Given the similarities between torso gain of function mutations (torgof) and cic1 phenotypes, the expression patterns of tailless and huckebein were examined in capicua1 (cic1) embryos. Expression of both genes expands toward the center of such embryos, predominantly in the posterior domain. The expanded expression of tll and hkb is very similar to that observed in torgof and groucho mutant embryos. The expression of a lacZ transgene under the control of a tor-RE from the tll promoter that drives terminal-specific transcription has also been examined. In cic1 mutant embryos, expression of this construct is derepressed toward the middle of the embryo. Together, these results suggest that the cic gene is normally required to restrict tll and hkb expression to the embryonic poles (Jimenez, 2000).
cic could affect tll and hkb expression by restricting Tor signaling to the embryonic poles (e.g., by limiting the domain of Tor receptor activation, or the domain of Tor signal transduction inside of the embryo). Alternatively, cic could function, like gro, as a repressor of tll and hkb downstream of the Tor pathway. To help distinguish between these possibilities, epistasis analyses were performed using loss-of-function mutations in tor, Draf, and Dsor (encoding a Drosophila MAPK kinase homolog) that normally cause a phenotype complementary to that of cic1, that is, absence of terminal structures. Embryos from females homozygous for cic1 and tor are identical to those from cic1 females alone. Likewise, cic1 females carrying loss-of-function clones of Draf or Dsor in the germ line produce embryos that display the cic phenotype. Thus, cic acts genetically downstream of Draf and Dsor. In addition, the domain of Tor signal activity was examined directly using a monoclonal antibody against the active, diphosphorylated form of Drosophila MAPK (known as Erk) and a normal pattern of Erk activation was found in cic1 embryos. This shows that derepression of tll and hkb in cic1 mutant embryos is not due to an expanded domain of Tor signaling, suggesting that cic is part of the activity that represses tll and hkb in the central region of the embryo and is inhibited by Tor signaling at the embryonic poles (Jimenez, 2000).
The similar effects of cic and gro on terminal patterning raise the possibility that cic is necessary for Gro corepressor activity in general. However, two lines of evidence argue against this idea: (1) Gro participates in many developmental processes, whereas the role of cic appears restricted to terminal and dorsoventral patterning; (2) Gro-dependent repression by Hairy in a sex determination assay does not require cic function. These results indicate that cic does not generally affect Gro activity (Jimenez, 2000).
Tor signaling at the embryonic poles regulates repressor processes that operate during dorsoventral patterning. Such patterning depends on the Dorsal morphogen, a rel domain factor that accumulates in ventral nuclei of early embryos and acts as both an activator and repressor of transcription: it activates ventral-specific genes [for example, twist (twi)] and represses dorsal-specific genes, such as zen. Repression by Dorsal requires its association with Gro and other postulated corepressors that bind next to Dorsal in the zen promoter. This repressor complex is under negative regulation by Tor signaling at the embryonic termini, allowing zen expression at each pole of the embryo (Jimenez, 2000 and references therein).
The mechanism of repression by Dorsal is not fully understood. Dead-Ringer (Dri) and Cut (Valentine, 1998) function as corepressors that assist Dorsal (and Gro) in Dorsal's function as a repressor. However, the effects of removing either of these two factors appears weaker than those caused by the loss of Dorsal or Gro function, suggesting that other factors may also contribute to Dorsal repression. Because cic is involved in a Gro-mediated process that is inactivated by Tor signaling, it was of interest to see if cic could also be involved in Dorsal repression. Consistent with this idea, zerknullt expression is expanded ventrally in cic1 mutant embryos. Although this expansion is not as strong as in dorsal or gro mutants, ectopic zen transcripts are clearly detected in lateral and ventral regions of the embryo, especially in its posterior half. In contrast, activation of twi by Dorsal is normal in cic1 embryos, suggesting that cic only participates in repression, not activation, by Dorsal (Jimenez, 2000).
To test further the role of cic in ventral repression of zen, an examination was carried out of a lacZ transgene carrying an even-skipped (eve) stripe 2 enhancer coupled to a silencer from the zen promoter: the zen Ventral Repression Element (VRE), which includes binding sites for Dorsal and adjacent regulatory sites. In wild-type embryos, lacZ expression directed by the eve stripe 2 enhancer is repressed ventrally by the VRE. This repression is clearly attenuated in cic1 mutant embryos, permitting stripe 2 activation in the ventral-most side of the embryo. In addition, significant ectopic lacZ expression is observed in ventral and lateral regions of the embryo, as expected if repression by Dorsal bound to the VRE is switched in favor of activation. These results suggest that cic encodes one of the cofactors required for VRE activity and the conversion of Dorsal from an activator to a repressor of transcription. Because Dri and Cut also function as Dorsal corepressors, it appears that this role is shared by several factors with overlapping activities (Jimenez, 2000).
In Drosophila, the maternal terminal system specifies cell fates at the embryonic poles via the localised stimulation of the Torso receptor tyrosine kinase (RTK). Signalling by the Torso pathway relieves repression mediated by the Capicua and Groucho repressors, allowing the restricted expression of the zygotic terminal gap genes tailless and huckebein. This study reports a novel positive input into tailless and huckebein transcription by maternal posterior group genes, previously implicated in abdomen and pole cell formation. Absence of a subset of posterior group genes, or their overactivation, leads to the spatial reduction or expansion of the tailless and huckebein posterior expression domains, respectively. The terminal and posterior systems converge, and exclusion of Capicua from the termini of posterior group mutants is ineffective, accounting for reduced terminal gap gene expression in these embryos. It is proposed that the terminal and posterior systems function coordinately to alleviate transcriptional silencing by Capicua, and that the posterior system fine-tunes Torso RTK signalling output, ensuring precise spatial domains of tailless and huckebein expression (Cinnamon, 2004).
Terminal gap gene expression must be tightly regulated for the correct specification of terminal cell fates at the nonsegmented poles. Clearly, the Tor pathway plays a key role in driving tll and hkb transcription, given that terminal gap genes are not expressed at the posterior end of terminal group mutants, and as a result terminal structures such as the terminal filzkorper (FK) do not form. In this paper, a novel biological role is unraveled for the maternal posterior system, showing that members of this group, in particular Nos, positively regulate transcription of the zygotic subordinate genes of the terminal system. Torso response elements (TREs) in the tll upstream regulatory region, which are derepressed in cic mutants, also respond to alterations in maternal osk dosage, and the Cic repressor is not excluded from the termini of posterior group mutants. These results are consistent with the posterior system feeding into the Tor signalling pathway, upstream of or at the level of the Cic repressor. It is suggested that the concerted activities of both the terminal and posterior systems, in their spatially overlapping zones of action, generate accurate domains of terminal gap gene expression at the posterior (Cinnamon, 2004).
It was originally proposed that the four maternal systems that pattern the early Drosophila embryo act largely independently of each other. Recent work, however, demonstrated interactions between the Tor pathway and the anterior and D/V systems. For example, tll has been shown to respond to the anterior determinant Bicoid (Bcd) even when Tor signalling is genetically blocked. Indeed, cis-acting DNA elements responsive to these three maternal systems have been found in the tll upstream regulatory region. The current results now link the terminal and posterior systems, previously thought to be independent of each other, in terminal gap gene regulation, reinforcing the idea that maternal systems that pattern the early embryo act in a coordinated manner (Cinnamon, 2004 and references therein).
Why has the positive input, by posterior group genes into terminal patterning, been largely overlooked to date? Classical segmentation studies mostly involved phenotypic analyses at the cuticular level. For this reason, and when taking into account the primary contribution of the terminal system, the delicate input by the posterior group has gone unnoticed. Thus, the unextended FK that develops in posterior group mutant background, which may arise from decreased terminal gap gene expression, had largely been attributed to pleiotropic effects arising from abdominal defects. It has been possible to detect the relatively subtle changes in tll and hkb gene expression patterns only by investigating terminal gap gene regulation at the molecular level. In fact, at least one other molecular study had previously reported reduced terminal gap gene expression in osk mutant embryos (Cinnamon, 2004 and references therein).
One emerging concept is that, for the refinement of the expression levels and spatial extents of RTK signalling targets, it is also imperative to integrate accurately information originating from other, non-RTK sources. In many cases this integration occurs at the level of target gene enhancers, with various effectors of distinct signalling pathways binding to specific DNA elements to regulate transcription. For example, D-Pax2 expression in the cone and pigment cells of the developing eye is regulated by effectors of the EGFR RTK pathway, such as Pointed P2 and Yan, and also by the Notch signalling component Suppressor of Hairless, as well as by the transcription factor Lozenge. The current study shows that terminal gap gene expression requires not only Tor RTK pathway activity but also a contribution from the posterior system. In this instance, inputs from these two maternal coordinate systems are interpreted and linked not at the level of terminal gap gene promoters but at the level of the Cic repressor. Thus, Cic functions as an integrator of multiple regulatory inputs, with both the posterior and terminal systems acting to relieve transcriptional silencing mediated by this repressor (Cinnamon, 2004).
Surprisingly, anterior tll and hkb expression is also reduced in posterior group mutants. Similarly, others have reported prolonged bcd expression and head defects in pum mutants. It is speculated that low levels of Osk and Nos, which escape translational repression, similarly regulate terminal gap gene expression via Cic removal at the anterior. In accordance with this, the dismissal of Cic from the anterior pole of posterior group mutants is also ineffective (Cinnamon, 2004).
How does Nos, which has been assigned the role of a translational repressor, positively regulate tll and hkb transcription? The results suggest that Nos does so indirectly, by downregulating the accumulation of the Cic repressor at the termini. The exact mechanism by which the Tor pathway mediates the exclusion of Cic from terminal regions has not been established, but one model argues that phosphorylation of Cic by MAPK causes degradation of the protein, as in the case of Yan. Thus, Nos could be affecting this process in one of several possible ways, at the level or downstream of MAPK. For example, Nos could be facilitating the translocation of phosphorylated MAPK into the nucleus. In posterior group mutants, then, activated MAPK would remain in the cytoplasm rather than enter the nucleus, impeding Cic phosphorylation and degradation. Alternatively, Nos may be modulating MAPK activity, or regulating adaptor proteins that promote Cic phosphorylation by nuclear MAPK. Nos may also be controlling the translation of factors that are involved in the nuclear trafficking (import/export) or degradation of Cic, or perhaps may even be acting on the cic message itself. Future studies will distinguish between these possibilities, and may shed new light on the molecular mechanisms underlying role of Nos in other developmental processes, for example, the establishment/maintenance of transcriptional quiescence in pole cells. The positive input by the posterior group genes is viewed as evolving to modulate terminal pathway activity, merging with other varied modes of Tor regulation to ultimately ensure accurate tll and hkb expression and, consequently, precise cell fate determination (Cinnamon, 2004).
The Tor signal transduction pathway is under multiple tiers of regulation, outside and inside the nucleus. For instance, internalisation and trafficking of the activated Tor receptor to the lysosome for degradation attenuates the signal, as evident by the spatial broadening and temporal prolonging of Tor activation in mutants for hrs, a component of the endosomal recycling machinery (Lloyd. 2002). Yet another level of control is provided by the tyrosine phosphatase corkscrew, which sharpens the gradient of Tor activity. Additionally, multiple cytoplasmic adaptor proteins take part in transducing the Tor signal, conceivably buffering against surplus or deficiency in signalling (Cinnamon, 2004).
In the nucleus, tll and hkb are subjected to silencing by several repressors. Derepression of tll is observed in grainy-head and tramtrack69 (ttk69) mutants, and the proteins encoded by these genes bind tll promoter sequences. Cic and Gro appear to play a leading role in terminal gap gene silencing, given that mutations in cic and gro bring about a significant expansion of the tll and hkb expression domains. Intriguingly, however, tll expression never reaches the middle of the embryo in these mutants. tll is uniformly expressed, albeit weakly, throughout the embryo only when both the developmental corepressors Gro and CtBP are removed concomitantly. This broadened tll expression likely stems from the fact that there is a redundancy in the activities that normally restrict terminal gap gene transcription from inappropriately spreading into the central portion of the embryo; by jointly removing the Gro and CtBP coregulators, activity of the above repressors is compromised. Alternatively, CtBP might be acting in conjunction with a novel, unidentified repressor that prevents tll transcription in the middlemost region of the embryo (Cinnamon, 2004).
So what is the purpose of the input by the posterior group genes into tll and hkb transcription? Quantitative differences in Tor receptor activity have to be eventually interpreted and translated into distinct cell fates at the termini. Strong Tor activation induces both hkb and tll expression, whereas weaker Tor activation only brings about tll expression. It is surmised that the precision endowed by the Tor RTK cascade may not suffice for the complex patterning of the termini, given that mere two-fold fluctuations in Tor signalling result in defective embryonic development. For example, mutants with reduced Tor RTK activity show partial tll expression and the complete loss of hkb. These mutants consequently develop incomplete terminal structures and die at the larval stage. Conversely, overactivation of the Tor pathway leads to anterior expansion of the posterior tll expression domain, perturbing segmentation in central body parts, likely as a result of downregulation of abdominal gap genes by the Tll protein. Thus, the precise spatial confinement of terminal gap gene expression domains requires the coordinated integration of regulatory inputs, coming from two maternal systems and converging on the same effector protein, Cic (Cinnamon, 2004).
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 torD4021 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 torD4021/+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 (torRL3) 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 torD4021 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 torRL3 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 torRL3 genotype was examined in a trunk (trk) background to eliminate ligand-induced activation. On its own, a single copy of torRL3 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 torRL3 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 torRL3/+; cic/cic have a much stronger phenotype that those from trk torRL3/+; cic/+. Therefore, the Cic protein present in trk torRL3/+; cic/+ embryos is still at least in part functional implying that the torRL3 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 torRL3/+; 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).
The dorsoventral axis of the Drosophila egg is established by dorsally localized activation of the epidermal growth factor receptor (Egfr) in the ovarian follicular epithelium. Subsequent positive- and negative-feedback regulation generates two dorsolateral follicle cell primordia that will produce the eggshell appendages. A dorsal midline domain of low Egfr activity between the appendage primordia defines their dorsal boundary, but little is known about the mechanisms that establish their ventral limit. This study demonstrated that the transcriptional repressor Capicua is required cell autonomously in ventral and lateral follicle cells to repress dorsal fates, and functions in this process through the repression of mirror. Interestingly, ectopic expression of mirror in the absence of capicua is observed only in the anterior half of the epithelium. It is proposed that Capicua regulates the pattern of follicle cell fates along the dorsoventral axis by blocking the induction of appendage determinants, such as mirror, by anterior positional cues (Atkey, 2006; full text of article).
In both cic homozygous egg chambers and cic mutant follicle cell clones, ectopic mirr expression is restricted to the anterior half of the epithelium, indicating that mirr is also regulated by positional information along the AP axis. Although in principle a posterior repressor could account for this effect, on the basis of prevailing models of follicular epithelium AP patterning, the hypothesis that expression of mirr requires positive input from an anterior positional cue is favored. It is propose that, in wild-type ovaries, Cic blocks the induction of mirr by this anterior signal. However on the dorsal side, where Cic becomes downregulated, this signal is not blocked, leading to the induction of mirr expression and appendage-producing fate. In cic mutant ovaries, the anterior signal induces mirr expression throughout the DV axis (Atkey, 2006).
A likely candidate for an anterior signaling molecule required for mirr expression is Dpp, which is produced by the anterior-most follicle cells and regulates gene expression along the AP axis. Coordinate regulation of mirr along the DV and AP axes provides a molecular explanation for the observation that appendage-producing fates are determined at the intersection of Egfr and Dpp signaling. Regulation of mirr by an anterior cue such as Dpp could also explain the observation that mirr expression in cic mutant ovaries is normal until stage 10B; although the cic mutant cells are competent to express mirr, detectable levels may not be induced until the posterior migration of anterior follicle cells in mid-oogenesis brings the source of Dpp to the anterior margin of the oocyte (Atkey, 2006).
In addition to invoking an anterior signal in the regulation of mirr, the data indicate that mirr is also positively regulated by dorsally restricted Egfr signaling, independent of Cic. cic mutant egg chambers exhibit ectopic mirr throughout their anterior circumference, but mirr levels remain highest dorsally. In grk;cic double mutant egg chambers this dorsal high point of mirr expression is abolished, suggesting that the wild-type dorsal anterior mirr expression pattern is the result of both dorsal and anterior inputs (Atkey, 2006).
Collectively, the data support a model in which Cic blocks the induction of mirr expression and appendage-producing fates in response to an anterior signal, for example Dpp. Egfr-mediated downregulation of Cic in dorsal anterior follicle cells therefore allows these cells to respond to Dpp, contributing to the dorsal anterior mirr expression pattern, whereas the presence of Cic in ventral and lateral follicle cells blocks their response to this cue. Within the dorsal Cic-free domain, the Rhomboid/Spi/Aos autocrine-feedback loop would regulate Egfr activity to resolve two distinct appendage primordia. In cic mutant egg chambers, all follicle cells would be competent to respond to the anterior signal, resulting in ectopic mirr expression and appendage-producing fate in the anterior follicle cells that receive the signal (Atkey, 2006).
Previous work has shown that the appendage primordia are determined at the intersection of dorsal and anterior signals, and the simplest interpretation has been that these signals function additively to specify appendage-producing fate. Instead, however, the demonstration that the distribution of Cic along the DV axis determines the competence of follicle cells to respond to AP patterning signals reveals unexpected crosstalk between DV and AP patterning signals, and indicates that Cic integrates these pathways. Along the DV axis, it is proposed that the pattern of the follicular epithelium is determined by the function of two Egfr targets, Cic and Aos, in distinct domains. High levels of Egfr activity induce production of Aos at the dorsal midline, where it antagonizes Spi, thus splitting the initial dorsal domain of Egfr activity and defining the dorsal limits of the appendage primordia. Lower levels of Egfr signaling are sufficient to downregulate Cic, defining a dorsal domain that lacks Cic and is therefore competent to adopt dorsal fates. Cic remains present in ventral and lateral follicle cells, where it blocks the induction of crucial transcriptional targets, such as mirr, by Dpp. The dorsal limit of the Cic domain thus defines the ventral limit of the appendage primordia. Cic-mediated repression of target genes may represent a general mechanism for the integration of multiple spatial inputs in a developing tissue (Atkey, 2006).
Capicua functions in two Gro-dependent repressor processes inactivated by Torso signaling. Therefore, it was asked whether Cic interacts with Gro in vitro. Three different fragments of Cic (amino-terminal, central, and carboxy-terminal) were expressed in bacteria as GST fusions and their ability to bind radiolabeled Gro protein was assayed. The carboxy-terminal portion of Cic interacts with Gro, whereas the amino-terminal and central regions of the protein show little or no binding. The binding of Cic to Gro is weaker than that of Hairy, but stronger than the Dorsal/Gro interaction in the same assay. The interaction of Cic with Gro does not depend on the conserved carboxy-terminal domain of Cic, indicating that this domain mediates another aspect of Cic function. Taken together, the results support the idea that Cic and Gro form a repressor complex inactivated by Torso signaling during terminal and dorsoventral patterning (Jimenez, 2000)
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).
cic expression was analyzed by in situ hybridization and CIC mRNA was found to be present at high levels in early blastoderm embryos (stage 1-3), consistent with a maternal expression of the gene. The cic transcripts decay rapidly so that they are barely detected by the onset of gastrulation and at later stages of embryogenesis. These results, together with the strictly maternal effect character of the cic1 mutation, argue that cic function is restricted to terminal and dorsoventral patterning of the early embryo (Jimenez, 2000).
cic was identified in a P-element screen for female sterile mutations that affect the anteroposterior embryonic pattern. Females homozygous for the cic mutation (cic1) are fully viable and produce embryos that form head and tail structures but lack most of the segmented trunk. Embryos lacking maternal cic function are referred to as cic mutant embryos. The phenotype of cic1 mutant embryos is rather uniform: Most embryos (>80%) retain only 1-3 partial abdominal denticle belts at 25°C, whereas the rest of the embryos do not show any signs of abdominal segmentation. This latter phenotype is shown by virtually all embryos from females carrying the cic1 allele in trans with a deficiency in the region, suggesting that cic1 is a strong hypomorph. The cic1 phenotype is similar to that of embryos from females carrying dominant gain-of-function mutations in tor (torgof) and other components of the Tor RTK pathway. These mutations cause constitutive Tor RTK signaling in all regions of the embryo, leading to ectopic tll and hkb expression, and the subsequent differentiation of the segmented trunk as terminal structures. However, the above results with deficiencies of the cic region indicate that cic1 is a recessive loss-of-function mutation (Jimenez, 2000).
The dorsal-ventral pattern of the Drosophila egg is established during oogenesis. Epidermal growth factor receptor (Egfr) signaling within the follicular epithelium is spatially regulated by the dorsally restricted distribution of its presumptive ligand, Gurken. As a consequence, pipe is transcribed in a broad ventral domain to initiate the Toll signaling pathway in the embryo, resulting in a gradient of Dorsal nuclear translocation. Expression of pipe RNA requires the action of fettucine (fet) in ovarian follicle cells. Loss of maternal fet activity produces a dorsalized eggshell and embryo. Although similar mutant phenotypes are observed with regulators of Egfr signaling, genetic analysis suggests that fet acts downstream of this event. The fet mutant phenotype is rescued by a transgene of capicua (cic), which encodes an HMG-box transcription factor. Cic protein is initially expressed uniformly in ovarian follicle cell nuclei, and is subsequently downregulated on the dorsal side. Earlier studies described a requirement for cic in repressing zygotic target genes of both the torso and Toll pathways in the embryo. cic controls dorsal-ventral patterning by regulating pipe expression in ovarian follicle cells, before its previously described role in interpreting the Dorsal gradient (Goff, 2001).
A class of dominant suppressors of a weakly ventralizing mutation (spzD1) were isolated in a dysgenic screen. These suppressors map to 92D and define a new locus that has been termed fettucine (fet). The fetE11 mutation is a representative allele caused by the insertion of a P element. In a subsequent screen, the EMS-induced alleles fetT6 and fetU6 were generated. While embryos laid by spzD1/+ females were ventralized and failed to hatch, about 10% of eggs laid by fet spzD1/++ females hatched. Flies carrying the fetE11 allele are viable as homozygotes and as transheterozygotes with fetU6 and fetT6, and these females exhibit a recessive maternal effect phenotype in which the eggshell and embryo are dorsalized. The fetU6 allele behaves genetically like a null allele and is larval lethal, while the fetT6 allele is a strong hypomorph, and homozygotes die as pharate adults (Goff, 2001).
The fet eggshell morphology is dorsalized, as assessed by a lateral shift of broadened dorsal appendages. In the strongest mutant combinations, ectopic dorsal appendage base material is secreted around the anterior circumference of the egg. Embryos produced by fet mutant females (referred to as fet embryos) exhibit an expansion of dorsal cell fates around the circumference of the embryo. These fet embryos fail to hatch. They secrete a cuticle which consistes entirely of dorsal epidermis lacking any structures derived from lateral or ventral regions. This cuticular phenotype is preceded at the cellular blastoderm stage by expanded expression of the dorsal marker zen around the circumference of the embryo at the expense of the expression of the ventrolateral and ventral markers sog and Twist (Goff, 2001).
Both the production of the Grk signal in the oocyte and initial activation of the Egfr in follicle cells appear unaffected in mutant fet ovaries. As a consequence of Egfr signaling, the follicular epithelium is normally partitioned into dorsal and ventral domains through the spatially restricted expression of mirror and fringe transcripts, respectively. Concomitant with this early response, Egfr modulates its own signaling during the course of oogenesis by inducing the expression of genes (e.g. rho, kek1 and Cbl) that encode regulators, which act at the level of receptor activation. At early stage 10, which corresponds to the period when Egfr activation leads to the transcription of target genes, the expression patterns of mirror, fringe, rho and kek1 in mutant fet ovaries are indistinguishable from wild type (Goff, 2001).
The dorsalized phenotype observed with the loss of fet activity in follicle cells is apparently caused by a requirement for fet in each branch of the Egfr pathway that separately patterns the embryo and eggshell. In establishing DV polarity of the embryo, fet is required as an essential transcriptional regulator of pipe RNA expression in the ventral follicle cells. By epistasis analysis, fet acts downstream of Egfr and causes a dorsalized phenotype even when Egfr signaling is reduced (Goff, 2001).
In establishing DV polarity of the eggshell, fet appears to be involved in refining Egfr activity later during oogenesis. Although mirror-lacZ is initially expressed correctly, the domain later expands around the anterior circumference of the egg chamber, suggesting a role for fet after the first round of Egfr signaling by Grk. This interpretation is supported by analysis of the double mutant. In contrast to the strictly linear relationship observed for establishing embryonic polarity, the eggshell phenotype produced by Egfr1; fet females shows contributions from each of the individual phenotypes. The distance between dorsal appendages is reduced, as observed for the ventralizing Egfr1 mutation, but the individual appendage structure is broadened, as seen for the dorsalizing fet mutation (Goff, 2001).
In the wild-type eggshell, dorsal appendage pattern is achieved through refinement of the Egfr activation profile by both positive and negative feedback regulation. Expression of rhomboid RNA is induced as a result of Egfr activation and positively regulates continued Egfr signaling. Induction of argos RNA expression leads to negative feedback on Egfr signaling at the dorsal midline, causing refinement of one domain of Egfr activation into two laterally symmetric domains that specify the placement of the paired dorsal appendages, This dorsal appendage pattern can be genetically altered in different ways. The K10 and squid mutant phenotype is characterized by an eggshell with a fused cylindrical dorsal appendage around the anterior circumference of the egg deposited around a dorsalized embryo. The fet and Cbl mutant eggshell phenotype appears distinct; rather than a single cylindrical structure, two laterally placed broadened dorsal appendages form, often associated with circumferential dorsal appendage base material. A change in either the strength or timing of Egfr signaling might translate into these different phenotypes (Goff, 2001).
In addition to the maternal effect eggshell and embryo phenotype, viable fet alleles exhibit wing phenotypes and strong fet alleles are lethal. This range of fet phenotypes, as compared with the cic1 phenotype, may be accounted for by the molecular nature of the mutations. The cic1 mutation is caused by a hobo mobile element insertion in the 5' untranslated region of the cic transcript, while the bwk8482 allele, which also has a maternal effect phenotype caused by a germline defect, is associated with a P-element insertion in the same region. These mobile elements may contain cryptic promoters that allow sufficient expression of the cic transcript to rescue the somatic but not the germline functions of this gene (Goff, 2001).
Signaling via the receptor tyrosine kinase (RTK)/Ras pathway promotes tissue growth during organismal development and is increased in many cancers. It is still not understood precisely how this pathway promotes cell growth (mass accumulation). In addition, the RTK/Ras pathway also functions in cell survival, cell-fate specification, terminal differentiation, and progression through mitosis. An important question is how the same canonical pathway can elicit strikingly different responses in different cell types. This study shows that the HMG-box protein Capicua (Cic) restricts cell growth in Drosophila imaginal discs, and its levels are, in turn, downregulated by Ras signaling. Moreover, unlike normal cells, the growth of cic mutant cells is undiminished in the complete absence of a Ras signal. In addition to a general role in growth regulation, the importance of cic in regulating cell-fate determination downstream of Ras appears to vary from tissue to tissue. In the developing eye, the analysis of cic mutants shows that the functions of Ras in regulating growth and cell-fate determination are separable. Thus, the DNA-binding protein Cic is a key downstream component in the pathway by which Ras regulates growth in imaginal discs (Tseng, 2007).
A genetic screen was performed, by using mitotic recombination in the developing eye, for mutations that allow homozygous mutant cells to outgrow their wild-type neighbors. In addition to mutations in genes, such as Tsc1, Tsc2, Pten, salvador, warts and hippo, that encode negative regulators of growth and result in grossly enlarged eyes, mutations were identified where the only observable abnormality was an overrepresentation of mutant over wild-type tissue. Four such mutations belonged to a single lethal complementation group. Eyes containing mutant clones showed an increased relative representation of mutant tissue over wild-type tissue. Eyes containing mutant clones also consistently contained more ommatidia (mean = 763 ommatidia) and were thus slightly larger than eyes containing clones that were homozygous for the parent chromosome (mean = 703 ommatidia). Otherwise, the eyes were normal in appearance (Tseng, 2007).
All four alleles failed to complement the lethality of cicfetU6 and cicfetE11, which are alleles of capicua (cic). Mutations in the cic locus (also known as fettucine and bullwinkle) have been isolated in screens for mutations that disrupt either embryonic patterning or patterning of the eggshell, but the role of cic as a negative regulator of growth has not been described previously. cic encodes a protein with a single high-mobility group (HMG)-box that localizes to the nucleus and that is likely to bind DNA via its HMG-box motif. Each of the four mutant chromosomes isolated in the screen has a mutation in the coding region of the cic gene (Tseng, 2007).
An antibody that recognizes the C-terminal portion of Cic stains nuclei throughout the eye imaginal disc. There is a stripe of increased expression immediately anterior to the morphogenetic furrow and reduced expression in the morphogenetic furrow itself. Staining is not detected in clones of cicQ474X cells, thus confirming that the antibody recognizes the C-terminal portion of the Cic protein (Tseng, 2007).
In the eye imaginal disc, loss-of-function mutations in cic appear to increase tissue growth but do not seem to perturb cell-fate specification or differentiation. cic mutant ommatidia were indistinguishable from wild-type ommatidia in terms of the size, number, and arrangement of photoreceptor cells in the adult retina and appear to develop normally at earlier stages. Discs containing cic clones also showed normal patterns of BrdU incorporation throughout the eye imaginal disc. However, cic clones anterior to the morphogenetic furrow contained a 2- to 3-fold higher density of cyclin-E-positive cells per unit of pixel area than wild-type clones, consistent with the increased rate of cell proliferation in mutant clones. As in wild-type discs, no BrdU incorporation was observed in cic mutant discs posterior to the second mitotic wave, and ectopic cyclin E protein was not observed in cic clones posterior to the second mitotic wave. The patterns of mitosis as assessed by staining with anti-phospho-histone H3 were also unchanged. Thus, cic cells maintain a relatively normal pattern of S phases and mitoses in the eye disc and are still able to exit from the cell cycle in a timely manner. In mature pupal eye discs, occasional extra interommatidial cells are observed in mutant clones, suggesting that cic cells may have a subtle defect in developmental apoptosis (Tseng, 2007).
To examine the growth characteristics of cic cells at greater resolution, cells from the eye and wing discs of early third instar larvae (120 hr AED) were dissociated and analyzed by flow cytometry. The distribution of mutant cells in the different phases of the cell cycle as assessed by their DNA content was very similar to that of wild-type cells, as was cell size as assessed by forward scatter in cells of the eye disc or the wing disc. As in the adult eye and the eye imaginal disc, the area occupied by mutant clones in the wing disc was larger than the corresponding wild-type twin spots, suggesting that the mutant cells collectively grow (accumulate mass) more quickly than their wild-type neighbors. Also, mutant clones typically contained more cells than their wild-type twin spots. The inferred population doubling time calculated from the median clone size was 10.3 hr in mutant clones compared to 12.3 hr in the wild-type twin spots. The simplest interpretation of all of these observations is that cic cells have an increased rate of growth (mass accumulation) compared to wild-type cells but maintain a normal size because of a commensurate acceleration of the cell cycle. These findings indicate that a normal function of cic is to restrict cell growth in both the eye and wing imaginal discs (Tseng, 2007).
Previous work has shown that the levels of Cic protein are responsive to the level of signaling via RTKs and Ras. In the embryo, the level of Cic protein in the terminal regions is decreased upon signaling via the Tor RTK. Activation of Ras in the cells of the wing imaginal disc also reduces Cic levels in those cells. In eye discs, loss-of-function clones of Egfr or Ras, although small, had clearly elevated levels of Cic protein. Conversely, clones of cells expressing the activated form of Ras, Ras (Val12), had reduced levels of Cic. Thus, as in other tissues, increased signaling via the Egfr/Ras pathway reduces Cic protein levels in the eye disc. Furthermore, studies with mutations in the effector domain of Ras suggest that Ras regulates Cic primarily via the Raf/MAPK pathway. This is consistent with a recent study that has shown a direct interaction between Cic and MAPK (Tseng, 2007).
In the eye imaginal disc, clones of RasΔC40b, a null allele of Ras, were much smaller than their wild-type twin spots. Strikingly, clones of cells that were mutant for both cic and RasΔC40b were indistinguishable from cic clones in that they were typically larger than their twin spots. Thus, the loss of cic function completely bypasses the requirement for Ras in promoting cell growth. In contrast to the result obtained with cic, clones that were doubly mutant for Ras as well as a different negative regulator of growth, Tsc1, were no larger than Ras clones. Hence, the ability of cic to suppress the growth defect of Ras clones is specific and not a general property of negative regulators of growth. Also, cic mutations did not suppress the growth defect resulting from mutations in the Insulin Receptor (InR), Akt, and Rheb. Thus, cic mutations appear capable of rendering cell growth independent of Ras-mediated signaling but not independent of InR/PI3K- or Tor-mediated signaling. Taken together, these findings support the notion that the ability of Cic to restrict cell growth is specific to its function as a downstream component of the Ras pathway (Tseng, 2007).
In addition to promoting tissue growth, the recruitment of photoreceptor cell precursors to the developing ommatidia occurs via reiterated use of the EGFR/Ras pathway. Clones of cells that are mutant for RasΔC40b do not contain clusters of cells expressing the neural marker Elav, and instead they contain only the regularly spaced single Elav-positive nuclei that belong to the R8 photoreceptor cells. Although clones doubly mutant for cic and RasΔC40b are of normal size, they, like Ras clones, contain single nuclei that stain with anti-Elav and express the R8-specific marker Senseless. Thus, loss of cic function does not bypass the requirement for Ras function in the specification of photoreceptor cells R1-R7. Mutations in Tsc1 suppress the requirement for Ras neither in growth nor in photoreceptor differentiation. Thus, adult eyes containing clones doubly mutant for cic and RasΔC40b have large patches of tissue lacking any recognizable ommatidia. In retinal sections, there are no photoreceptor cells in the cic Ras double-mutant clones, and all the photoreceptor cells at the borders of the clone are wild-type for Ras. Thus, although they exhibit impaired photoreceptor differentiation, cic Ras double-mutant clones are not impaired in their growth and, unlike Ras clones, are not outcompeted by neighboring cells. Indeed, the phenotype of cells doubly mutant for Ras and cic is extremely similar to that of large Ras clones that are generated in a Minute background, suggesting that cic mutations primarily rescue the growth disadvantage of Ras clones (Tseng, 2007).
Thus, in the eye disc, there may be a branching of the Egfr/Ras pathway. One branch, functioning via Cic, appears important for growth regulation, whereas the other branch, acting via Pnt, appears important for photoreceptor cell-fate specification. In contrast to Ras clones, clones of pnt in the eye imaginal disc do not show a marked growth defect, suggesting that pnt has a minor role in regulating tissue growth in the eye disc (Tseng, 2007).
In mammalian cells, several extracellular growth factors that act via RTKs increase the activity of cyclin D/Cdk4 or cyclin D/Cdk6 complexes that can phosphorylate and inactivate the retinoblastoma protein (pRb) and thus promote S phase entry. However, it is still unclear how inactivation of pRb can cause cell growth (mass accumulation). At least in Drosophila, the role of Cic appears distinct from cyclin D because neither are cyclin D protein levels elevated in cic clones nor is the growth advantage of cic cells over wild-type cells compromised in flies that completely lack Cdk4/6 function. Other studies suggest that Ras can promote cell growth by stabilizing Myc protein via MAPK-mediated phosphorylation. This mode of Ras function also appears to be dispensable under conditions where cic function is inactivated but may still be relevant at physiological levels of Ras signaling (Tseng, 2007).
Notably, these data also show that Cic also functions as a negative regulator of tissue growth in the wing disc. However, in this tissue, Cic has a role in specifying cell fates as well because others have shown that cic mutations result in the formation of ectopic vein tissue. Thus, although the role of Cic as a regulator of growth in imaginal discs appears to be general, the importance of Cic in pathways that regulate cell-fate determination may vary from one tissue to another (Tseng, 2007).
The human and mouse genome each appear to have a single cic ortholog whose function in the regulation of growth has not been addressed to date. However, a recent study that determined the DNA sequence of 13,023 genes from 11 breast and 11 colorectal cancers found missense mutations in the human cic ortholog in three of the breast cancers. Although the functional consequences of these mutations have not been evaluated, these data suggest that Cic may indeed function in restricting cell growth in human cells (Tseng, 2007).
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date revised: 16 January 2008
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