The epidermal growth factor receptor (EGFR) and Hippo signaling pathways control cell proliferation and apoptosis to promote tissue growth during development. Misregulation of these pathways is implicated in cancer. Understanding of the mechanisms that integrate the activity of these pathways remains fragmentary. This study identifies bantam microRNA as a common target of these pathways and suggests a mechanistic link between them. The EGFR pathway acts through bantam to control tissue growth. bantam expression is regulated by the EGFR pathway, acting via repression of the transcriptional repressor Capicua. Thus EGFR signaling induces bantam expression by alleviating the effects of a repressor. bantam in turn acts in a negative feedback loop to limit Capicua expression. bantam appears to be a transcriptional target of both the EGFR and Hippo growth control pathways. Feedback regulation by bantam on Capicua provides a means to link signal propagation by the EGFR pathway to activity of the Hippo pathway and may play an important role in integration of these two pathways in growth control (Herranz, 2012).
The ability of the EGFR pathway to drive tissue growth resides in its ability to coordinately stimulate cell proliferation and suppress apoptosis. Understanding how coordinated control is achieved depends on identification of the effector mechanisms that mediate these outputs along with the connections to other growth regulatory pathways. The results show that the bantam miRNA is a critical target of the EGFR pathway. Further, a mechanism is outlined by which bantam serves as a link between the EGFR and Hippo pathways (Herranz, 2012).
In Drosophila, EGFR pathway effectors include the transcription factors Pointed, Tramtrack, and Yan, and the HMG-box repressor Capicua. Capicua has an important role in early embryonic patterning and as a negative growth regulator. Although several Capicua targets involved in embryonic patterning have been identified, how Capicua regulates tissue growth was unknown. These results identify bantam as an important target of Capicua required to mediate EGFR-dependent tissue growth (Herranz, 2012).
A key finding of this study is the regulatory feedback relationship between bantam and Capicua. Each represses the activity of the other. Viewed from the perspective of the EGFR/MAPK pathway alone, the outcome of this relationship would be signal amplification, with downregulation of Capicua levels by bantam reinforcing direct MAPK-induced turnover of Capicua protein. This adds a new mechanism to the repertoire of positive and negative feedback loops affecting EGFR pathway activity. These feedback mechanisms are thought to be important in disease, and their regulation is complex. Relatively little is known about Capicua in cancer, although one recent study reports mutants in the human Cic protein in oligodendroglioma (Bettegowda, 2012; Herranz, 2012 and references therein).
An alternative logic for the relationship between bantam and Capicua may be seen in the fact that it links the output of the EGFR pathway to the output of the Hippo pathway, mediated through transcriptional regulation of bantam by Yorkie. EGFR signaling via MAPK and bantam cooperate to downregulate Capicua protein levels. Thus the transcriptional output of the Hippo pathway via Yorkie can be seen as potentiating EGFR signaling by 'lowering' the effective threshold of MAPK activity needed to reduce Capicua to a given level. Alternatively, the lack of sufficient Yorkie activity would lower bantam activity and thereby raise the threshold of EGFR activity required to reach an effective level of Capicua downregulation. This provides a mechanism to ensure coordination of the growth regulatory pathways. Signaling via the Hippo pathway has also been shown to induce the EGFR ligand amphiregulin to promote tissue growth in a nonautonomous manner. Thus, there appear to be multiple levels of crosstalk between these pathways (Herranz, 2012).
Considerable evidence is emerging linking miRNAs to robustness of regulatory feedback networks. It is intriguing that miRNAs are now implicated in regulation of all three of the known transcriptional effectors of EGFR signaling. miR-7 acts in two feed-forward loops downstream of EGFR to control photoreceptor specification and differentiation in the Drosophila eye. EGFR acts via the transcription factors Yan and Pointed. Yan is a direct target of miR-7. Yan also represses miR-7 transcription directly as well as indirectly. In the same cells, the ETS-1 factor Pointed-P1 activates miR-7 to repress Yan as well as acting directly to repress Yan. Use of interlinked motifs is thought to provide stability to the cell differentiation program controlled by EGFR. The current findings link bantam to regulation of a third EGFR transcriptional effector, Capicua, in addition to its regulation by the Hippo pathway. Coordination of diverse growth control inputs by miRNAs might contribute to robustness (Herranz, 2012).
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).
The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind), a gene expressed in a stripe along the DV axis in lateral regions of the embryo. This study shows that a short 12 base pair sequence ('the A-box') present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, affinity chromatography using the A-box element was conducted and a number of DNA-binding proteins and chromatin-associated factors were found using mass spectroscopy. Only Grainyhead (Grh), a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. The results suggest that maternally expressed Grh acts as an activator to support expression of ind, which was surprising since this factor was identified using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic) also binds to the A-box sequence. While Cic was not identified through the A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator) and Cic (repressor) may also support a 'switch-like' response that helps to sharpen the ind dorsal boundary. Furthermore, the results also demonstrate that TGF-beta signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo (Garcia, 2011).
Other studies have shown combinatorial interactions are necessary to support patterns of gene expression along the DV axis. For instance, one study showed Dorsal and Zelda function together to produce the broad lateral domain of sog. Mutation of either the Dorsal sites or the Zelda sites in the sog CRM produced a pattern that was narrower than the wild-type expression pattern. It was concluded that both Dorsal and Zelda must be present to produce a proper Sog pattern. It is also well appreciated that Dorsal can act cooperatively with the bHLH transcription factor Twist to support expression in ventral and ventrolateral regions of the embryo. It is proposed that Grh and Dorsal act together to support the ind expression pattern. While the ind CRM containing a mutant Dorsal site did support some expression, the expression pattern contained a gap and was weaker in posterior regions; in contrast, in Dorsal mutants, ind expression is completely absent. This result may be explained if both indirect as well as direct functions for Dorsal are required to support ind expression. For instance, Dorsal has other target genes including rho, which is required to support Egfr signaling. Furthermore, mutation of the A-box/Grh binding site within the ind CRM caused expression of the reporter that was expanded dorsally and weak, suggesting this site mediates repression and also activation. Similar to Dorsal mutants, the phenotype observed when the A-box sites were mutated is different than the phenotype in the Grh mutants, thus it cannot be ruled out that Grh may act through other sites as well as the A-box and/or that Grh may act indirectly to influence ind expression by regulating the expression of other transcription factors. A model is proposed that is most consistent with the current data which is that ind is activated in regions where Dorsal is present as well as optimal levels of Grh; it is then refined by Snail and Vnd in ventral regions and Cic and Schnurri/Mad/Medea (SMM) in dorsal regions (Garcia, 2011).
grh and cic genes are both maternal and ubiquitously expressed, thus, another input is necessary to explain how localized expression of ind is supported. This positional information could be provided in part by competition between Grh and Cic proteins for the A-box binding site and in part by ventrolaterally-localized Egfr signaling. A model in which Egfr signaling supports activation of ind via inhibition of a ubiquitous repressor (e.g. Cic) is supported by the results which demonstrate that A-box mediated repression is expanded in Egfr mutants. A recent study also showed expanded expression of an ind CRM fragment reporter in ras cic double mutants in which neither Egfr signaling or Cic repressor is present, suggesting that Egfr may function by inhibition of an 'inhibitor' to promote activation. This data suggests that the putative A-box repressor, Cic, may not be dorsally localized but that its activity is regulated by Egfr signaling which provides the positional information necessary for a sharp boundary. However, the domain of dpERK activation (as detected by anti-dpERK, an antibody to the dual-phosphorylated from of ERK) does not exactly overlap with the ind expression domain at cellularization, as would be expected in the simplest model (Garcia, 2011).
Ajuria (2011) suggested that Egfr signaling supports ind expression through inhibition of Cic, and it is added that it is also plausible Egfr signaling impacts activation of ind through Grh. In fact, a recent study showed that Grh activity during wound response is modulated by ERK signaling. Specifically, both unphosphorylated and phosphorylated Grh were shown to be able to bind DNA and act as an activator. The former is used during normal development of the epidermal barrier and the latter is used to overcome a semi-dormant state during wound response. Another study showed the tyrosine kinase Stitcher activates Grh during epidermal wound healing. In the early embryo Grh may be phosphorylated by Egfr signaling to support activation of ind through the A-box binding site. It is suggested that phosphorylation of both Grh as well as Cic by Egfr signaling can act as a switch to help fine-tune the expression of ind (Garcia, 2011).
Whether a relationship between Grh activation and Cic repression was used in regulation of other genes containing A-box or Cic binding sites was investigated. One other Cic target gene, hkb, was unaffected in Grh mutants. As the A-box site (WTTCATTCATRA) is larger than the Cic consensus binding sequence [T(G/C)AATGAA, complement TTCATT(G/C)A] defined by Ajuria (2011) it is possible that Grh needs the full A-box site to bind. The full A-box sequence is not present in the hkb CRM, but Cic binding may be facilitated by a partial sequence (i.e., TGAATGAA). Alternatively, it is possible that a role for Grh and/or Cic at the A-box is context dependent. For instance, Grh-mediated activation may be a necessary input to support ind expression but not for the support of hkb, which also receives activation input from Bicoid and Hunchback transcriptional activators and is expressed in the pre-cellularized embryo (Garcia, 2011).
Other studies have suggested that Grh acts to repress transcription of fushi tarazu (ftz), dpp, and tll in the Drosophila embryo, but this study is the first to identify a role for Grh-mediated gene activation in the early embryo, in support of dorsoventral patterning. Previous studies had shown that Grh can function as an activator at later embryonic stages. One analysis identified Grh (also called NTF-1 or Efl-1) biochemically using an element from the dpp early embryonic CRM, however the dpp expression domain was unchanged in the grh mutants (Garcia, 2011).
Another recent study also showed Grh binds to sites that are similar to Zelda binding sites (Harrison, 2010). Zelda and Grh each showed stronger affinity for different variations of the shared consensus sequence, but in vitro studies showed they also competed for binding. Harrison (2010) proposed that as levels of Zelda increase it is able to compete against Grh for binding sites and cause activation of the first zygotic genes. Competition at the same binding sites results in a cascading effect in which ubiquitous activators regulate genes in a temporally related manner. It was proposed that Grh functions first to silence gene expression; while, alternatively, the current data is more consistent with a model in which Grh mediated activation follows that of Zelda. ind is considered a 'late' response gene as it appears at mid stage 5 (nc 14), at the onset of cellularization, whereas Zelda was shown to support gene expression earlier at nc 10 (Garcia, 2011).
It is possible that Grh competes for binding to a variety of sites (not only those recognized by Zelda), and that this competition influences gene activation/repression. At the A-box sequence, Cic and Grh may compete to help establish a sharp boundary; unfortunately, the Cic binding to the A-box sequence demonstrated previously in vitro was quite weak (Ajuria1, 2010), so this competition is best examined in vivo in future studies (Garcia, 2011).
This study found there is yet another tier of repression activity that is independent of the A-box mediated repression. Analysis of the eve.stripe3/7-ind-mutant-A-box reporter construct revealed that, while dorsal-lateral repression was lost, there was still repression in the dorsal-most part of the embryo. This led to the idea that other binding sites in the ind CRM, independent of the A-box binding site, mediate repression. Previous research showed ectopic TGF-beta/Dpp signaling can repress ind expression, and therefore it is hypothesized that the repression activity observed in dorsal-most regions of the embryo may be regulated by Dpp signaling (Garcia, 2011).
The results suggested that the Dpp dependent repression supports repression in the dorsal most part of the embryo and not in dorsal lateral regions of the embryo. An expansion of the ind domain in the mutants affecting only this dorsal-most repressor would not be expected, thus the SMM site was mutated in the context of two mutant A-boxes and it was found that the expression pattern was expanded into dorsal regions of the embryo. However, when the A-box sites were mutated, expansion of ind more dorsally into dorsal-lateral regions was seen, but expression was absent in dorsal-most regions. It is possible the embryo can tolerate a slight expansion of ind into dorsal lateral regions of the embryo but expansion of ind into the non-neurogenic ectoderm is detrimental. Thus, two tiers of repression have developed to insure that expression of ind is limited to the neurogenic ectoderm. It is suggested that partially redundant repressor mechanisms are more common than appreciated, because in contrast to activation it is difficult to track repression activity (Garcia, 2011).
Epigenetic changes to DNA and chromatin remodeling have been shown to be vital in repression and activation of genes that define structures in late stages of Drosophila development. For example, Polycomb group genes silence the homeotic genes of the Bithorax complex, which control differentiation of the abdominal segments. To date, little is known regarding how/if chromatin factors play a role in early development of Drosophila embryos. This study has presented evidence that several chromatin-related factors bound an A-box affinity column but did not bind a column containing the mutant A-box element. Although several of these factors did not bind to the A-box element alone when tested by EMSA, it is possible that they bind indirectly via a larger complex. One of these factors Psq has been implicated in both silencing and activation via the Polycomb/Trithorax response elements. Independently, Psq was recently found to positively regulate the Torso/RTK signaling pathway in the germline, while being epistatic to cic a negative regulator of the Torso signaling. It is possible that some of these factors play a role in regulating ind via the A-box element, which would suggest a role for chromatin remodeling early in development - an avenue which is worth pursuing in future studies (Garcia, 2011).
The homeodomain (HD) protein Bicoid (Bcd) is thought to function as a gradient morphogen that positions boundaries of target genes via threshold-dependent activation mechanisms. This study analyzed 66 Bcd-dependent regulatory elements, and their boundaries were shown to be positioned primarily by repressive gradients that antagonize Bcd-mediated activation. A major repressor is the pair-rule protein Runt (Run), which is expressed in an opposing gradient and is necessary and sufficient for limiting Bcd-dependent activation. Evidence is presented that Run functions with the maternal repressor Capicua and the gap protein Kruppel as the principal components of a repression system that correctly orders boundaries throughout the anterior half of the embryo. These results put conceptual limits on the Bcd morphogen hypothesis and demonstrate how the Bcd gradient functions within the gene network that patterns the embryo (Chen, 2012).
This study identified 32 enhancers that respond to Bcd-dependent activation and form expression boundaries at different positions along the AP axis of fly embryos. Adding these elements to the 34 previously known enhancers constitutes the largest data set of in vivo-tested and -confirmed enhancers regulated by a specific transcription factor in all of biology (Chen, 2012).
The 32 confirmed enhancers were identified among 77 tested genomic fragments, which were selected because they showed in vivo-binding activity, or they conformed to a stringent homotypic-clustering model for predicted Bcd-binding sites, or both. All seven previously unknown fragments showing in vivo binding and a predicted site cluster directed Bcd-dependent transcription in the early embryo. Other fragments from the top 50 ChIP-Chip signals (which do not conform to the clustering model) were also very likely (21 of 26) to test positive in the in vivo test, but this likelihood drops significantly (9 of 25) in a set of fragments from lower on the list of ChIP-Chip fragments. Interestingly, of 19 tested fragments that contain clusters of predicted sites, but no in vivo binding activity, not a single one tested positive in vivo. These results suggest that in ;vivo binding assays are much better predictors of regulatory function than simple site-clustering algorithms alone (Chen, 2012).
One explanation for the failure of these predicted site clusters to bind Bcd in vivo is that they lie in heterochromatic regions of the genome that prevent site access. However, because they fail to function when taken out of their normal context (in reporter genes), whatever is preventing activation must be a property of the fragment itself and not its location in the genome. Interestingly, a number of Bcd site cluster-containing fragments drive expression later in development. It is proposed that these fragments fail to bind Bcd because they lack sites for cofactors that facilitate Bcd binding. In preliminary experiments it was observed that Bcd-activated fragments contain on average more binding sites for the ubiquitous activator protein Zelda (Zld) than those that fail to activate. Zld has been shown to be critical for timing the zygotic expression of hundreds of genes in the maternal to zygotic transition (Chen, 2012).
These results suggest strongly that a gradient of Run protein plays a major role in limiting Bcd-dependent activation. Run seems to work as part of a repression system that also includes Cic and possibly Kr. Expression boundaries in the region anterior to the presumptive cephalic furrow shift toward the posterior in run and cic mutants, and the double mutant causes boundaries that are normally well separated to collapse into a single position (Chen, 2012).
The use of multiple repressors permits flexibility in binding site architecture within enhancers that establish boundaries at similar positions. For example type I enhancers show overrepresentations of both Run and Cic sites, but 27% lack strong matches to the Cic PWM, and 12% lack strong matches to the Run PWM. Importantly, however, all type I enhancers lacking Cic sites contain Run sites, and those lacking Run sites contain Cic sites. Multiple Kr sites were observed in a large number of Bcd-dependent enhancers, which suggests that Kr is also a major component of the repression system that orders Bcd-dependent expression boundaries. Taken together, these data suggest that antagonistic repression of Bcd-mediated activation is a key design principle of the system that organizes the AP body plan. The repressors identified so far (Run, Cic, and Kr) are expressed in overlapping domains with gradients at different positions, consistent with the formation and ordering of a relatively large number of boundaries throughout the anterior half of the embryo (Chen, 2012).
The close linkage between repressor sites and Bcd sites within discrete enhancers suggests that repression occurs via short-range interactions that interfere directly with Bcd binding or activation. Interestingly, Cic also shows repressive effects that seem to be binding site independent. For example some type I enhancers do not contain recognizable Cic sites, but their expression boundaries expand posteriorly in cic mutants. This could be caused by the reduced expression of run and Kr in cic mutants. However, genetically removing both Kr and run causes a less dramatic expansion than that seen in the absence of cic. This suggests that Cic binds these enhancers via suboptimal sites or that it is required for the correct patterning of another unknown repressor. Another possibility is that these expansions are caused indirectly by changing the balance of MAPK phosphorylation events that control terminal patterning (Chen, 2012).
These results do not strictly falsify the Bcd morphogen hypothesis, but they support the idea that the Bcd gradient can establish only a 'rough framework that is elaborated by the interaction of the zygotic segmentation genes'. What is the nature of this framework, and what role does it play in the network that precisely positions target gene boundaries (Chen, 2012)?
One component of the system, the Cic repression gradient, is maternally produced and formed by downregulation at the poles via the terminal patterning system. This gradient is formed independently of Bcd but is critical for establishing boundaries of Bcd-dependent target genes. In contrast, Bcd is involved in activating the expression patterns of run and Kr and in repressing them in anterior regions. Both run and Kr expand anteriorly in bcd mutants. There is no evidence that Bcd functions directly as a transcriptional repressor, so these repressive activities are probably indirect. Previous work showed that the Bcd target gene gt is involved in setting the anterior Kr boundary, and it is hypothesized that another Bcd target gene, slp1, encodes a forkhead domain (FKH) protein that sets the anterior boundary of the early run pattern. slp1 is expressed in a pattern reciprocal to the run pattern and was previously shown to position the anterior boundaries of several pair-rule gene stripes including run stripe 1 (Chen, 2012).
These results suggest that a major function of the Bcd gradient is the differential positioning of two repressors, Slp1 and Gt, which set the positions of the Run and Kr repression gradients, which then feedback to repress Bcd-dependent target genes. How are slp1 and gt differentially positioned? One possibility is that slp1 and gt enhancers respond to specific concentrations within the Bcd gradient, consistent with the original model for morphogen activity. However, the fact that the slp1 and gt expression domains form boundaries at the same positions in embryos lacking the Cic and Run repressors argues against this model for these genes (Chen, 2012).
It was also shown that Bcd target genes normally expressed in cephalic regions form and correctly position posterior boundaries in embryos containing flattened Bcd gradients. Run is still expressed in these embryos, specifically in a domain that consistently abuts the boundaries of the anterior Bcd target genes, regardless of copy number. This suggests that a mutually repressive interaction between Slp1 and Run is maintained in these embryos but does not explain how these boundaries are consistently oriented perpendicularly to the AP axis. The answer might lie in the fact that the flattened Bcd gradients in these embryos are not completely flat but are present as shallow gradients with slightly higher levels in anterior regions. In these embryos the slight changes in concentration along the AP axis might cause a bias that enables the orientation of the mutual repression interaction. In wild-type embryos, Bcd is much more steeply graded, which makes this bias stronger and the boundary between these mutual repressors more robust (Chen, 2012).
These results suggest that antagonistic repression precisely orders Bcd-dependent expression boundaries. However, repression may not be required for the activity of all morphogens. For example the extracellular signal activin has been shown to activate target genes in a threshold-dependent manner in isolated animal caps from frog embryos. Also, a gradient of the transcription factor Dorsal (Dl) is critical for setting boundaries between different tissue types along the dorsal-ventral (DV) axis of the fly embryo. It is thought that the major mechanism in Dl-specific patterning is threshold-dependent activation, which is quite different from the system described in this paper. One major difference between Bcd and Dl is the number of boundaries specified: three for Dl and more than ten for Bcd. It is proposed that the robust ordering of more boundaries simply requires a more complex system (Chen, 2012).
In general, though, it seems that antagonistic mechanisms are involved in controlling the establishment or interpretation of most morphogen activities. For example in the Drosophila wing disc, the TGF-N2 signal Dpp forms an activity gradient that is refined by interactions with multiple extracellular factors. Also, in vertebrates the signaling activity of the extracellular morphogen Sonic hedgehog (Shh) is affected by positive and negative interactions with specific molecules on the surfaces of receiving cells (Chen, 2012).
There is some evidence that transcriptional repression is also used for refining the patterning activities of extracellular molecules. Dpp acts as a long-range morphogen that activates two major target genes (optomotor blind [omb] and spalt [sal]) in nested patterns with boundaries at different positions with respect to the source of Dpp. Although these boundaries could in theory be formed by differential responses to the morphogen, it is clear that the transcriptional repressor Brinker (Brk), which is expressed in an oppositely oriented gradient, also plays an important role. The Brk gradient is itself positioned by Dpp activity in a manner analogous to positioning of the Run and Kr repressor gradients by Bcd. Also, a similar transcriptional network functions in Shh-mediated patterning of the vertebrate neural tube, where a series of spatially oriented repressors feeds back to limit the expression boundaries of Shh-mediated cell fate decisions (Chen, 2012).
Conceptually, these more complex systems are reminiscent of the reaction-diffusion model proposed by Turing, in which a localized activator would activate a repressor, which would diffuse more rapidly than the activator, and feed back on its activity. These systems strongly suggest that the patterning activity of a single monotonic gradient is insufficiently robust for establishing precise orders of closely positioned expression boundaries. By integrating gradients with repressive mechanisms that refine gradient shape or influence outputs, systems are generated that ensure consistency in body plan establishment while still maintaining the flexibility required for complex systems to evolve (Chen, 2012).
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).
The maternal transcription factor Dorsal (Dl) functions as both an activator and a repressor in a context-dependent manner to control dorsal-ventral patterning in the Drosophila embryo. Previous studies have suggested that Dl is an intrinsic activator and its repressive activity requires additional corepressors that bind corepressor-binding sites near Dl-binding sites. However, the molecular identities of the corepressors have yet to be identified. This study presents evidence that Capicua (Cic) is involved in Dl-mediated repression in the zerknullt (zen) ventral repression element (VRE). Computational and genetic analyses indicate that a DNA-binding consensus sequence of Cic is highly analogous with previously identified corepressor-binding sequences and that Dl failed to repress zen expression in lateral regions of cic mutant embryos. Furthermore, electrophoretic mobility shift assay (EMSA) shows that Cic directly interacts with several corepressor-binding sites in the zen VRE. These results suggest that Cic may function as a corepressor by binding the VRE (Shin, 2014).
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).
Astigarraga, S., (2007). A MAPK docking site is critical for downregulation of Capicua by Torso and EGFR RTK signaling. EMBO J. 26(3): 668-77. PubMed ID: 17255944
Krivy, K., Bradley-Gill, M. R. and Moon, N. S. (2013). Capicua regulates proliferation and survival of RB-deficient cells in Drosophila. Biol Open 2: 183-190. PubMed ID: 23429853
Mutations in rbf1, the Drosophila homologue of the RB tumour suppressor gene, generate defects in cell cycle control, cell death, and differentiation during development. Previous studies have established that EGFR/Ras activity is an important determinant of proliferation and survival in rbf1 mutant cells. This study reports that Capicua (Cic), an HMG box transcription factor whose activity is regulated by the EGFR/Ras pathway, regulates both proliferation and survival of RB-deficient cells in Drosophila. cic mutations allow rbf1 mutant cells to bypass developmentally controlled cell cycle arrest and apoptotic pressure. The cooperative effect between Cic and RBF1 in promoting G1 arrest is mediated, at least in part, by limiting Cyclin E expression. Surprisingly, evidence was also found to suggest that cic mutant cells have decreased levels of reactive oxygen species (ROS), and that the survival of rbf1 mutant cells is affected by changes in ROS levels. Collectively, these results elucidate the importance of the crosstalk between EGFR/Ras and RBF1 in coordinating cell cycle progression and survival (Krivy, 2013).
Both Dap and Cic are negative regulators of proliferation downstream of the EGFR/Ras pathway. However, EGFR/Ras activity promotes Dap expression while inhibiting Cic expression (Astigarraga, 2007). Accordingly, their expression patterns at the MF show that Cic expression drops where Dap expression is most prominent. Immunostaining with anti-phospho-MAPK, which is a marker for EGFR activity and cells initiating differentiation processes, shows a similar expression pattern to that of Dap. Perhaps, once cells start to differentiate, Dap plays a predominant role over Cic to maintain differentiating cells in the G1 phase. This would also explain the absence of EdU-positive cells in rbf1 cic double mutant clones that express Atonal. It was noticed that Dap expression is slightly increased in rbf1 mutant clones. This is likely in response to the Cyclin E activation. In contrast to the MF, co-expression of Dap and Cic proteins was detected in the anterior region of the eye disc. Perhaps, in this region, EGFR/Ras is activated to a level at which both proteins can coexist. Nevertheless, the results indicate that, at least at the MF, the cellular context in which Dap and Cic act to restrict proliferation is distinct (Krivy, 2013).
In cic homozygous mutant eye discs generated in the rbf1120a heterozygous background, Cyclin E levels are specifically increased at the MF. In fact, this is the location where dE2F1 proteins are most highly expressed in the eye disc. While this observation suggests a strong cooperation between dE2F1 and Cic, no changes were observed in the dE2F1 protein level nor its activity in cic mutant clones. This result indicates that cic mutations do not affect dE2F1 activity in general. A previous study demonstrated that increases in both Cyclin E and E2F activities are necessary to overcome the cell cycle arrest imposed at the MF. This likely explains why ectopic S-phase cells were not observed in cic mutant cells generated in rbf1120a heterozygous eye discs despite the elevated level of Cyclin E expression. E2F target genes are consistently expressed at a lower level in cic mutant eye discs generated in the rbf1120a heterozygous background than rbf1120a homozygous mutant eye discs. Nevertheless, it cannot be excluded that dE2F1 is required for the effect of cic mutations on Cyclin E expression. It is interesting to note that both Dap and Cic act on Cyclin E/CDK2 and that their mutations cooperate with rbf1 mutations to cause uncontrolled proliferation. Perhaps, such a context in which either E2F1 or Cyclin E/CDK2 activity is elevated represents a sensitised genetic background where regulators of the other protein can be identified. Indeed, haploinsufficiency of rbf1 is shown to be sufficient to dominantly modify the rough eye phenotype induced by p21 overexpression, the mammalian inhibitor of Cyclin E/CDK2 (Krivy, 2013).
The molecular mechanism by which RBF1 and Cic cooperatively regulate Cyclin E expression remains unclear. RT-qPCR did not reveal that cic mutations produce any discernable changes in cyclin E RNA levels, indicating that the effect of cic mutations on Cyclin E expression is post-transcriptional and likely to be indirect. One interesting observation is that heterozygosity of rbf1 seems to have a general effect on the expression level of RBF1 target genes. Transcript levels of mcm2 and rnrS are elevated in the rbf1 heterozygous background compared to the wild type. This raises the possibility that increased expression of RBF1 target genes in general provides a specific context that allows the cic mutation to have an effect on Cyclin E expression. The transcriptional changes that are induced by cic mutations in control and rbf1 mutant backgrounds are currently being investigated to determine if Cic regulates different transcriptional programs depending on the status of RBF1 (Krivy, 2013).
Whether the alteration of Cyclin E levels is the only molecular mechanism by which cic and rbf1 mutations cooperate to promote ectopic S phase is still unclear. Oddly, no discernible change was observed in Cyclin E levels when cic mutant clones were generated in an rbf1120a homozygous background despite the presence of ectopic S-phase cells. One possible explanation for this is that rbf1 homozygous mutations increase Cyclin E expression to the level higher than what is achieved by cic mutations in rbf1120a heterozygous backgrounds. This increase is perhaps near, but not over, the threshold that can overcome the G1 arrest at the MF. In this context, cic mutations would provide the additional Cyclin E expression that is required to actually surpass this threshold. However, once cells enter S phase, Cyclin E is rapidly targeted for degradation, making it difficult to detect the increase in Cyclin E level. The lack of an increase in Cyclin E level in the rbf1120a homozygous background could also indicate that cic mutations can result in additional molecular changes that can cooperate with rbf1 mutations. While it is unclear what these changes might be, it is known that Cic's ability to regulate ROS is not likely to contribute to the cell cycle defect. No changes were observed in the EdU staining pattern when expression levels of SOD2 were altered in an rbf1 mutant background. Presently, transcriptional changes induced by cic mutations are being compared in wild-type and rbf1 mutant backgrounds in order to postulate a molecular mechanism (Krivy, 2013).
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date revised: 15 July 2013
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