torso

Promoter Structure

The promoter contains a TATA box upstream from the start site (Sprenger, 1989).

Malignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. The requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) has been investigated using a genetic approach in Drosophila. The JAK/STAT pathway plays little or no role in signaling by wild-type Tor. STAT, encoded by marelle (mrl; Stat92E), is essential for the gain-of-function mutant Tor (Tor^GOF) to activate ectopic gene expression. These findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation (Li, 2002).

How does Tor^GOF RTK activate Mrl? There are at least three possible mechanisms through which STAT activation by RTK could occur. RTK could directly bind and activate STAT proteins. Alternatively, STAT could be indirectly activated by the RTK, either via JAK or MAPK. Genetic evidence allows the possibilities that Tor^GOF activates Mrl via JAK or MAPK to be ruled out. (1) Whether or not removal of Hop activity modifies the tor^GOF phenotype was examined. Surprisingly, a hop null mutation does not suppress tor^GOF, indicating that unlike Mrl, Hop is not required for ectopic tll expression. (2) Removal of mrl does not suppress rl^Sevenmaker (rl^Sem), which encodes a GOF mutant form of Drosophila MAPK, suggesting that Mrl is not essential for the effects of GOF mutation in MAPK. To test for a physical interaction between Mrl and Tor^GOF, Tor was immunoprecipitated from wild-type and tor^GOF embryos, respectively, with anti-Tor antibody, and the presence of Mrl was examined in the immune complexes. A specific band corresponding to Mrl was detected in the immunoprecipitates. The Tor-Mrl association, however, is only observed in the presence of vanadate (a general tyrosine phosphatase inhibitor), suggesting that this interaction takes place only when the cytoplasmic protein phosphorylation status is preserved, or when Tor and/or Mrl have been activated by the presence of vanadate. Altogether, these results are consistent with a direct activation of Mrl by Tor^GOF, possibly following recruitment of Mrl to phosphotyrosine residues on the Tor RTK via SH2-phosphotyrosine peptide interaction (Li, 2002).

Since Mrl activation is required for ectopic tll expression induced by Tor^GOF, whether Mrl-binding sites (TTCNNNGAA) are present in the regulatory region of the tll gene was examined. A search in the tll regulatory region revealed two putative Mrl-binding sites with the consensus TTCNNNGAA located at –2357 (site 1) and –2462 (site 2) upstream of the tll transcription start site. These two sites are able to bind Mrl, although site 2 shows a much lower affinity. Interestingly, the two Mrl sites are located 105 bp apart in the tll regulatory region. This configuration is reminiscent of that existing in the eve stripe 3 enhancer, where cooperative binding of two Mrl homodimers has been demonstrated. To assess the functional relevance of the two Mrl sites in tll expression, transgenes containing the 5.9 kb regulatory fragment upstream of the tll transcription start site fused to the lacZ gene were introduced into flies. This 5.9 kb fragment had been shown previously to drive lacZ expression in a pattern almost identical to that of the endogenous tll gene. Accordingly, lacZ expression is greatly expanded in a tor^GOF background. A 5.9 kb fragment with the two Mrl binding sites mutated, showed wild-type activity for lacZ expression in wild-type embryos, suggesting that these Mrl-binding sites are dispensable for tll expression under normal Tor signaling. However, in a tor^GOF background, the mutant 5.9 kb fragment shows greatly diminished ability to drive lacZ expression in an expanded domain compared to the situation when the Mrl binding sites are wild type. These results are consistent with the genetic results that Mrl is required for the full activity of gain-of-function, but not wild-type Tor (Li, 2002).

To account for the involvement of Mrl in tll regulation, it is proposed that a hyperactivated RTK requires a downstream pathway that is not essential for wild-type RTK under normal physiological situations. In wild-type embryos, Tor is activated only in the two terminal regions and defines the spatial limits of tll expression domains by relieving the transcriptional repressors bound to the tll promoter. Mrl is not an essential factor for tll activation in the terminal regions, although it remains to be determined whether Mrl contributes to the activation of tll expression redundantly with other yet unidentified factors. In tor^GOF mutant embryos, Tor^GOF is constitutively active in all regions of the embryo and causes ectopic tll expression. In this case, Mrl activation is indispensable for the ectopic tll expression in the central regions of the embryo. The differential requirement for Mrl in central and terminal regions might be due to the lack of other activators of tll and/or the presence of additional repressors in the central region of the embryos. Consistent with this idea, in the absence of Tor signaling (such as in tor mutant embryos), tll can be induced by uniformly expressing activated forms of downstream signaling components (such as Ras^V12 or 14-3-3). The resulting induction of tll expression happens preferentially in the terminal regions. Thus tll expression could be determined by the balance between repressors and activators that can bind to the tll promoter (Li, 2002).

These findings may explain some of the conflicting observations on the role of STAT in RTK signaling in mammals. For example, thanatophoric dysplasia type II (TD II) dwarfism in humans is caused by mutations that lead to constitutive activation of a human RTK FGF receptor 3 (FGFR3). Similar to Tor^GOF activating Mrl, it has been shown that an activated mutant FGFR3 specifically activates STAT1 in both human patient tissues and mouse models. The activated STAT1 in this case induces expression of the cell-cycle inhibitor p21^WAF1/CIP1, resulting in growth inhibition of bone tissues. However, STAT1 is not known to be required for bone development. STAT1 knockout mice have perfect bones, although they exhibit defective immune systems. This might be explained by a redundancy among different STAT proteins. Alternatively, STAT1 may not be required for normal FGFR3 signaling in bone development. The presence of several STAT genes in mammals makes it technically difficult to distinguish between the above two possibilities using the mouse as a genetic model. In contrast, the presence of a single JAK and a single STAT gene in Drosophila allows the relationship between RTK and JAK/STAT signaling to be examined, without being limited by gene redundancy. These observations in Drosophila suggest that the TD II syndrome in humans could be explained if STAT1 is not normally required for FGFR3 signaling, but it becomes essential only for the activating mutant FGFR3 (Li, 2002).

Transcriptional Regulation

Early embryogenesis in Drosophila is controlled by maternal gene products, which are deposited in the egg during oogenesis. It is not well understood how maternal gene expression is controlled during germline development. pipsqueak (psq) is a complex locus that encodes several nuclear protein variants containing a PSQ DNA-binding domain and a BTB/POZ domain. Psq proteins are thought to regulate germline gene expression through epigenetic silencing. While psq was originally identified as a posterior-group gene, this study shows a novel role of psq in embryonic terminal patterning. A new psq loss-of-function allele, psq^rum, was identified that specifically affects signaling by the Torso (Tor) receptor tyrosine kinase (RTK). Using genetic epistasis, gene expression analyses, and rescue experiments, it was demonstrated that the sole function impaired by the psq^rum mutation in the terminal system is an essential requirement for controlling transcription of the tor gene in the germline. In contrast, the expression of several other maternal genes, including those encoding Tor pathway components, is not affected by the mutation. Rescue of the psq^rum terminal phenotype does not require the BTB/POZ domain, suggesting that the PSQ DNA-binding domain can function independently of the BTB/POZ domain. The finding that tor expression is subject to dedicated transcriptional regulation suggests that different maternal genes may be regulated by multiple distinct mechanisms, rather than by a general program controlling nurse-cell transcription (Grillo, 2001).

psq has functions in many developmental stages, and presumptive null mutants are lethal. This study reports that a set of psq mutations unveils a specific role in tor transcription. Why is this phenotype only observed associated with these particular psq mutations? Among the psq alleles that allow adult survival, strong mutations block oogenesis at early stages. Thus, in those cases, an early requirement in oogenesis would mask a later requirement for tor transcription. Psq proteins are present in multiple isoforms. Only a few psq alleles have been molecularly characterized and among those many are due to transposon insertions. Therefore, it is difficult on the basis of the molecular analysis of the psq mutations to assign distinct functions to the isoforms generated by the different transcripts (Grillo, 2001).

A number of reasons argue for the psq^rum mutation unveiling a physiological function of psq in tor transcription, rather than the rum phenotype being a neomorphic effect caused by a special truncated Psq protein. First, the terminal phenotype of the psq^rum mutation is observed in homozygosity, as well as in trans-heterozygous combinations with several other psq loss-of-function alleles. Second, the terminal phenotype of the psq^rum mutation arises in association with a mild posterior phenotype, a well-known psq loss-of-function phenotype. Third, a transheterozygous combination of other psq loss-of-function alleles also produces embryos showing reduced tor expression and terminal defects. And finally, expression of psq rescues the psq^rum terminal phenotype (Grillo, 2001).

The psq^rum mutation causes a decrease in the wild-type splicing at one specific site. Nevertheless, since all known isoforms share this splicing, it cannot be inferred whether a particular isoform is responsible for tor transcriptional regulation. However, the rescue experiments indicate that both a long Psq isoform containing the BTB/POZ domain and a short isoform lacking this domain are capable of providing the psq function controlling tor transcription that is missing in psq^rum mutants. Thus, on the one hand, the BTB/POZ domain appears to be dispensable for this psq function, but on the other hand, a long isoform can substitute for a short isoform, arguing against separate functions of these psq isoforms in the context of Tor signaling. Thus, an overall decrease of many psq isoforms in the psq^rum mutant could be affecting tor transcription (Grillo, 2001).

In this regard, it is worth considering together the terminal and posterior defects associated with the psq^rum mutation. tor is affected more strongly than vas in psq^rum mutants, while the opposite is true for other psq mutants (e.g., psq^HK38, psq²⁴⁰³, and psq^fs1, in which vas is strongly affected, but not tor, according to their cuticle phenotype. These data argue against a simple model in which tor and vas transcription would be impaired below different thresholds of psq activity (Grillo, 2001).

As Psq is thought to repress gene expression through epigenetic silencing, Psq could activate tor expression indirectly, through the repression of a still unidentified tor repressor. Alternatively, Psq could activate tor expression directly. Indeed, genetic interaction studies suggest that psq and Trithorax-like (Trl) act together in transcriptional activation as well as in transcriptional silencing of homeotic genes (Grillo, 2001).

Not much is known about how transcription is regulated in Drosophila nurse cells. One possibility is that spatially and temporally coexpressed genes share a common mode of transcriptional regulation. Indeed, enrichment of specific core motifs in the promoters of genes with female germline expression is consistent with such a hypothesis. The multiple effects of psq mutations during oogenesis might argue for such a general role of psq. However, and in spite of psq's multiple requirements in the germline, tor transcription appears to be distinctly regulated. A similar case appears to apply to bcd transcription, which was found to be specifically controlled by Serendipity-δ (Sry-δ), a zinc finger protein. sry-δ null alleles block oogenesis, and only a particular genetic combination revealed the specific requirement of sry-δ for bcd transcription. Thus, both psq and sry-δ have a basic function in oogenesis, probably through the transcriptional control of other germline genes, and a specific function in the control of tor and bcd, respectively. This similarity is particularly intriguing considering the peculiarities of early Drosophila embryogenesis and the fact that anterior patterning by bcd seems to be restricted to Diptera and that tor-dependent terminal patterning appears in Diptera and Coleoptera, but not in Hymenoptera. Thus, the regulation of bcd and tor transcription by a specific function of more general germline transcription factors might be related to their particular recruitment to embryonic patterning. Interestingly, in the case of terminal patterning, tor transcription appears to be regulated independently from that of genes encoding the other elements of the signaling pathway (e.g., Ras and Raf), as induced expression of tor is sufficient to rescue the psq^rum terminal phenotype. However, the only essential function of these other elements of the Tor pathway in the oocyte is to transmit the Tor signal, as indicated by the phenotype of mutant germline clones. Thus, in the absence of tor activity, these products appear to be silent in the Drosophila germline. Altogether, these data suggest a possible multiple-step way to acquire new regulatory mechanisms in a given set of cells. This possibility appears particularly suggestive in the light of recent results pointing to Tor as the receptor for prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone. Could this be a more ancient role of tor that would subsequently have been recruited for embryonic terminal patterning in some insects? Such a scenario appears to apply to the Toll signaling pathway, which shares some similarities with the Tor pathway, and has a widely conserved function in immunity in many animals and whose components are also transcribed by the Drosophila female germline to specify the embryonic dorsoventral pattern (Grillo, 2001).

The histone demethylase KDM5 is essential for larval growth in Drosophila

In Drosophila, the larval prothoracic gland integrates nutritional status with developmental signals to regulate growth and maturation through the secretion of the steroid hormone ecdysone. While the nutritional signals and cellular pathways that regulate prothoracic gland function are relatively well studied, the transcriptional regulators that orchestrate the activity of this tissue remain less characterized. This study shows that lysine demethylase 5 (KDM5) is essential for prothoracic gland function. Indeed, restoring kdm5 expression only in the prothoracic gland in an otherwise kdm5 null mutant animal is sufficient to rescue both the larval developmental delay and the pupal lethality caused by loss of KDM5. These studies show that KDM5 functions by promoting the endoreplication of prothoracic gland cells, a process that increases ploidy and is rate limiting for the expression of ecdysone biosynthetic genes. Molecularly, it was shown that KDM5 activates the expression of the receptor tyrosine kinase torso, which then promotes polyploidization and growth through activation of the MAPK signaling pathway. Taken together, these studies provide key insights into the biological processes regulated by KDM5 and expand understanding of the transcriptional regulators that coordinate animal development (Drelon, 2019).

This study demonstrates that KDM5 is essential for the function of the ecdysone-producing prothoracic gland during Drosophila larval development. Crucial to this conclusion was the finding that expressing kdm5 in the prothoracic gland was sufficient to rescue the lethality and developmental delay phenotypes of kdm5¹⁴⁰> null allele homozygous mutant animals. Consistent with this observation, prothoracic gland function was defective in kdm5 mutants, with mutant larvae having low levels of ecdysone and reduced expression of downstream hormone-responsive target genes. Demonstrating the importance of KDM5-mediated regulation of ecdysone production, dietary supplementation of 20E restored normal developmental timing to kdm5 mutant larvae. At the cellular level, loss of KDM5 slowed the prothoracic gland endoreplicative cycles that increase the ploidy of these cells and are important for ecdysone biosynthesis. Restoring these endocycles by expressing cyclin E re-established normal developmental timing to kdm5¹⁴⁰ mutants but was not able to rescue their lethality. In contrast, ectopic activation of the Torso/MAPK pathway that functions upstream of cyclin E was able to restore both developmental timing and rescue the lethality caused by loss of KDM5. It is therefore proposed that KDM5-mediated activation of the Torso/MAPK pathway in the prothoracic gland is important for larval growth regulation through its role in promoting polyploidization, and for adult eclosion by mechanisms that remain to be determined (Drelon, 2019).

Consistent with an important developmental role for KDM5-mediated regulation of Torso/MAPK, mutations in torso or ablation of the neurons that produce its ligand PTTH cause a 5-day delay to larval development similar to that observed for kdm5¹⁴⁰. Because loss of KDM5 reduces, but does not eliminate, torso expression, the kdm5¹⁴⁰ phenotype cannot be accounted for solely by the twofold change to Torso/MAPK signaling observed. The dysregulation of additional KDM5-regulated genes is therefore likely to contribute to the prothoracic gland phenotypes of kdm5 mutant larvae. Little is known about the transcriptional regulation of torso and other genes that make up the upstream pathways that regulate ecdysone production. One possibility is that KDM5 functions as a direct transcriptional activator of the torso gene: thus, decreased expression of this receptor would be expected in kdm5 mutant animals. Because of the small size of the prothoracic gland, it is not currently feasible to carry out ChIP experiments to examine KDM5 promoter binding in this tissue. It is, however, notable that the torso promoter was not bound by KDM5 in existing ChIP-seq datasets from larval wing imaginal discs or from whole adult flies. This could be because torso expression is largely restricted to the prothoracic gland during larval development and so may not be expected to have promoter-bound KDM5 in the tissues examined to date. Alternatively, KDM5 might regulate torso indirectly. In the silkworm Bombyx mori, expression of the torso gene is repressed in response to starvation conditions. Although the mechanism by which this occurs is unknown, it does indicate that other cellular defects caused by loss of KDM5 could lead to changes to torso transcription and subsequent decrease in MAPK activity and ecdysone production. Whether the regulation of torso by KDM5 is direct or indirect, it occurs in a demethylase-independent manner, as larvae lacking enzymatic activity show a normal developmental profile (Drelon, 2018). Consistent with this observation, components of KDM5 complexes that regulate gene expression through demethylase-independent mechanisms also affect developmental timing. For example, a development delay similar to that of kdm5¹⁴⁰ is caused by RNAi-mediated knockdown of the histone deacetylase HDAC1 or the NuRD complex components asf1 and Mi-2. KDM5 could therefore interact with these proteins to regulate the expression of genes that are crucial to the regulation of larval development (Drelon, 2019).

Increased ploidy of prothoracic gland cells is important for optimal expression of steroidogenic genes and can be induced by activation of the Torso/MAPK pathway. Because the genes required for ecdysone biosynthesis are among the most abundantly expressed in the prothoracic gland, their mRNA levels may be entirely limited by gene copy number. A similar requirement for copy number amplification to produce peak gene expression levels has been observed in other cell types in Drosophila, including chorion gene expression in ovarian follicle cells. However, it is not known how Torso/MAPK activation promotes prothoracic gland cell cycle progression. One mechanism might be by affecting levels of cell cycle regulators such as the transcription factor E2f1, which is essential for both mitotic and endoreplicative cell cycles. This model is based on studies of the polyploid enterocytes of the adult midgut, in which activation of the MAPK pathway via the EGF receptor stabilizes E2f1 protein, leading to transcription activation of cyclin E. Although restoring correct endocycling in kdm5 mutant prothoracic glands was able to rescue their developmental timing, it did not impact their eclosion defect. This could be because loss of KDM5 leads to additional defects within the prothoracic gland that are ultimately detrimental to the function of this endocrine tissue, such as increased oxidative stress, which this study has shown to be affected in kdm5 hypomorphic mutant wing discs. Alternatively, KDM5 could have a cell cycle independent role in maintaining ecdysone levels during pupal development. kdm5¹⁴⁰ mutant animals die as pharate adults that have no obvious morphological abnormalities but fail to eclose (Drelon, 2018). Nevertheless, these animals could have significant defects in, for example, nervous system development, which requires ecdysone and is important for eclosion (Drelon, 2019).

The observed role of KDM5 in the growth and polyploidization of larval prothoracic gland raises the possibility that it might play key roles in other cell types that use endoreplicative cycles. This could have broad consequences for understanding of KDM5 biology, as polyploidization is observed in many plant and animal cell types and is widely used during Drosophila larval development. In addition, while the role of polyploid cells in the etiology or maintenance of cancers remains a topic of ongoing research, KDM5-regulated endocycling could contribute to its tumorigenic activities in humans. Regulation of polyploidization in cells of the nervous system could also contribute to the link between KDM5 protein dysregulation and intellectual disability. This could, for example, be mediated by KDM5 function in glial cells, as polyploidization of superineurial glial cells in Drosophila is required for normal brain development. Although it is not clear the extent to which a similar phenomenon occurs during human brain development, it is interesting to note that glial cell types contribute to the severity of intellectual disability disorders such as Rett syndrome. Thus, although there is still much to be learned regarding the contribution of polyploid cells to normal development and to clinically relevant disorders, KDM5-regulated transcriptional programs could be key to the function of cells that use this variant cell cycle (Drelon, 2019).

Targets of Activity

There are least two separate but synergistically interacting promoter regions that mediate transcriptional activation of tailless (tll) by the terminal system. This functional synergism between regulatory elements may play a role in the translation of the Torso morphogen gradient into the sharp boundary of tll gene activity (Liaw, 1993).

Activation of the Torso RTK at the poles of the embryo sets off a phosphorylation cascade that leads to the spatially specific transcription of the tailless (tll) gene. The TOR response element (TOR-RE) has been mapped to an 11-bp sequence. The gene activator GAGA and the repressor Grainyhead/NTF-1 bind to the TOR-RE. NTF-1 can be phosphorylated by rolled, also known as MAPK (mitogen-activated protein kinase), a protein in the Ras pathway activated by Torso. Thus activation of the TOR RTK at the poles of the embryos leads to inactivation of a repressor and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo (Liaw, 1995).

Anterior repression of orthodenticle is carried out by Huckebein which in turn receives input for the torso system, from Dorsal and from Bicoid. Dorsal functions in the anterior repression of otd expression. The repression function of Dorsal is mediated, at least in part, through Huckebein, since anterior hkb expression is lost in dorsal mutants. Contrary to early models of embryonic pattern formation, high levels of Bicoid are not required for otd activation or for the establishment of anterior head structures (Gao, 1996).

sloppy paired, in addition to behaving like a segment polarity gene and as a pair rule gene, acts like a gap gene in the head. All three maternal systems active in the cephalic region are required for proper slp expression. High levels of the terminal system (Torso) inhibit slp through bicoid. Low levels of terminal system activity seem to potentiate bcd as an activator of slp in more posterior positions. Dorsal, the morphogen of the dorsoventral system, and the head-specific gap gene empty spiracles, act as repressor and corepressor respectively in the regulation of slp (Grossnicklaus, 1994).

The anterior, the dorsoventral and terminal systems, are required for the activation of crocodile expression and for the spatial control of the anterior cap domain, while the posterior system is not required for the regulation of the croc expression pattern. In the absence of bcd activity, croc fails to be expressed in the anterior cap domain. Although Bcd acts in a concentration-dependent manner, croc expression can only be expanded ventrally in the absence of dorsal activity. In fact, the lack of dorsal activity causes a strong reduction of the croc expression domain to a single spot, corresponding in position to the peak of bicoid activity at the anterior pole. Conversely, Dorsal activity along the entire dorsoventral axis, as in embryos laid by Toll mutant females, causes an expansion of the croc expression domain towards the dorsal-most position. In embryos lacking tor activity, croc expression is abolished in the dorsal region. However, if tor is activated ectopically due to a dominant tor mutation, the croc expression domains are expanded significantly on the ventral side. Thus Dorsal postively regulates croc and Bcd requires Dl to set the spatial limit of the croc anterior expression domain (Häcker, 1995).

In the Drosophila embryo, cell fate along the anterior-posterior axis is determined by maternally expressed genes. The maternal gene torso is required for the development of the unsegmented region of the head (acron). otd expression responds to the activity of the maternal tor gene at the anterior pole of the embryo (Finkelstein, 1990).

The closely linked POU domain genes pdm-1 and pdm-2 are first expressed early during cellularization in the presumptive abdomen; the initially broad domain soon resolves into two stripes. This expression pattern is regulated by the same mechanisms that define gap gene expression domains. The borders of pdm-1 expression are set by the terminal system genes torso and tailless, and the gradient morphogen encoded by hunchback. (Cockerill, 1993).

The three maternal systems (anterioposterior (bicoid); terminal (torso); dorsoventral (dorsal) control the early expression of Goosecoid. The GSC stripe never appears in bicoid mutants, the stripe is shifted anteriorly in torso mutants and the ventral repression of the stripe is abolished in dorsal mutants (Goriely, 1996).

Transcription of Bicoid target genes is repressed at the anterior pole owing to the activity of torso . Both activation by BCD and repression by TOR can be reproduced by a minimal promoter containing only BCD-binding sites upstream of a transcriptional start site. Repression requires the D-raf kinase and is associated with phosphorylation of BCD protein. Repression requires neither tailless nor huckebein, previously thought to constitute the sole zygotic output of the TOR signaling system. Thus phosphorylation of BCD down-regulates transcriptional activation by BCD (Ronchi, 1993).

The Torso receptor tyrosine kinase cascade represses expression of Bicoid targets at the most anterior tip of the embryo. Neither the homeodomain (HD) nor the activation domain of BCD is targeted by Torso in the anterior repression of BCD targets. When a BCD mutant protein whose HD has been replaced by the Gal4 DNA-binding domain is expressed in early embryos, a reporter gene driven by Gal4 DNA-binding sites is first activated in an anterior domain and then repressed from the anterior pole. The down-regulation of Bcd-Gal4 activity requires torso function but does not depend on endogenous bcd activity, indicating that the Bcd protein alone and none of its targets is required to mediate the effect of torso. Functional analysis of a chimeric protein, whose activation domain has been replaced by a generic activation domain, indicates that the activation domain of BCD is also not specifically required for its down-regulation by Torso. It is propose that Torso does not affect the ability of BCD to bind DNA, but instead directs modification of BCD or of a potential BCD co-factor, which renders the BCD protein unable to activate transcription (Bellaiche, 1996).

capicua (cic) is involved in gene repression in Drosophila terminal and dorsoventral patterning. Tor signaling proceeds via the Ras/Raf/MAPK pathway to regulate expression of the zygotic genes tll and hkb, which are specifically expressed at each pole of the embryo. These genes encode transcription factors that initiate the developmental programs leading to differentiation of head and tail structures. Tor signaling does not activate terminal gene expression directly; rather, it functions by antagonizing at the poles a uniformly distributed repressor activity, allowing other maternal factors to activate transcription locally. Evidence for this view comes from the identification of regulatory elements in the tll promoter (called tor response elements, tor-REs) that confer terminal-specific expression and that, when mutated, cause severe derepression of tll transcription (Jimenez, 2000 and references therein).

Additional evidence for the regulation of tll and hkb by relief of repression derives from the role of the Groucho (Gro) corepressor in this process. Gro is a nuclear WD-repeat protein that does not bind DNA but interacts with a variety of DNA-bound transcriptional repressors. These associations recruit Gro to target promoters, bringing about transcriptional repression. Gro has been shown to participate in terminal development by restricting the expression of tll and hkb to the embryonic termini: embryos deprived of maternal Gro function show derepression of tll and hkb toward the middle of the embryo (Jimenez, 2000 and references therein).

What is the actual target of Tor signal inactivation at the embryonic poles? The Drosophila Yan Ets-like repressor factor is known to be degraded in response to RTK signaling during eye development. Thus, it is possible that the target of Tor signaling is similarly inactivated at the embryonic poles. The Gro protein is uniformly distributed in the blastoderm embryo and does not show down-regulation at the termini. Also, Gro corepressor activity during sex determination is not inhibited by Tor signaling, arguing that Gro is not the target of the Tor signal. To monitor the pattern of Cic distribution in embryos, a polyclonal antibody was raised against an HMG box-containing fragment of the protein. This antibody reveals a distinctive nuclear signal in wild-type but not cic¹ blastoderm embryos, confirming both that Cic is a nuclear protein and that the cic¹ allele is a strong loss-of-function mutation. Remarkably, Cic is distributed asymmetrically in blastoderm embryos, being present in nuclei from the presumptive trunk but absent at each embryonic pole. Because CIC mRNA is uniformly distributed in the embryo, the exclusion of the protein from the poles argues that Cic is under negative post-transcriptional regulation by the Tor signal transduction pathway (Jimenez, 2000).

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterized Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity (Janody, 2000).

The results indicate that early anterior expression of a labral determinant, cnc, and of two foregut determinants, wg and hh, is repressed when zygotic expression of hb is allowed to persist at the anterior pole of the Drosophila blastoderm embryo. Expression of cnc, wg and hh is under the positive regulation of bcd and torso but no zygotic gene has yet been implicated in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and that torso-induced anterior repression of hb is necessary for their positive control by torso. To determine whether the positive control of cnc, wg and hh by torso could be the result of a double negative control involving hb, expression of these genes was analysed in hb zygotic mutant embryos derived from torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb at the pole, expression of these genes should be recovered in hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is not recovered in hb minus embryos derived from torso females whereas it is normal in hb minus embryos. This indicates that, although necessary, the anterior repression of hb is not sufficient to mediate Torso positive control on cnc, wg and hh early anterior expression (Janody, 2000).

The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo. The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb). Since these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. A well-conserved sequence repeat, (GTTT)₄ has been identified that is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors. Further genetic analysis suggests that the (GTTT)₄-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip. Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position. Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer. These results suggest a common mechanism for preventing anterior activation of three different eve enhancers (Andrioli, 2002).

The eve2Delta(GTTT)₄-lacZ transgene is repressed at the anterior tip, even in gt mutants, suggesting that yet another mechanism prevents activation in this region. This mechanism could work through another localized repressor activity, or by modifying Bcd, the major activator of eve 2. Consistent with the latter possibility, it has been previously shown that Bcd-dependent activation of hb and orthodenticle (otd) is downregulated by the Tor phosphorylation cascade at the anterior tip. To test whether tor controls the ability of Bcd to activate eve 2, the eve2Delta(GTTT)₄-lacZ transgene was crossed into embryos lacking tor activity. This causes a significant derepression at the anterior tip, suggesting that tor-mediated modification of bcd activity is important for preventing activation in this region. A similar derepression is not detected with the wild-type eve2-lacZ transgene in tor mutants, suggesting that Tor-mediated repression is dependent on the (GTTT)₄-binding activity. In summary, these results suggest that multiple activities are required for anterior repression of eve 2, and that three different mechanisms prevent activation in different anterior regions (Andrioli, 2002).

RTK signaling modulates the Dorsal gradient

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. This study demonstrates that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, it was shown that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, the results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo (Helman, 2012).

Specification of body axes in all metazoans is initiated by a small number of inductive signals that must be integrated in time and space to control complex and unique patterns of gene expression. It is therefore of utmost importance to unravel the mechanisms underlying crosstalk between different signaling cues that concur during early development. This study has elucidated a novel signal integration mechanism that coordinates RTK signaling pathways with the Dl nuclear gradient, and thus with terminal and DV patterning of the Drosophila embryo (Helman, 2012).

Previous work had identified an input by Torso signaling into specific transcriptional effects of Dl. The current results establish a general mechanism, which involves RTK-dependent control of the nuclear Dl gradient itself, and thus affects a large group of Dl targets. This regulatory input is based on RTK-dependent derepression of wntD, a Dl target that encodes a feedback inhibitor of the Dl gradient. Thus, Dl activates wntD effectively only when accompanied by RTK signaling, enabling region-specific negative-feedback control of the nuclear Dl gradient. In the absence of RTK signaling, wntD is not expressed and the levels of nuclear Dl are elevated. Consequently, Dl target genes are ectopically expressed, both at the poles and along the DV axis (Helman, 2012).

Torso RTK signaling depends on maternal cues and is independent of the Dl gradient. Thus, it can be viewed as a gating signal that operates only at the embryonic poles, where it controls Dl-dependent gene regulation. However, the activity of the EGFR RTK pathway later on in development crucially depends on Dl, which induces the neuroectodermal expression of rhomboid, a gene encoding a serine protease required for processing of the EGFR ligand Spitz. In this case, EGFR-dependent induction of WntD represents a negative feedback loop that reduces nuclear levels of Dl laterally and, consequently, limits the expression of multiple Dl targets along the DV axis (Helman, 2012).

It should be noted that the regulatory interactions that have been characterized do not preclude the existence of other mechanisms modulating nuclear Dl concentration or activity. For example, the progressive dilution or degradation of maternal components involved in Toll receptor activation upstream of Dl should cause reduced Dl nuclear accumulation and retraction of its targets as development proceeds. It is also possible that Torso- or EGFR-induced repressors block transcription of Dl target genes directly. Accordingly, the ectopic sna expression observed in embryos mutant for components of the Torso pathway such as DSor and trunk probably reflects both loss of WntD activity on Dl and loss of Hkb-mediated repression of sna. In this context, it is interesting to note that sna expression expands and colocalizes with Hkb at the poles of wntD mutants; perhaps repression of sna by Hkb is not sufficient to override increased Dl activation in this genetic background. Thus, the Torso pathway probably employs more than one mechanism to exclude Dl target expression from the termini. Furthermore, the existence of such additional regulatory mechanisms could explain why wntD mutants do not have a clear developmental phenotype, despite the broad effects on Dl-dependent gene expression patterns caused by the genetic removal of wntD. It is proposec that corrective mechanisms are present, which make the terminal and DV systems robust with respect to removal of the WntD-based feedback, such as RTK-induced repressors. Understanding the basis of this robustness will require additional studies (Helman, 2012).

This work shows that RTK-dependent relief of Gro- and Cic-mediated repression is essential for transcriptional activation of wntD by Dl. Correspondingly, in the absence of cic or gro, the early expression of wntD expands ventrally throughout the domain of nuclear Dl. The early onset of this derepression, and the presence of at least one conserved Cic-binding site in the proximal upstream region of wntD, indicate that repression of wntD may be direct. Interestingly, it is thought that Gro and Cic are also involved in assisting Dl-mediated repression of other targets such as dpp and zen, as gro and cic mutant embryos show derepression of those targets in ventral regions. However, as ectopic wntD expression in these mutants leads to reduced nuclear localization of Dl along the ventral region, it is conceivable that decreased Dl activity also contributes to the derepression of dpp and zen (Helman, 2012).

In conclusion, the data presented in this study demonstrate RTK-dependent control of nuclear Dl via wntD, based on multiple regulatory inputs, including negative gating, feed-forward loops and negative feedback control. Together, these mechanisms provide additional combinatorial tiers of spatiotemporal regulation to Dl target gene expression. Future studies will show whether other signal transduction cascades and/or additional developmental cues also impinge on the Dl morphogen gradient (Helman, 2012).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.