torso

DEVELOPMENTAL BIOLOGY

Embryonic

TOR protein is uniformly expressed along the surface membrane of early embryos despite its localized activity at both poles. Polarized activity of this protein depends on other terminal gene functions, one of which may be a localized extracellular ligand generated during oogenesis. Different levels of active TOR protein can specify distinct portions of the terminal pattern. Thus, TOR functions as a ubiquitous surface receptor that is activated by a spatially restricted ligand. Localized activity of the TOR kinase may generate one or more gradients of intracellular signals that control body pattern (Casanova, 1989).

A MAPK docking site is critical for downregulation of Capicua by Torso and EGFR RTK signaling

Early Drosophila development requires two receptor tyrosine kinase (RTK) pathways: the Torso and the Epidermal growth factor receptor (EGFR) pathways, which regulate terminal and dorsal-ventral patterning, respectively. Previous studies have shown that these pathways, either directly or indirectly, lead to post-transcriptional downregulation of the Capicua repressor in the early embryo and in the ovary. This study shows that both regulatory effects are direct and depend on a MAPK docking site in Capicua that physically interacts with the MAPK Rolled. Capicua derivatives lacking this docking site cause dominant phenotypes similar to those resulting from loss of Torso and EGFR activities. Such phenotypes arise from inappropriate repression of genes normally expressed in response to Torso and EGFR signaling. These results are consistent with a model whereby Capicua is the main nuclear effector of the Torso pathway, but only one of different effectors responding to EGFR signaling. Finally, differences in the modes of Capicua downregulation by Torso and EGFR signaling are described, raising the possibility that such differences contribute to the tissue specificity of both signals (Astigarraga, 2007).

Nuclear trapping of ERK downstream of Torso shapes the terminal gradient in the Drosophila embryo

Patterning of the terminal regions of the Drosophila embryo relies on the gradient of phosphorylated ERK/MAPK (dpERK), which is controlled by the localized activation of the Torso receptor tyrosine kinase. This model is supported by a large amount of data, but the gradient itself has never been quantified. This study presents the first measurements of the dpERK gradient and establishes a new intracellular layer of its regulation. Based on the quantitative analysis of the spatial pattern of dpERK in mutants with different levels of Torso as well as the dynamics of the wild-type dpERK pattern, it is proposed that the terminal-patterning gradient is controlled by a cascade of diffusion-trapping modules. A ligand-trapping mechanism establishes a sharply localized pattern of the Torso receptor occupancy on the surface of the embryo. Inside the syncytial embryo, nuclei play the role of traps that localize diffusible dpERK. It is argued that the length scale of the terminal-patterning gradient is determined mainly by the intracellular module (Coppey, 2008).

This study identifies the nuclear trapping of dpERK as a mechanism responsible for the intracellular spatial processing of the terminal signal. This conclusion is based on the analysis of the dynamics of the wild-type dpERK gradient. Between nuclear cycles 10 and 14, the dpERK levels are amplified at the termini and attenuated in the subterminal regions of the embryo. The observed dynamics of the dpERK gradient is consistent with a model where dpERK is a diffusible molecule, which is trapped and dephosphorylated by the nuclei. A uniform increase in the nuclear density would increase the trapping of the dpERK molecules at the poles and prevent their diffusion to the middle of the embryo. This is consistent with biochemical and imaging data showing that dpERK rapidly translocates to the nucleus, which can also serve as a compartment of dpERK dephosphorylation. In addition, the model makes a testable prediction about the dynamics of the nucleocytoplasmic (N/C) ratio of phosphorylated MAPK (Coppey, 2008).

The nuclear and cytoplasmic levels of dpERK were quantified in cycle 13 and 14 embryos. Plotting the nuclear and cytoplasmic profiles against each other gives a clear linear relationship, as predicted by a simple formula. Furthermore, the nucleocytoplasmic ratio clearly increases between these two nuclear cycles: the N/C ratio is ~1.4 and ~2 at nuclear cycles 13 and 14, respectively. These measurements show that the nuclear trapping rate is indeed an increasing function of the nuclear density, as predicted by the model (Coppey, 2008).

The observed N/C ratios show that a significant fraction of total dpERK nuclear. As a consequence, defects in the nuclear density should generate clear defects in the gradient. This can be tested in mutants with 'holes' in the nuclear density in blastoderm embryos. For example, in shakleton (shkl) embryos, the migration of nuclei to the poles is delayed and a number of embryos exhibit major disruptions in nuclear density. As predicted by the model, shkl embryos show striking disruptions in the dpERK gradient. The quantified posterior gradient of this particular mutant embryo shows a clear local correlation with the nuclear distribution, emphasizing the role of the nuclei at this stage. In early embryos, the gradient is more extended, presumably reflecting the lack of the nuclei at the poles. Similar defects were found in the giant nuclei (gnu) mutant embryos, which show a different type of defect in nuclear organization. These results support the model in which the syncytial nuclei play an important role in shaping the dpERK gradient (Coppey, 2008).

To summarize, it is proposed that the dpERK gradient is controlled by a cascade of at least two diffusion-trapping modules. In the extracellular compartment, a ligand-trapping mechanism, identified in previous studies, establishes a sharp gradient of Torso receptor occupancy. A similar mechanism regulates the dpERK gradient inside the embryo, where syncytial nuclei act as traps that localize diffusible dpERK. At this time, it cannot be ruled out that the observed sharpening of the dpERK gradient can be modulated also by changes in the spatial distribution of the Torso ligand, but currently there are no data in support of this mode of regulation (Coppey, 2008).

The dynamics of the dpERK gradient is qualitatively different from that of the Bicoid gradient, which remains stable during the last five nuclear divisions. It has been proposed that a stable gradient of Bicoid can be established in the absence of Bicoid degradation, because of the reversible trapping of Bicoid by an exponentially increasing number of nuclei. The differences between the dynamics of the Bicoid and dpERK gradients are attributed to two effects. The first effect is due to the differences in the 'chemistries' of the two systems: the morphogen in the terminal system is degraded (MAPK is dephosphorylated), whereas the anterior morphogen is stable (it is proposed that Bicoid is not degraded on time scale of the gradient formation). The second effect is due to the differences in the initial conditions: by the 10th nuclear division, which is the starting point of the activation of the terminal system, Bicoid gradient is essentially fully established. Thus, a common biophysical framework can describe the Bicoid and dpERK gradients. It remains to be determined whether the nuclear export affects the length scale of the Dorsal gradient, which patterns the dorsoventral axis of the embryo (Coppey, 2008)

Effects of Mutation or Deletion

The formation of the unsegmented terminal regions of the Drosophila larvae, acron (the terminal head structure including the brain) and telson (the terminal tail structure) requires the function of at least five maternal genes (terminal genes class). In their absence, the telson and acron are not formed. One of them, torso, has gain-of-function alleles that have an opposite phenotype to the lack-of-function (tor-) alleles: the segmented regions of the larval body, thorax and abdomen, are missing, whereas the acron is not affected and the telson is enlarged. In strong gain-of-function mutants, the pair-rule gene fushi tarazu (ftz) is not expressed, demonstrating the suppression of the segmentation process in an early stage of development. The tor gain-of-function effect is neutralized, and segmentation is restored in double mutants with the zygotic gene tailless (tll) that has a phenotype similar (but not identical) to that of tor-. This suggests that TOR acts through tll, and that in the gain-of-function alleles of tor, the TLL gene product is ectopically expressed in the middle regions of the embryo, where it inhibits the expression of segmentation genes like ftz (Klingler, 1988).

14-3-3 proteins have been shown to interact with Raf-1 and cause its activation when overexpressed. However, their precise role in Raf-1 activation is still enigmatic, as they are ubiquitously present in cells and found to associate with Raf-1 in vivo regardless of Raf's activation state. The function of the Drosophila 14-3-3 gene leonardo (leo) has been analyzed in the Torso (Tor) receptor tyrosine kinase (RTK) pathway. In the syncytial blastoderm embryo, activation of Tor triggers the Ras/Raf/MEK pathway that controls the transcription of tailless (tll). In the absence of Tor, overexpression of leo is sufficient to activate tll expression. The effect of leo requires D-Raf and Ras1 activities but not KSR or DOS, two recently identified essential components of Drosophila RTK signaling pathways. Tor signaling is impaired in embryos derived from females lacking maternal expression of leo. It is proposed that binding to 14-3-3 by Raf is necessary but not sufficient for the activation of Raf and that overexpressed Drosophila 14-3-3 requires Ras1 to activate D-Raf (Li, 1997).

Hypoactivity in torso results in the loss of the most posterior domain of fushi tarazu expression and the terminal cuticular structures. In contrast, a torso hyperactivity mutation causes the loss of central fushi tarazu expression and central cuticular structures. This effect is caused by abnormal persistence of the Torso product in the central region of the embryo during early development. Thus, the amount and timing of torso activity is key to distinguishing the central and terminal regions of the embryo. Mutations in the tailless terminal gene act as dominant maternal suppressors of the hyperactive torso allele, indicating that the torso product acts through, or in concert with, the tailless product (Strecker, 1989).

Injecting eggs with in vitro synthesized Torso mRNAs revealed that torso activation is governed by an extracellular molecule produced at terminal regions of the egg during early embryogenesis. Mutant ligand-binding Torso proteins can suppress telson formation in a dominant negative manner, suggesting that the ligand is limited in amount. Analysis of torso mutations indicates that Torso functions as a tyrosine kinase and that gain-of-function mutations causing ligand-independent activation are located in the extracellular domain (Sprenger, 1992).

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).

hindsight expression in the midgut is controlled by the maternal and zygotic members of the torso mediated terminal pathway. Embryos produced by homozygous torso loss-of-function mutant females lack Hnt protein in the posterior midgut, which lies within the domain of torso function. Instead of extending their germ bands dorsoanteriorly, most such embryos form spiralled germ bands. Reciprocally, embryos carrying torso gain of function mutations lack dorsal expression (that is, in the presumptive amnioserosa), consistent with conversion of central cell fates to more terminal ones. These embryos also show expanded expression of Hnt protein in the enlarged posterior midgut primordium and a twisted gastrulation phenotype (Yip, 1997).

Eight alleles of Dsor1 encoding a Drosophila homolog of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppress mutations for receptor tyrosine kinases (RTKs) torso, sevenless, and to a lesser extent, Drosophila EGF receptor. Furthermore, the Dsor1 alleles show no significant interaction with gain-of-function mutations of Egfr. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrates that Dsor1 can activate yeast MAP kinase homologs if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes reveal that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors rather than constitutive activity (Lim, 1997).

To investigate a Ras-independent means of activating the Mapk cascade, mutations have been isolated that suppress the lethality of a Drosophila Raf mutation [also referred to as l(1) pole hole]. Six extragenic Su(Raf) loci have also been identified. These mutations not only suppress Raf^C110 but also other partial loss-of-function Raf alleles that do not impair Ras-Raf binding. This suggests that the suppression of Raf^C110 by the extragenic Su(Raf) mutations does not necessarily involve the restoration of Ras-Raf binding. Developmental analyses have shown that all six extragenic Su(Raf) mutations promote signaling in the Sevenless (Sev) and Egfr RTK pathways. Su(Raf)34B is a gain-of-function mutation in the Dsor1 locus that encodes the fly Mek. Recently, Su(Raf)1 has been shown to encode Src42A. The isolation of mutations that suppress the suppressor activity of Su(Raf)1 is reported in this paper. These mutations define two known genes, Egfr and rolled (rl; also referred to as Mapk) and two previously uncharacterized loci. In addition, two alleles of Src42A were also isolated in the screen, although these mutations are not true suppressors of Su(Raf)1 (Zhang, 1999).

One of the novel suppressor loci was named semang (sag). sag is required during both embryonic and imaginal disc development. Mutations in sag cause zygotic lethality. To identify developmental pathways where sag functions, the phenotypes associated with sag mutations were examined with particular attention to those processes controlled by known Drosophila RTKs. The results of these analyses show that sag participates in the Torso (Tor) and Drosophila DFGF-R1 RTK (Breathless) pathways during embryonic development. sag also disrupts the embryonic peripheral nervous system. During imaginal disc development, sag mutations affect two processes known to require Egfr signaling: the recruitment of photoreceptor cells and wing vein formation. Thus sag functions broadly in several RTK-mediated processes. This role of sag in RTK signaling is further supported by the genetic interaction between sag and other known RTK signaling genes. sag dominantly enhances the phenotypes caused by reductions of RTK signaling in loss-of-function Raf or rl mutants. Consistent with this, sag dominantly suppresses the formation of supernumerary R7 cells caused by the activated sev-Ras1^V12 mutation. The sag mutations analyzed are likely to be loss-of-function mutations. These results suggest that sag may have a positive role in RTK signaling (Zhang, 1999).

Focus was placed on processes known to be controlled by RTKs. At the beginning of embryogenesis, the Tor RTK pathway specifies the embryonic terminal cell fates. Activation of Tor at the embryonic poles triggers the Ras-Mapk signaling cascade, resulting in the expression of two transcription factors: tailless (tll) and huckebein (hkb). tll and hkb in turn activate genes required for terminal development. Whenever there are reductions of tll and hkb expression due to reduced levels of Tor signaling, deletions of terminal structures occur. In the absence of Tor, the mutant embryos lack the anterior acron and all structures posterior to abdominal segment seven (A7). In sag homozygote mutant embryos derived from sag GLC eggs crossed to sag heterozygote males, terminal defects similar to those of the Raf^PB26 allele are observed. The head skeletal structure is collapsed; the tail region contains a partial deletion of the abdominal segment eight; the size of the anal pads is reduced and associated structures appear abnormal. The expressions of tll and hkb at the posterior embryonic pole are solely activated by tor signaling, whereas the anterior expressions are also activated by the bicoid morphogene. In wild-type cellular blastoderm embryos, tll is expressed posteriorly from 0% to 15% egg length (EL; 0% EL is at the posterior pole); hkb is expressed posteriorly from 0% to 9% EL. In sag mutant embryos, posterior tll expression is reduced to 10% EL; posterior hkb expression is reduced to 6% EL. In other words, there is an ~30% reduction of the posterior expressions of both tll and hkb in sag mutants. The anterior expression of these two genes appears grossly normal, with perhaps a slight broadening of tll and a slight reduction of hkb. Consistent with this, the anterior head defect appears variable and is only observed in 50% of the embryos. These results suggest that sag is involved in tor signaling, although the mutation blocks tor signaling to a lesser extent than a Ras1 gene deletion mutation. In embryos lacking maternal Ras1⁺, the posterior tll expression domain is reduced to 5% EL and hkb is not expressed at the posterior. The residual tll expression in the Ras1 mutant embryos reflects the functioning of the Ras1-independent pathway that activates the Mapk cascade (Zhang, 1999).

Drosophila has two other Src family members, Src64 and Tec29, both of which are involved in ring canal development during oogenesis. Src64 does not affect viability when mutated. The isolation of Su(Raf)1 as a mutation in Src42A that restores the viability of Raf mutants and the isolation of Egfr, rl, and sag as extragenic suppressors of Su(Raf)1 provides the first in vivo evidence that both Src42A and sag are modulators of RTK signaling. At this moment, it is not known where Src42A and sag fit into the known RTK signaling cascade. An Src42A cDNA driven by a ubiquitously expressing promoter rescues the lethality of both Su(Raf)1 homozygotes and Su(Raf)1/Df hemizygotes. Based on this, Su(Raf)1 has loss-of-function characteristics, suggesting that Src42A is, unexpectedly, a negative modulator of RTK signaling. However, the genetics of Su(Raf)1 suggest that the suppression of Raf^C110 may be attributed to a dominant-interfering effect because the Raf^C110 lethality is not suppressed in Src42A hemizygotes of genotype Df(2R)nap⁹/+. Because of this, the role of Src42A in RTK signaling is still being investigated. However, the genetic interaction as revealed by the modifying screen suggests that Egfr and other RTKs may possibly regulate Src42A and sag, which in turn modulate the Mapk cascade (Zhang, 1999).

Transcriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFB homolog Dorsal (Dl). Dl acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), Dl is converted into a repressor. Gro is recruited to the enhancer element in the vicinity of Dl by sequence-specific transcription factors such as Dead Ringer (Dri). The interplay between Dl, Gro and Dri on the hkb enhancer was examined and it was shown that when acting over a distance, Gro abolishes rather than converts Dl activator function. However, reducing the distance between Dl- and Dri-binding sites switches Dl into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro -- quenching and silencing of transcription -- are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with Dl. RTK signaling provides a way of modulating Dl function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer (Hader, 1999).

The cis-acting element has been identified that mediates expression of the Drosophila gene hkb, which is necessary for terminal pattern formation and to size the mesoderm anlage in the blastoderm embryo. Deletion analysis of this element reveals a 162 base pair (bp) sub-element that integrates the activities of the Tor-dependent RTK signaling cascade and the morphogen Dl. This element, termed hkb ventral element (VE), comprises a 112 bp ventral activator element (VAE) and a 50 bp ventral repressor element (VRE) (Hader, 1999).

The VAE contains a Dl-binding site, identified in vitro, and mediates gene activation along the ventral side of the embryo. VAE-mediated gene expression is absent in embryos lacking Dl activity and extends throughout Toll^10b mutants, in which Dl is present in all nuclei of the embryo. The expression pattern is not altered in embryos lacking snail and twist, the zygotic mediators of Dl. It is also not affected in embryos that lack Tor or express constitutively active Tor^Y9, which causes RTK signaling throughout the embryo. In contrast, the VE fails to activate in the absence of Tor and mediates broad ventral expression in tor^Y9 embryos not seen in the absence of Dl activity. This indicates that VAE mediates transcriptional activation by Dl, that the VRE, which by itself fails to activate transcription, is necessary to prevent Dl-dependent activation in the central region of the embryo, and that the activity of the unknown repressor, mediated by the VRE, is relieved by RTK signaling (Hader, 1999).

To investigate whether this action of Gro on Dl is determined by the arrangement of Dri- and Dl-binding sites in the VE, the transcription patterns driven by a modified VE-kni-element were examined in which the normal distance of 91 bp between the binding sites was reduced to 45 bp. This reduction results in Dl-dependent repression along the ventral side of wild-type embryos. Repression is not observed in the absence of Gro or Dl or in embryos expressing the constitutively active Tor^Y9 protein. In contrast, the repression domain expands anteriorly in tor mutant embryos, which lack RTK signaling, and is found to be Dl-dependent. This suggests that the spatial arrangement of the Dl- and Dri-binding sites dictates the mechanism by which Gro and Dl act within the enhancer element. In one case, Dl is suppressed by Gro, in the other, Dl is converted into a potent silencer of transcription that can override activation by Bcd and Cad. Both modes of repression are controlled by Tor-dependent RTK signaling (Hader, 1999).

These results establish that the cooperation between two maternal signaling systems, which determines the spatial limits of the Drosophila mesoderm anlage through hkb expression, is based on the management of the ubiquitously distributed factors Gro and Dri by local RTK signaling and that Gro can act through different modes on Dl. Lack of dead ringer (dri) activity does not result in an overt expansion of hkb expression on the ventral side of the embryo. However, as has been observed for VE-dependent gene expression, it causes only weak defects in mesoderm formation as compared with Gro-deficient embryos or embryos that express hkb under the control of the VAE. Thus, the interactions shown here represent only the Dri-dependent aspect of Gro's effect on hkb expression. The full picture of hkb control is likely to involve additional and redundantly acting factor(s) that recruit Gro to sites flanking the VE within the hkb control region (Hader, 1999).

Overactivation of receptor tyrosine kinases (RTKs) has been linked to tumorigenesis. To understand how a hyperactivated RTK functions differently from wild-type RTK, a genome-wide systematic survey was conducted for genes that are required for signaling by a gain-of-function mutant Drosophila RTK Torso (Tor). Chromosomal deficiencies were screened for suppression of a gain-of-function mutation tor (tor^GOF); this screen led to the identification of 26 genomic regions that, when in half dosage, suppress the defects caused by tor^GOF. Testing of candidate genes in these regions revealed many genes known to be involved in Tor signaling (such as those encoding the Ras-MAPK cassette, adaptor and structural molecules of RTK signaling, and downstream target genes of Tor), confirming the specificity of this genetic screen. Importantly, this screen also identified components of the TGFß (Dpp) and JAK/STAT pathways as being required for Tor^GOF signaling. Specifically, it was found that reducing the dosage of thickveins (tkv), Mothers against dpp (Mad), or STAT92E (aka marelle), respectively, suppress tor^GOF phenotypes. Furthermore, it has been demonstrated that in tor^GOF embryos, dpp is ectopically expressed and thus may contribute to the patterning defects. These results demonstrate an essential requirement of noncanonical signaling pathways for a persistently activated RTK to cause pathological defects in an organism (Lia, 2003).

High Bicoid levels render the terminal system dispensable for Drosophila head development

In Drosophila, the gradient of the Bicoid (Bcd) morphogen organizes the anteroposterior axis while the ends of the embryo are patterned by the maternal terminal system. At the posterior pole, expression of terminal gap genes is mediated by the local activation of the Torso receptor tyrosine kinase (Tor). At the anterior, terminal gap genes are also activated by the Tor pathway but Bcd contributes to their activation. Evidence is presented that Tor and Bcd act independently on common target genes in an additive manner. Furthermore, the terminal maternal system is shown not to be required for proper head development, since high levels of Bcd activity can functionally rescue the lack of terminal system activity at the anterior pole. This observation is consistent with a recent evolution of an anterior morphogenetic center consisting of Bcd and anterior Tor function (Schaeffer, 2000).

The terminal maternal system directly modifies Bcd by phosphorylation at several MAPK sites in a Ser/Thr (S/T)-rich region located between the homeodomain and the identified transcriptional activation domains. A deletion variant of Bcd that lacks all these activation domains but still contains the S/T-rich region (BcdDeltaQAC) is able to rescue to viability bcd loss-of-function mutants. Hence, it is conceivable that the ability of the tor pathway to create negative charges through phosphorylation of this region of Bcd might result in an acidic-rich transcriptional activation domain that compensates for the loss of all the other activation domains. If this were the case, then the transcriptional activity of the BcdDeltaQAC deletion variant should be highly dependent on tor function. To test this hypothesis, the ability of a BcdDeltaQAC transgene to rescue the bcd phenotype in embryos derived from bcd;tsl double mutant mothers was assayed. BcdDeltaQAC rescues the bcd phenotype of the bcd;tsl double mutant similarly to a wild-type bcd transgene, resulting in a tsl only phenotype. Since BcdDeltaQAC is functionally independent of the tor pathway, it is concluded that the terminal system is not responsible for BcdDeltaQAC's activation potential. This result is also consistent with the notion that, in transient transfection experiments and transgenic studies, Bcd transcriptional activity is not significantly modified by mutations of the putative MAPK consensus sites. Thus, the described direct modification of Bcd by the tor pathway does not appear to be necessary for Bcd's function (Schaeffer, 2000).

tor function is necessary to allow a normal expression pattern of most Bcd target genes: many Bcd target genes such as otd are expressed in a reduced anterior domain in tor mutants. Furthermore, the expression domain of these genes is expanded in tor gain-of-function backgrounds, again suggesting that the tor pathway potentiates Bcd function. This effect could be direct, as Bcd transcriptional activity might be enhanced by direct modification of the protein (for instance by phosphorylation). Alternatively, the effect might be indirect, since most Bcd target promoters might also be responsive to Tor through distinct elements (Schaeffer, 2000).

A direct effect should be detectable with simply organized Bcd target promoters that only contain Bcd-response elements and no Tor-response elements. The proximal hb promoter (P2) resembles such a simple Bcd-response element, which uses activators to set an expression border without the assistance of repressors. The hb P2 promoter is not directly responsive to the terminal pathway; in the absence of Tor activity, the posterior border of hb expression moves only very slightly towards the anterior and, in tor gain-of-function embryos (tor⁴⁰²¹), the posterior expression border does not respond significantly to ubiquitously activated Tor (Schaeffer, 2000).

However, the hb P2 promoter is still 300 bp long and might contain elements that are not well defined, and the hb pattern is very dynamic. Therefore, in addition an artificial Bcd responder gene was used whose promoter elements are all known. This promoter contains only Bcd and Hb binding sites (Bcd3Hb3-LacZ), and its expression is reminiscent of the hb P2 promoter, with an anterior cap expression domain from 100% to 65% EL. If Bcd were a direct target of Tor, the posterior border of the reporter gene expression domain should move in response to a tor gain-of-function allele. However, the expression pattern does not change in a tor⁴⁰²¹ background. This argues for a Bcd activator function that is not under direct control of the terminal system. Thus, Bcd and Tor seem to be part of two independent pathways, which share common target genes (Schaeffer, 2000).

When a complete series of Bcd deletion variants was assayed for their ability to rescue the bcd loss-of-function phenotype in the absence of terminal system activity, one transgenic line was found that not only rescues the bcd phenotype but also the anterior part of the tsl phenotype (labrum and dorsal bridge), resulting in a posterior terminal mutant phenotype only. This particular transgenic line carries a bcd variant that deletes an alanine-rich domain (BcdDeltaA) and has been shown to activate the bcd target gene hb in a widely enlarged expression domain. Using Bcd immunostaining, it has been shown that this transgenic line exhibits levels of Bcd that are approximately 2- to 3-fold higher than wild type. Since other BcdDeltaA lines did not exhibit the same ability to rescue the tsl phenotype, it is concluded that the higher expression level of this particular line rather than the lack of a specific negative protein element (alanine-rich domain) is responsible for overcoming the requirement for the terminal pathway at the anterior (Schaeffer, 2000).

To further address whether high levels of bcd activity are sufficient to rescue the anterior terminal system phenotype or, if only a particular Bcd deletion variant is capable thereof, the ability of increased doses of wild-type bcd transgenes to rescue several terminal mutant backgrounds was tested. Since the previous experiments were performed with the tsl¹ allele, which might only represent a strong hypomorphic allele rather than a null, another tsl mutant, tsl⁴ , was included that is among the strongest in the allelic series, as well as null mutant alleles of the terminal genes trk and tor. To increase the Bcd expression level, flies containing an X chromosome or a third chromosome each carrying two wild-type bcd rescue constructs were used; these flies carry up to six copies of bcd. The phenotypes of all terminal mutants (tsl, trk or tor) are similar: lack of labrum and dorsal bridge in the anterior and deletion of all structures posterior to A7. Four copies of the bcd gene were able to rescue anterior structures including labrum and dorsal bridge in about 40% of all embryos derived from a tsl⁴ mutant background, while the posterior terminal phenotype is unaffected. Six copies of bcd are necessary to obtain the same anterior rescue in about 15% of all embryos derived from trk mutants and in about 5% of all embryos derived from tor mutants. However, not all embryos with rescued labrum and dorsal bridge had a perfectly aligned head skeleton. This might be due to incomplete rescue, but it could also be due to Bcd-mediated overexpression of hb at the anterior pole, which results in terminal-like phenotypes (Schaeffer, 2000).

Actually 50%, 70% or 85% of the head cuticles of tsl, trk or tor mutants, respectively, could not be analyzed for rescue due to severe anterior defects, which seemed more severe than normal terminal phenotypes. Nonetheless, some of the rescued embryos (less than 2%) were able to hatch and move around, which suggests complete anterior rescue. These probably represent embryos where just enough Bcd was present to overcome the lack of the terminal system but not too much to induce the phenotype due to high ectopic expression of hb. All larvae died within 2 hours, likely due to the posterior terminal defects. It should be noted that very few embryos exhibited the type of abdominal segment fusions that have been described for embryos derived from mothers carrying excess copies of the bcd gene. This might be due to the lack of terminal system function at the posterior pole in these experiments. Since no tail is made, there is probably more space for fate-map shifts towards the posterior, resulting in the correct establishment of abdominal segments A1 to A6. The rescue of the anterior terminal phenotype by high levels of bcd further indicates that the major role of the anterior terminal system is the potentiation of Bcd activity (Schaeffer, 2000).

In the posterior region of the embryo, the tor pathway activates the zygotic effectors tll and hkb, which are sufficient to specify the most posterior anlagen and the gut of the larva. At the anterior, the function of the terminal system is more difficult to interpret and, in tor mutants, hkb expression is only reduced. It actually requires bcd;tsl double mutants to lose all anterior hkb expression, which indicates additive functions of the anterior and terminal systems on this common target gene. hkb seems particularly interesting in this context, as its function is required for the formation of the labrum: reduction of hkb expression, as observed in terminal mutant background leads to the deletion of this particular structure (Schaeffer, 2000).

Therefore, it was asked whether the rescue of anterior structures (e.g. the labrum) mediated by high levels of Bcd in terminal system mutants is correlated with the restoration of the hkb expression pattern. Expression of hkb is first detected in the terminal regions (anterior and posterior) of the syncytial blastoderm. In terminal mutant embryos, the posterior domain is absent, whereas the anterior domain is reduced. In a tsl background with four or six copies of bcd, however, hkb expression extends further towards the posterior. Hence, the level of hkb expression can be regained by increasing the levels of Bcd in a terminal system mutant, even though its exact expression domain cannot be restored. It is likely that fate-map shifts are able to absorb the slightly changed expression domain of hkb. This suggests that the lack of terminal system activity at the anterior can simply be overcome by another system through enhancement of transcriptional activation of common target genes (Schaeffer, 2000).

Tor has been shown to antagonize Groucho-mediated repression of genes such as hkb and tll, probably by acting on the HMG-box transcription factor Capicua. Therefore, it is likely that Tor enhances Bcd activity by derepression, i.e. the inactivation of potential repressors of Bcd target genes, and thereby rendering any transcriptional activator more potent. As the cis-regulatory control regions of most developmental genes comprise both repressor and activator sites, the inactivation of potential repressors should lead to enhanced expression, or enlarged expression domains, as observed for several bcd target genes in a tor gain-of-function background (Schaeffer, 2000).

Since bcd and tor appear to function independently of each other, it is conceivable that anterior tor activity can also enhance the function of other transcriptional activators through derepression. Therefore, in long-germband insects that might lack a true bcd homolog, the anterior terminal system could also assist other activators, like homologs of Otd or Hb. Moreover, the fact that, in certain situations in the Drosophila embryo, anterior Tor activity can be dispensable for proper head development, is consistent with the observation that a posterior morphogenetic center is more frequently found in insects than an anterior center. Although flies accumulate BCD mRNA and tor activity at the anterior pole of the egg, this might not be useful for most short-germband insects as their embryos develop in the posterior part of the egg. In this case, the anterior of the embryo is far away from the anterior of the egg and the morphogenetic role of bcd and tor would not be effective in patterning the head. In Tribolium castaneum, an intermediate-germband beetle, the activity of the terminal system is conserved at the anterior of the embryo, but it does not appear to have a specific function for pattern formation. Correspondingly, the tll homolog of Tribolium is only expressed at the posterior pole of the embryo. This is in contrast to Drosophila, where tll is expressed at both poles of the early embryo. Moreover, in spite of arguments supporting the presence of a bcd-like function in Tribolium, no bcd homologous gene has been identified outside higher diptera notwithstanding Bcd's homeodomain and bcd's location in the Hox cluster. It is therefore reasonable to assume that an anterior morphogenetic center consisting of Bcd and anterior Tor activity is not a general feature of insects (Schaeffer, 2000).

Coactivation of STAT and Ras by Torso is required for germ cell proliferation and invasive migration in Drosophila

Primordial germ cells (PGCs) undergo proliferation, invasion, guided migration, and aggregation to form the gonad. In Drosophila, the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development, and coactivation of STAT and Ras is required for PGC proliferation and invasive migration. Embryos mutant for stat92E or Ras1 have fewer PGCs, and these cells migrate slowly, errantly, and fail to coalesce. Conversely, overactivation of these molecules causes supernumerary PGCs, their premature transit through the gut epithelium, and ectopic colonization. A requirement for RTK in Drosophila PGC development is analogous to the mouse, in which the RTK c-kit is required, suggesting a conserved molecular mechanism governing PGC behavior in flies and mammals (Li, 2003).

STAT92E plays an essential role in mediating the phenotypic effects of gain-of-function mutations of Torso, TorGOF, but is only minimally required for wild-type Tor function in patterning the terminal structures of the Drosophila embryo. To investigate whether wild-type Tor nevertheless activates STAT92E, an antibody was used that recognizes the phosphorylated, or active form of STAT92E (pSTAT92E) to examine the activation status of STAT92E in different genetic backgrounds. In early embryos, pSTAT92E is detected in the anterior and posterior terminal regions in a pattern reminiscent of Tor activation. By analyzing embryos mutant for loss- or gain-of-function mutations of tor as well as those lacking JAK, encoded by hopscotch (hop), it was concluded that the early STAT92E activation is dependent on Tor but not Hop, suggesting that Tor may activate STAT92E independent of Hop. Because STAT92E contributes only marginally to the expression of the Tor target gene tailless (tll), it was of interest to find whether the early activation of STAT92E by Tor had any other biological functions. It was evident that Tor activation correlates temporally and spatially with the formation of PGCs, which are localized at the posterior pole of the early embryo. Tor-dependent activation of STAT92E as well as that of the Ras-MAPK signaling cassette, as detected by an antibody against activated ERK/MAPK (diphospho-ERK), persists in pole cells at this stage. STAT92E activation was detected in PGCs during their migration and in the gonads of late embryos, that are formed following the migration of pole cells through a complex route. These observations indicate that STAT92E and Ras1/Draf activation may play a role in PGC development (Li, 2003).

So far the only known function of Tor has been in pattern formation, since Tor protein is present only transiently in early embryos. Therefore, the finding that Tor is involved in germ cell migration was initially unexpected. However, there is a precedent for the requirement of an RTK in germ cell migration in the mouse. Mutations in the mouse genes dominant white-spotting (W) cause migration and proliferation defects in germ cells as well as a few other cell types. W encodes the protooncoprotein c-kit, an RTK that is expressed on the membrane of mouse PGCs. Sl encodes the c-kit ligand termed stem cell factor (SCF), which is localized on the membrane of somatic cells associated with PGC migratory pathways. Interestingly, c-kit and Tor share structural similarities and both are structurally similar to the platelet derived growth factor (PDGF) receptor, in which an insert region separates the intracellular kinase domain. Moreover, similar to Tor and the PDGF receptor, c-kit is able to activate STAT molecules as well as the Ras-MAPK cascade. Although true molecular homologs of c-kit and SCF are not yet found in the Drosophila genome, the functional and structural similarities between Tor and c-kit suggest that flies and mice share molecular mechanisms for regulating primordial germ cell proliferation and migration (Li, 2003).

In addition to germ cells, the ovarian border cells of Drosophila are also capable of invasive and guided migration. Border cells of the Drosophila ovary are follicle cells that, during oogenesis, delaminate as a cluster six to ten cells from the anterior follicle epithelium, invade the nurse cells, and migrate toward the oocyte. Interestingly, it has been shown that the detachment and guided migration of these cells require STAT92E activation. Mutations in components of the Hop/STAT92E pathway cause border cell migration defects. In addition, border cell migration also requires RTK signaling. An RTK related to mammalian PDGF and VEGF receptors, PVR, is required in border cells for their guided migration toward the oocyte. PVR appears functionally redundant with another fly RTK, EGFR, in guiding border cells. Taken together, these results indicate that the invasive behavior and guided migration of Drosophila ovarian border cells require both STAT92E and RTK activation. In light of the results from analyzing PGC migration, it is proposed that activation of both STAT and components downstream of RTK signaling may serve as a general mechanism for invasive and guided cell migration (Li, 2003).

It has been shown that actin-based cytoskeletal reorganization plays a crucial role in cell shape changes and movements. The identification of STAT and Ras coactivation as an essential requirement for germ cell migration raises an interesting question of how activated STAT and Ras coordinate the cytoskeletal reorganization required for germ cell migration. STAT92E has been shown to be involved in the transcriptional activation of many signaling molecules as well as key transcription factors. A recent systematic search for STAT92E target genes has revealed a plethora of genes that might be directly activated by STAT92E, among which are those involved in the regulation of cytoskeletal movements and actin reorganization. Upregulation of such genes in response to spatial cues should facilitate cell movements. In addition, Ras and other small GTP proteins have been implicated in multiple cellular processes that require cytoskeletal reorganization. It remains to be determined how these two signaling pathways coordinate germ cell movements in response to guidance cues from surrounding somatic tissues (Li, 2003).

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).

Neuroendocrine control of Drosophila larval light preference

Animal development is coupled with innate behaviors that maximize chances of survival. This study shows that the prothoracicotropic hormone (PTTH), a neuropeptide that controls the developmental transition from juvenile stage to sexual maturation, also regulates light avoidance in Drosophila melanogaster larvae. PTTH, through its receptor Torso, acts on two light sensors (the Bolwig's organ and the peripheral class IV dendritic arborization neurons) to regulate light avoidance. PTTH was found to concomitantly promote steroidogenesis and light avoidance at the end of larval stage, driving animals toward a darker environment to initiate the immobile maturation phase. Thus, PTTH controls the decisions of when and where animals undergo metamorphosis, optimizing conditions for adult development (Yamanaka, 2013)

Animal development is associated with multiple primitive, innate behaviors, allowing inexperienced juveniles to choose an environment that maximizes their survival fitness before the transition to adulthood. In insects, this transition is timed by a peak of ecdysone production induced by the prothoracicotropic hormone (PTTH). In the larval brain of Drosophila, PTTH is produced by two pairs of neurosecretory cells projecting their axons onto the prothoracic gland (PG), where ecdysone is produced. Transition to adulthood is associated with drastic changes in larval behavior: Feeding larvae remain buried in the food, whereas wandering larvae (at the end of larval development) crawl out and find a spot where they immobilize and pupariate. Mechanisms allowing proper coordination of these behavioral changes with the developmental program remain elusive (Yamanaka, 2013)

Two pairs of neurons in the central brain were recently reported to control larval light avoidance. Using specific antibodies to PTTH, this study established that these neurons labeled by the NP0394-Gal4 and NP0423-Gal4 lines correspond to the PTTH-expressing neurons. Moreover, silencing the ptth gene by using NP0423-Gal4 or a ubiquitous driver (tub-Gal4) impaired light avoidance, indicating that PTTH itself controls this behavior. PTTH activates Torso, a receptor tyrosine kinase whose knockdown in the PG prevents ecdysone production and induces a developmental delay. In contrast, knocking down torso in the PG did not cause any change in light avoidance, indicating that the role of PTTH in ecdysteroidogenesis is functionally distinct from its role in light avoidance behavior (Yamanaka, 2013)

Because in Drosophila the PTTH-producing neurons only innervate the PG, it was reasoned that PTTH is secreted into the hemolymph and reaches the cells or organs involved in light avoidance. Consistent with this, inactivation of PTTH-expressing neurons affects light avoidance with 8 to 10 hours delay, arguing against PTTH neurons projecting directly on their target cells to control light avoidance. PTTH peptide is present in the PTTH-expressing neurons throughout larval development and shows a marked increase before wandering, correlating with the rapid increase of ecdysteroidogenesis at this stage. Using an enzyme-linked immunosorbent assay (ELISA), it was found that PTTH is readily detected in the hemolymph with a fluctuation pattern similar to that of its accumulation in the PTTH-expressing neurons. Furthermore, hemolymph PTTH levels were significantly decreased upon RNA interference (RNAi)–mediated knockdown of ptth in the PTTH-expressing neurons, suggesting that in addition to the paracrine control of ecdysteroidogenesis in the PG, PTTH also carries endocrine function (Yamanaka, 2013)

Pan-neuronal knockdown of torso (elav>torso-RNAi^GD) recapitulates the loss of light avoidance observed upon torso ubiquitous knockdown (tub>torso-RNAi^GD), suggesting that PTTH acts on neuronal cells to control light avoidance. The potential role of torso was specifically tested in two neuronal populations previously identified as light sensors in Drosophila larvae: (1) the Bolwig's organ (BO) and (2) the class IV dendritic arborization (da) neurons tiling the larval body wall. An enhancer trap analysis of torso, as well as in situ hybridization using a torso antisense probe, confirmed torso expression in class IV da neurons. In parallel, torso transcripts were detected by means of quantitative reverse transcription polymerase chain reaction in larval anterior tips containing the BO, and their levels were efficiently knocked down by using the BO-specific drivers Kr5.1-Gal4 and Rh5-Gal4, demonstrating torso expression in the BO. The knockdown of torso in the BO (Kr5.1>torso-RNAi^GD and GMR>torso RNAi^GD) or in the class IV da neurons (ppk>torso-RNAi^GD) abolished larval light avoidance (motoneurons serve as a negative control: OK6>torso-RNAi^GD). Knocking down torso in both neuronal populations (ppk>, GMR>torso-RNAi^GD) mimicked the effect observed with the BO driver or class IV da neuron driver alone. A similar loss of light avoidance was observed when these neurons were separately inactivated by expressing the hyperpolarizing channel Kir2.1 (GMR>Kir2.1 and ppk>Kir2.1), suggesting that both of these light sensors are necessary for light avoidance behavior. Down-regulation of PTTH/Torso signaling did not lead to any neuronal morphology or locomotion defect, further indicating its direct effect on light sensing. The knockdown of torso in class IV da neurons or in the BO had no effect on the pupariation timing. Taken together, these results indicate that PTTH/Torso signaling is required for light avoidance behavior in two distinct populations of light-sensing neurons and that this function is separate from its role in controlling developmental progression (Yamanaka, 2013)

Drosophila light-sensing cells use photosensitive opsins that upon exposure to light, activate transient receptor potential (TRP) cation channels, thus depolarizing the membrane and triggering neural activation. Although the BO and class IV da neurons use different photosensitive molecules and TRP channels, one can assume that PTTH/Torso signaling regulates the phototransduction pathway through a similar mechanism in both types of neurons. Immunohistochemical detection of Rh5, the opsin involved in light avoidance behavior in the BO, showed no difference in protein level in torso mutant background. PTTH/Torso signaling knockdown did not change the expression level of Gr28b, a gustatory receptor family gene that plays an opsin-like role in class IV da neurons. These results strongly suggest that PTTH affects signaling components downstream of the photoreceptors (Yamanaka, 2013)

The neural activity of the light sensors was investigated using the calcium indicator GCaMP3 for live calcium imaging. torso mutant class IV da neurons showed a 25% reduction of their response to light as compared with that of control. This was accompanied by a loss of light avoidance, indicating that such partial reduction of the GCaMP3 signal corresponds to a reduction of neural activity strong enough to exert a behavioral effect. Indeed, blocking the firing of class IV da neurons by using TrpA1-RNAi caused a similar 25% reduction of the GCaMP3 signal and behavioral effect. This suggests that in da neurons, PTTH/Torso signaling exerts its action upstream of TrpA1 channel activation. Accordingly, a strong genetic interaction was observed between torso and TrpA1 mutants for light preference. A genetic interaction between torso and Rh5 mutants was also detected, further supporting that PTTH/Torso signaling affects a step in phototransduction between the photoreceptor molecule and the TRP channel. Collectively, these data are consistent with the notion that PTTH/Torso signaling acts to facilitate TRP activation downstream of photoreceptor-dependent light sensing (Yamanaka, 2013)

A previous study suggested that larval photophobic behavior diminishes at the end of larval development, perhaps facilitating larval food exit and entry into the wandering phase. The present finding and the increase of PTTH at the beginning of the wandering stage appear to contradict such a hypothesis. Indeed, a sustained larval light avoidance mediated by PTTH was detected that persisted through the wandering stage. These results imply that wandering behavior is triggered by a signal distinct from light preference. Consistent with this notion, the timing of wandering initiation in ppk>torso-RNAi^GD or Kr5.1>torso-RNAi^GD larvae was found comparable with that of control animals, despite the fact that these animals are not photophobic (Yamanaka, 2013)

As found in other insects, wandering is either directly or indirectly triggered by PTTH- induced ecdysone production. Therefore, concomitant PTTH-mediated photophobicity could ensure that wandering larvae maintain a dark preference for pupariation site, providing better protection from predators and dehydration during the immobile pupal stage. To test this hypothesis, a light/dark preference assay was developed for pupariation. When exposed to a light/dark choice, larvae indeed showed a strong preference to pupariate in the dark. This behavior was abolished either by inactivating PTTH-expressing neurons (ptth>Kir2.1), by silencing ptth in the PTTH-expressing neurons (NP0423>ptth-RNAi, dicer2), or by introducing a torso mutant background (torso[e00150]/[1]). Dark site preference for pupariation was observed in Drosophila populations collected in the wild, confirming that this innate behavior was selected in a natural environment (Yamanaka, 2013)

This work illustrates the use of a single biochemical messenger, PTTH, for the concomitant control of two major functions during larval development. PTTH establishes a neuroendocrine link between distinct neuronal components previously shown to be involved in light avoidance. In contrast to previous interpretations but consistent with another study, this study showed that wandering is independent of light preference and that PTTH maintains a strong light avoidance response through to the time of pupariation. High levels of circulating PTTH during the wandering stage could reinforce the robustness of light avoidance, which might otherwise be compromised by active roaming. This eventually promotes larvae to pupariate in the dark, a trait potentially beneficial for ecological selection. PTTH is thus at the core of a neuroendocrine network, promoting developmental progression and appropriate innate behavioral decisions to optimize fitness and survival (Yamanaka, 2013)

Torso-like functions independently of Torso to regulate Drosophila growth and developmental timing

Activation of the Drosophila receptor tyrosine kinase Torso (Tor) only at the termini of the embryo is achieved by the localized expression of the maternal gene Torso-like (Tsl). Tor has a second function in the prothoracic gland as the receptor for prothoracicotropic hormone (PTTH) that initiates metamorphosis. Consistent with the function of Tor in this tissue, Tsl also localizes to the prothoracic gland and influences developmental timing. Despite these commonalities, in studies of Tsl it was unexpectedly found that tsl and tor have opposing effects on body size; tsl null mutants are smaller than normal, rather than larger as would be expected if the PTTH/Tor pathway was disrupted. It was further found that whereas both genes regulate developmental timing, tsl does so independently of tor. Although tsl null mutants exhibit a similar length delay in time to pupariation to tor mutants, in tsl:tor double mutants this delay is strikingly enhanced. Thus, loss of tsl is additive rather than epistatic to loss of tor. It was also found that phenotypes generated by ectopic PTTH expression are independent of tsl. Finally, this study shows that a modified form of tsl that can rescue developmental timing cannot rescue terminal patterning, indicating that Tsl can function via distinct mechanisms in different contexts. It is concluded that Tsl is not just a specialized cue for Torso signaling but also acts independently of PTTH/Tor in the control of body size and the timing of developmental progression. These data highlight surprisingly diverse developmental functions for this sole Drosophila member of the perforin-like superfamily (Johnson, 2013).

Terminal patterning in the Drosophila embryo involves secretion of the membrane attack complex/perforin-like (MACPF) protein Torso-like (Tsl) from specialized follicle cells at the anterior and posterior ends of the oocyte into the perivitelline space. Following its secretion, Tsl remains at the embryo poles through association with the vitelline membrane. Through a poorly understood mechanism that involves the eggshell proteins fs(1)Nasrat, fs(1)Polehole, and Closca, Tsl likely permits localized activation of the cysteine knot-like growth factor Trunk (Trk), possibly by proteolytic cleavage. Activated Trk then binds to Tor and activates signaling at the embryo poles (Johnson, 2013).

Tsl is the only MACPF-like protein that can be identified in the Drosophila genome (Rosado, 2007). The majority of MACPF proteins characterized to date play roles in pore formation in mammalian immunity (including perforin itself and Complement C9) or in bacterial pathogenesis. Currently, it is unclear how Tsl functions at the embryo poles, and a simple pore forming function is difficult to reconcile with a central role in activation of the Tor-signaling pathway (Johnson, 2013).

Tor has a second major developmental role, in the prothoracic gland (PG), where it functions as the receptor for prothoracicotropic hormone (PTTH), a brain-derived neuropeptide hormone required for initiation of metamorphosis (Rewitz, 2009). Recently, it was shown that tsl is also expressed in the PG and that RNAi knockdown of tsl results in a significant (48 h) developmental delay (Grillo, 2009). Given that PTTH and Trk belong to the same superfamily of cysteine knot-like growth factors (Rewitz, 2009), it seemed likely that Tsl plays a role in Tor activation in the PG. This study confirms that loss of tsl leads to a delay in development. Remarkably, however, it was discovered that Tsl regulates body size and developmental timing independently of Tor. The results show that Tsl has diverse functions, some of which are independent of Torso, and is thus not just a specialized cue for Torso signaling (Johnson, 2013).

The data presented in this study show that the genes required for activation of Tor in the early embryo are not used in its activation in the PG. Ectopic expression of PTTH in cells that do not normally produce it, in the early embryo and the PG, results in active PTTH that is capable of activating Tor. These data suggest that these cells produce all proteins necessary for activation of PTTH. In contrast, ectopic Trk driven in the PG (where it is not normally produced) has no effect on developmental timing or adult size despite expression of Tsl in this tissue. Taken together, it is concluded that the activation requirements of Trk and PTTH are quite different. Specifically, tsl expression is unnecessary for PTTH activity and insufficient for ectopic Trk activity (Johnson, 2013).

Why would Tor be activated differently in the embryo and in the PG? It was reasoned that the answer to this question possibly lies with differences in the two ligands. During early embryogenesis Trk is secreted in an inactive form that requires Tsl and other terminal class genes for activation. In this situation Tor activation requires spatial constraint that is achieved by restricted Tsl expression controlling spatially localized Trk activation. In contrast, because the PG is directly innervated by PTTH-producing neurons that synapse within the gland, spatial control of Tor activation might not be necessary in this context. It is hypothesized that PTTH may simply be secreted from these neurons in a form that does not require further local activation and thus does not require Tsl (Johnson, 2013).

How might Tsl act in controlling body size and development timing? The tsl mutant phenotypes presented in this study resemble those observed when insulin signaling is reduced. Specifically, overexpression of a dominant negative form of the insulin-like receptor (InR) causes reduced size and delayed development. Additionally, larvae carrying heat-sensitive alleles of InR also display developmental delays and effects of size. However, despite these phenotypic similarities, experiments have not yielded evidence for interactions between tsl and the InR pathway (Johnson, 2013).

Another possibility is that tsl regulates developmental timing and growth earlier in development or in a tissue distinct from the PG. The growth rate defect that were observe in the tsl mutants may indicate that it acts earlier in development than tor. It must also be noted that the data do not implicate the PG as the source of timing and growth defects observed in tsl null mutants. Consistent with the latter possibility, it has not been possible to rescue the tsl null phenotypes by specifically expressing tsl in the PG using phm-Gal4. In addition, it was not possible to replicate the delay observed by Grillo (2013) using RNAi knockdown of tsl in the PG. As technical difficulties and experimental variations between laboratories can underlie such differences, however, the PG remains a candidate tissue given its crucial role in regulation of developmental transitions in response to nutritional inputs. Further experiments to manipulate Tsl function in different tissues and at different times during development will be required to determine the specific tissues and pathways underlying these phenotypes. Taken together, however, the current results reveal the surprising finding that the function of Tsl in its maternal patterning role is mechanistically distinct from its zygotic role in the developing larva (Johnson, 2013).

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date revised: 15 April 2020

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