor. It is therefore proposed that the restricted activation of the torso receptor is defined by the spatial control of the proteolytic processing of the trunk protein (Casali, 2001).

The C-terminal domain of Trk is sufficient to activate the Tor receptor but its activity appears to be inhibited by the presence of the amino-terminal domain of the protein. Although the deletion mutations that have been generated may not represent the actual in vivo protein, the hypothesis is favored that the truncated proteins could mimic the in vivo active protein and relieve the C-terminal region from such an inhibition. In particular, the observation that a mutation in a putative cleavage site eliminates the biological function of the Trk protein suggests that cleavage is physiologically relevant for Trk activity and weakens the possibility that the designed truncated forms would artificially alter the normal process of Trk protein activation (Casali, 2001).

The analysis of trk has unveiled the existence of two putative cleavage sites. Indeed, two different deletions generated at each site that retain 159 and 108 amino acids respectively are both able to activate Tor signaling in the embryos upon RNA injection. The favored hypothesis is that the smaller fragment could be similar to the in vivo active Trk fragment whereas the larger one could correspond to an intermediate form. In that case, the in vivo processing of the Trk protein could involve more that one cleavage event. Several observations support this model. First, Trk^C-108 is sufficient to activate Tor signaling as indicated by the expression of both tll and hkb, suggesting that the additional amino acids in Trk^C-158 are dispensable for Tor activation. In addition, Trk^C-108 fits very well with the other known active cystine-knot factors, both in its overall size and in the small amino acid chain just before the first cysteine of the knot. Finally the putative cleavage site at the basis of Trk^C-158 appears to be functionally important since a specific mutation at this site renders the protein inactive. Thus, both cleavage events could be taking place in the in vivo processing of Trk (Casali, 2001).

DEVELOPMENTAL BIOLOGY

Embryonic

During oogenesis TRK mRNA can be detected in nurse cells but not follicle cells. Transcripts are uniformly distributed in early embryos (Casanova, 1995).

Effects of mutation or deletion

Females mutant for either torso or trunk produce embryos with deletion in various anterior head structures (labrum, chitinous mouth plates) and the most posterior abdominal structures (posterior midgut, telson, abdominal segment 8). These effects are due to maternal deficiency in trunk (Schupbach, 1986).

Mutations in torso and trunk that express low levels of the respective protein have differential affects on the expression of tailless and huckebein. For example a reduced amount of TRK can trigger signaling of TOR to levels required to activate tll but not hkb. For a given number of TOR receptors, an increase in the amount of TRK results in the appearance of more structures of the most posterior segment (A8) (Furriols, 1996).

Regulated activation of receptor tyrosine kinases depends both on the presence of the receptors at the cell surface and on the availability of their ligands. In Drosophila, the Torso (Tor) tyrosine kinase receptor is distributed along the surface of the embryo but it is only activated at the poles by a diffusible extracellular ligand (generated at each pole), which is trapped by the receptor, thereby impeding further diffusion. However, it is not well understood how this signal is generated, although it is known to depend on the activity of many genes such as torso-like (tsl) and trunk (trk). To further investigate the mechanism involved in the local activation of the Tor receptor, the normal expression of the Tsl protein has been altered by generating females in which the tsl gene is expressed in the oocyte under the control of the tor promoter, rather than in the ovarian follicle cells. Analysis of the phenotypes generated by this hybrid gene and its interactions with mutations in other genes in the pathway has enabled dissection of the mechanism of Tor receptor activation and a more precise definition of the role of the different genes acting in this process. Uniform expression of tsl in the oocyte is able to trigger unrestricted activation of the Tor receptor. Observed is an expansion of the terminal portions of the body at the expense of the middle body segments. Specifically, tailless expression expands in mutant embryos. Triggering of Tor activation by Tsl expressed in the germ-line requires the product of the trk gene. Triggering of Tor activation by Tsl in the germ-line requires the product of fs(1)ph and fs(1)Nas. These two gene products are thought to be involved in the process of transference of the Tsl product from the follicle cells to the germ-line. But these two gene products are still required for Tor receptor activation, even when Tsl is expressed in the germ-line (Furriols, 1998).

Spatially distinct downregulation of Capicua repression and Tailless activation by the Torso RTK pathway in the Drosophila embryo

Specification of the terminal regions of the Drosophila embryo depends on the Torso RTK pathway, which triggers expression of the zygotic genes tailless and huckebein at the embryonic poles. However, it has been shown that the Torso signalling pathway does not directly activate expression of these zygotic genes; rather, it induces their expression by inactivating, at the embryonic poles, a uniformly distributed repressor activity. In particular, it has been shown that Torso signalling regulates accumulation of the Capicua transcriptional repressor: as a consequence of Torso signalling Capicua is downregulated specifically at the poles of blastoderm stage embryos. Extending the current model, it is shown that activation of the Torso pathway can trigger tailless expression without eliminating Capicua. In addition, analysis of gene activation by the Torso pathway and downregulation of Capicua unveil differences between the terminal and the central embryonic regions that are independent of Torso signalling, hitherto thought to be the only system responsible for confering terminal specificities. These data provide new insights into the mode of action of the Torso signalling pathway and on the events patterning the early Drosophila embryo (de las Heras, 2006).

While the Tor pathway is normally activated only at the embryonic poles, tor constitutive mutations trigger its activation over the entire embryo in a ligand-independent manner. In these cases, expression of the tor target genes is expanded too much broader domains and embryos develop head and tail structures lacking most of the segmented trunk. According to the current model one would expect that tll domain expansion in these mutations would be accompanied by an expansion of the Cic downregulation domain (de las Heras, 2006).

Embryos from mutant females bearing the tor^D4021 constitutive mutation (a strong gain-of-function mutation that acts as a dominant female sterile) have been analyzed and instead it was found that Cic protein is still downregulated only at the poles, as in the wild-type embryos. Therefore, while in the wild-type the posterior tll domain is complementary to the domain of Cic accumulation, in embryos from tor^D4021/+females these domains overlap and tll is expressed in spite of the presence of nuclear Cic. This behaviour is not allele-specific since embryos from homozygous females for another tor constitutive mutation (tor^RL3) display the same kind of Cic distribution and tll expression (de las Heras, 2006).

It has been postulated that wild-type Tor receptors and Tor receptors activated by ligand-independent constitutive mutations could signal through distinct downstream effectors. Therefore, whether the persistent accumulation of Cic in embryos from tor constitutive mutant females could be due to a distinct property of these mutations was analyzed. Alternatively, the persistent Cic accumulation could reflect a difference in response between Tor activation in the middle versus the terminal embryonic regions. To test these possibilities, ligand-dependent activation of the Tor receptor was triggered over the entire embryo by general expression of the torso-like (tsl) gene. tsl is the only known gene in the Tor pathway whose expression is locally restricted. Indeed its restricted expression in a group of cells at each end of the developing oocyte is the determinant for the local activation of the Tor pathway, since its ectopic expression is sufficient to induce widespread activation of the Tor receptor. Accordingly, it was found that driving tsl expression with a tubGAL4 driver in the oocyte gives rise to an expansion of the tll expression domain and to the generation of embryos with a tor-gain-of-function phenotype, in that they develop head and tail structures and lack most of the segmented trunk. However, and similarly to what is described above for tor constitutive mutations, in these embryos Cic downregulation is not expanded to a broader domain, indicating that even ligand-induced activation of the Tor pathway is unable to inhibit Cic protein accumulation in the embryonic middle regions (de las Heras, 2006).

In the experiments described above, activation of the Tor pathway over the whole embryo did not result in an expansion of Cic downregulation. Paradoxically, activated Tor could trigger downstream targets in the middle region even though Cic was still present. These observations raise the question of whether under these circumstances Cic is still able to act as a transcriptional repressor. Alternatively, Tor signalling could impair cic activity without removing Cic protein from the nuclei. To address this issue, the contribution of cic function was analyzed in embryos from tor constitutive mutants (de las Heras, 2006).

The strong transformations associated with the ectopic activation of the Tor pathway due to tor^D4021 mutations and tubGAL4 driven expression of tsl make it difficult to assess the operational state of the Cic repressor under these circumstances. To overcome this difficulty use was made of the weaker tor^RL3 constitutive mutation and cuticular transformations, which are more sensitive to small changes in the expression of tor targets genes than what can be visualized by whole mount in situs, were scored. Besides, in the following experiments the tor^RL3 genotype was examined in a trunk (trk) background to eliminate ligand-induced activation. On its own, a single copy of tor^RL3 gives rise to a very mild phenotype, in which occasionally one abdominal segment is deleted. In contrast, removing just one copy of the cic gene does not affect the embryonic pattern. However, a single copy of the tor^RL3 mutation combined with the removal of just one copy of the cic gene gives rise to prominent transformations; embryos from such females display variable phenotypes but in every case they show major deletions of the embryonic segments. Accordingly, there is an expansion of the domain of tll expression, which also in that case overlaps with the domain where Cic accumulates. In this situation, whether nuclear Cic protein is still functional can be assessed by removing the remaining copy of the cic gene and comparing the two phenotypes. Indeed, embryos from trk tor^RL3/+; cic/cic have a much stronger phenotype that those from trk tor^RL3/+; cic/+. Therefore, the Cic protein present in trk tor^RL3/+; cic/+ embryos is still at least in part functional implying that the tor^RL3 mutation is able to trigger tll activation without eliminating all cic repression activity (de las Heras, 2006).

What mechanisms are activated by Tor signalling that could bypass the need for Cic downregulation to activate terminal target genes? It has been suggested that the Stat92E transcription factor plays a role as a mediator of Tor signalling elicited by a Tor constitutive mutant receptor, but not in Tor signalling promoted by ligand-dependent activation of the receptor at the poles. The role of Stat92E was assessed in the tor constitutive mutant background. A reduction was found in the transformations associated with the trk tor^RL3/+; cic/+ genotype by removing a single copy of the stat92E gene. Whether this could also apply in the case of ectopic activation of the Tor pathway through ligand binding was analyzed; also in this case it was found that there is a reduction of the strength of the phenotype. In this case, however, the reduction is smaller, which could be due to the fact that the original transformation generated by the tubGAL4/UAStsl combination is much stronger and/or to a weaker involvement of stat92E in ligand-induced Tor signalling. Regardless, the results suggest that there is no fundamental difference in the role of stat92E between ligand-induced or constitutive activation of the Tor receptor. In support of this conclusion there is the recent observation that Stat92E is specifically phosphorylated at the poles by ligand-induced Tor signalling. Therefore, similarly to what was observed in the embryonic middle regions, it is proposed that Tor could also induce tll activation in the poles, and this occurs by a Cic downregulation-independent mechanism via stat92E. Altogether these results suggest that Tor signalling could normally trigger tll expression at the poles of wild-type embryos by two kinds of regulatory mechanisms, relief of cic repression and positive activation of tll expression. The positive effect of Tor signalling on tll expression could have been obscured by the fact that there is also a still unidentified Tor-independent activator, since terminal fate is specified in embryos lacking both Tor signalling and Cic repression. Accordingly, it has to be noted that stat92E mutants suppress ectopic activation of tll in the middle embryonic regions but not tll activation at the poles, which suggests that the role of stat92E on Tor signalling could be somehow redundant at the poles but absolutely required when Tor signalling is triggered in the embryonic middle regions (de las Heras, 2006).

The following conclusions can be drawn from these results. First, while activation of the Tor pathway at the embryonic poles downregulates Cic, Tor signalling appears to be necessary but not sufficient to eliminate Cic protein, as it can do so only at the embryonic poles. In this regard, it has to be noted that recent results indicate that the posterior maternal system can also affect Cic downregulation. Second, impairment of Cic repressor function is not an absolute requirement for tll expression, since tll can be expressed in situations where Cic repressor is still functional. In this regard, tll expression appears to be the result of a balance between repressor and activator factors and Cic repression might be overcome provided that activation is enhanced. And finally, there are differences between the terminal and the central embryonic regions that are independent of Tor signalling, as judged by the spatially restricted capacity of the Tor pathway to inhibit Cic accumulation and by the apparently distinct regional redundancy of stat92E function in Tor-dependent patterning. These results suggest that the Tor signalling pathway is not the only system that establishes a difference between the terminal and the central regions of the Drosophila embryo (de las Heras, 2006).

REFERENCES

Casali, A. and Casanova, J. (2001). The spatial control of Torso RTK activation: a C-terminal fragment of the Trunk protein acts as a signal for Torso receptor in the Drosophila embryo. Development 128: 1709-1715. 11290307

Casanova, J., et al. (1995). Similarities between trunk and spätzle, putative extracellular ligands specifying body pattern in Drosophila. Genes Dev 9: 2539-2544

de las Heras, J. M. and Casanova, J. (2006). Spatially distinct downregulation of Capicua repression and Tailless activation by the Torso RTK pathway in the Drosophila embryo. Mech. Dev. 123(6): 481-6. 16753285

Furriols, M., Sprenger, F. and Casanova, J. (1996). Variation in the number of activated torso receptors correlates with differential gene expression. Development 122: 2313-2317. 8681811

Furriols, M., Casali, A. and Casanova, J. (1998). Dissecting the mechanism of torso receptor activation. Mech. Dev. 70(1-2): 111-118. 9510028

Schupbach, T. and Wieschaus, E. (1986). Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila. Dev. Biol. 113: 443-448

date revised: 20 December 2006
 

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