Embryonic

scylla and charybdis are both expressed during embryogenesis in dynamic, partially overlapping patterns. In contrast to the broadly expressed scylla mRNA, charybdis transcripts are predominantly restricted to neurons of the CNS and PNS as assessed by mRNA in situ hybridization. During late larval stages scylla mRNA is uniformly expressed without apparent tissue-specific distribution, whereas charybdis mRNA expression could not be detected in third instar imaginal discs (Reiling, 2004).

Effects of Mutation and Overexpression

In order to identify novel genes involved in growth regulation by the Inr/TOR pathway, an EP overexpression screen was performed using a double-headed EP element. A genetically sensitized system involving coexpression of PKB and PDK1 (achieved by using EP837 that drives endogenous PDK1) was used in the eye; this leads to a big eye phenotype. Pilot experiments demonstrated that overexpression of PTEN or a dominant-negative version of the catalytic subunit of Drosopohila PI3K, Dp110, were not able to suppress the PKB/PDK1-dependent phenotype. Thus, the screening system is likely to identify components acting downstream of or in parallel to PKB/PDK1. For example, coexpression of Tsc1/Tsc2 strongly suppresses the phenotype of the tester flies (Reiling, 2004).

Two EP insertions (EP22.1, hereafter named EPscy, and EP9.85) were identified in the scylla locus as suppressors of the PKB/PDK1 bulging eye phenotype. BLASTP search with the Scylla amino acid sequence revealed another homologous protein encoded in the Drosopohila genome termed Charybdis. scylla (scy, CG7590) and charybdis (char, CG7533) are separated by ~232 kb of genomic DNA. Their gene products share a high degree of homology (38% identity, 49% similarity), suggestive of a gene duplication event. Whether charybdis overexpression would behave similarly to scylla was examined in the PKB/PDK1 overexpression assay using EP1035 (hereafter named EPchar). Indeed, the big eye phenotype of the tester system is also suppressed by EPchar. UAS transgenes with either the scylla or charybdis cDNA recapitulate the suppression phenotype of the corresponding EP element. Coexpression of scylla and charybdis further ameliorates the suppression phenotype to a nearly wild-type situation. Notably, scylla or charybdis overexpression on their own using a panel of different eye/wing Gal4 drivers reduces adult organ size. Coexpression of the caspase inhibitors p35 or DIAP1 does not rescue the small eye phenotype induced by expression of either scylla or charybdis in the eye. Moreover, no elevated cell death in eye imaginal discs overexpressing scylla/ charybdis under control of the GMR-Gal4 driver was observed by acridine orange staining. This suggests that apoptosis is not the cause for the eye size reduction. Thus, scylla and charybdis overexpression antagonizes the growth-promoting effects of PKB/PDK1 and is sufficient to negatively regulate growth (Reiling, 2004).

To investigate the function of Scylla and Charybdis in more detail, loss-of-function mutations were generated in both genes and the analysis was complemented with overexpression studies. Partial scylla deletions were obtained by imprecise excisions of EP9.85, which is integrated in the scylla open reading frame (ORF) and therefore already represents a scylla allele (hereafter named scy^EP9.85). For charybdis, a local hop strategy of EPchar was used to obtain char¹⁸⁰, constituting a new EP insertion (EP1035^*) in the charybdis 5'-untranslated region (UTR) plus the original EPchar. Quantitative real time-PCR showed that in char¹⁸⁰ homozygotes, charybdis mRNA expression is decreased to ~25% of wild-type levels. It is assumed that this charybdis allele is a strong hypomorph, since mRNA levels were only slightly more reduced (to 23% or 15%, respectively) when RNA was extracted from flies heterozygous for char¹⁸⁰ over either one of two independent deficiencies uncovering charybdis and scylla (Reiling, 2004).

All scylla mutant combinations and the char¹⁸⁰ homozygotes are viable and fertile without apparent mutant phenotype. scylla and char¹⁸⁰ mutant animals have the same weight as control flies. Measurement of wing size and hair density in the adult wing of scylla mutants revealed no differences in cell size and cell number as compared to control animals. scylla loss-of-function clones were created in imaginal disks using FLP/FRT-mediated mitotic recombination to test the effect on growth properties of the mutant tissue. One would expect a growth advantage of cells in clones lacking a bona fide negative growth regulator, as is the case for PTEN. However, larval scylla mutant clones were the same size as their wild-type sister clones. Likewise, clones obtained in adult eyes revealed no increase in cell size of scylla or char¹⁸⁰ mutant ommatidia. Thus, loss of Scylla or Charybdis function is dispensable for growth under normal conditions. It is conceivable that Scylla and Charybdis act in a redundant manner. Therefore, it was important to create scylla charybdis double mutants (Reiling, 2004).

char¹⁸⁰ were combined with three of scylla alleles by meiotic recombination. All double-mutant scylla charybdis combinations produced viable adult flies. Weight analysis of heteroallelic scylla charybdis flies demonstrated that simultaneous loss of Scylla and Charybdis significantly increases body weight. Consistent results were obtained by combining one copy of scy^{EP9.85/31/113} char¹⁸⁰ and the deficiency Df(3L)vin4 uncovering scylla and charybdis. Mutant females were on average 6%-23% and males 9%-17% heavier than control flies. Conversely, ubiquitous scylla/charybdis overexpression using the Act5CGal4 driver generated flies that are decreased in size and weight (15.1% using EPscy and 14.3% using UAS-char#53). The majority of scylla/charybdis-overexpressing flies eclosed with a minor delay (0.5-1 d) (Reiling, 2004).

To assess whether charybdis/scylla overexpression affects cell size, scylla and charybdis gain-of-function flip-out clones in the eye, marked by the absence of the red pigment, were generated. A moderate reduction in cell size was observed in cells overexpressing scylla or charybdis. Ommatidia overexpressing scylla or charybdis exhibit no patterning defects. In contrast, simultaneous removal of Scylla and Charybdis in clones of photoreceptor cells resulted in slightly enlarged cells. Consistently, when most of the head capsule and the eyes were made homozygous by means of the eyflp/FRT system in an otherwise heterozygous mutant background, a mild big head phenotype was generated in the double mutant but not in either single mutant (Reiling, 2004).

The gain-of-function clonal analysis in the eye showed that cell size is reduced upon forced scylla/charybdis expression, but it did not address the question whether cell number is affected. To clarify this issue, the wing size and cell number of flies that ubiquitously overexpress scylla were measured. The insect wing is a double-layered epithelial structure, and each cell in the wing secretes a single hair (trichome). Therefore, by counting the number of trichomes per defined area, hair density can be taken as a measure for cell number. Overall wing size was reduced by ~25% in males or ~15% in females overexpressing scylla. By extrapolating the number of cells per measured area, it was found that cell size is decreased by ~29% in males and ~21% in females. It is concluded that the size reduction brought about by overexpressing scylla is caused by a reduction in cell size. Cell density is even slightly increased (5.8% in males, 7.6% in females). The data show that Scylla and Charybdis have a growth-inhibitory role and that they share some functional redundancy (Reiling, 2004).

Zinke (2002) performed a whole-genome DNA microarray analysis of 2-d-old larvae (48 h AEL) grown on normal food that were subsequently subjected to a starvation regime for different time periods. Under these conditions, upon 12 h of starvation, scylla and charybdis expression were found to be on average 6.3 times and 4.6 times up-regulated, respectively. Therefore, the effects of starvation were tested on the viability of scylla and charybdis mutants, as well as on flies overexpressing both genes by exposing adult flies to a water-only diet. Various scylla heteroallelic combinations did not show elevated susceptibility to starvation. However, char¹⁸⁰ mutants lived significantly shorter lives than control flies, suggesting that Charybdis has a protective effect for the animal under nutrient-deprived conditions. Strikingly, forced expression of scylla/charybdis extends mean life span by up to twofold. Lipid and glycogen content of these flies were analyzed to see whether energy stores were altered. Indeed, flies overexpressing scylla and/or charybdis showed significantly elevated lipid levels. Glycogen levels were also measured but no statistically significant changes could be detected, although there was a tendency toward increased glycogen content (Reiling, 2004).

scylla and charybde are required for head involution in Drosophila

The similarities in the expression profiles of scyl and chrb (both spatially and temporally) led to the hypothesis that the two closely related genes share overlapping functions in the developing embryo. No mutant alleles of chrb exist to test this hypothesis. With respect to scyl, the EY03729 fly line harbors a P-element insertion 253 bp from the scyl transcription start site; flies homozygous for the EY03729 insertion are, however, fully viable and exhibit no detectable phenotypes. Thus, to directly assess the functional contributions of scyl and chrb to embryonic development, RNA interference (RNAi) techniques were used to disrupt expression of one or both genes and the effects of interference were monitored throughout embryogenesis (Scuderi, 2006).

Injection of either scyl or chrb dsRNA resulted in no observable phenotype. Injected embryos were monitored for alterations in their developmental program throughout gastrulation, as well as by cuticle assay. Absence of an observable phenotype was reproducible using up to 5 μM dsRNA, 10-fold more than the amount required to produce a phenotype in engrailed RNAi control experiments. In contrast, coinjection of both scyl and chrb dsRNAs (either dorsally or posteriorly) led to defects in head involution that were clearly visible at the level of the larval cuticle. The scyl chrb co-RNAi embryonic lethal phenotype is reminiscent of that exhibited by homozygotes harboring hypomorphic mutations in the Zen transcription factor that is required to pattern the embryonic head ectoderm. Even more notably, the scyl chrb co-RNAi embryonic lethal phenotype is identical to that exhibited by embryos harboring amorphic mutations in the Hid (Head involution defective) apoptotic activator that functions morphogenetically to sculpt the embryonic head (Scuderi, 2006).

The scyl chrb co-RNAi phenotype was next reproduced in genetic studies exploiting deficiencies that removed one or both genes and thereby confirmed the specificity of the RNAi-induced phenotype. Three deficiency lines that mapped cytologically to the region of 68B–68C were PCR-genotyped with respect to scyl and chrb. Whereas Df(3L)lxd6 deletes scyl only, Df(3L)vin4 and Df(3L)vin2 delete both genes. Although these deficiencies span multiple genes, embryonic lethal phenotypes will correspond to the loss-of-function phenotype of the earliest acting gene(s) uncovered by the deficiency -- in this case, presumably scyl and/or chrb (Scuderi, 2006).

The cuticle phenotypes observed in the deficiency lines are entirely consistent with results from RNAi assays. Cuticles derived from Df(3L)lxd6 homozygotes develop normally and exhibit no visible defects, consistent with the determination that scyl alone is deleted in this deficiency. In contrast, cuticles derived from Df(3L)vin4 and Df(3L)vin2 homozygotes, in which both scyl and chrb are missing exhibit clear defects in head involution, with the head structures compressed anteriorly. Structures that comprise the head skeleton include the mouth hooks, which are the most anterior structures, as well as the chitinous labrum and the episomal sclerites, which connect the mouth hooks to the pigmented structures of the cephalopharyngeal skeleton (CPS). In scyl chrb double mutants, mouth hooks are present, but the labrum and epistomal sclerites are missing; errors in head involution result in at least part of the CPS remaining at the surface of the embryo (Scuderi, 2006).

Finally, because deficiency and RNAi analyses revealed a requirement for both scyl and chrb in normal head development, whether these genes, like Hox genes, compensate for one another in a dose-dependent manner was assayed. In this regard, the effect of removing a total of three copies of the genes was examined. Cuticles derived from Df(3L)lxd6/Df(3L)vin4 transheterozygotes (scyl+/scyl chrb) revealed defects in head involution, indistinguishable from those observed in cuticles derived from embryos lacking all four copies of the sister genes. Notably, only 10 genes, none of which has a previously characterized embryonic lethal phenotype, map to the Df(3L)lxd6/Df(3L)vin4 region of overlap. These data demonstrate that quantitative changes in gene pair activity are of greater consequence than qualitative differences between the two gene products (Scuderi, 2006).

Based upon the observations that: (1) simultaneous loss of scyl and chrb function leads to a hid-analogous, cell death defective phenotype and (2) scyl and chrb are homologous to the mammalian apoptotic gene RTP801, it was postulated that the scyl and chrb gene products have pro-apoptotic functions in the embryonic Drosophila head. Two lines of experimentation were employed to test this hypothesis. (1) hid expression was examined in scyl chrb double mutant embryos in situ. The scyl and chrb gene products do not function as transcriptional modulators of hid since hid transcription is unaffected in scyl chrb double mutant embryos. (2) A Caspase-3 activity assay was employed to monitor apoptosis in wild-type and scyl chrb double mutant embryos. Activated Caspase-3 has been used previously to specifically label apoptotic cells in Drosophila. Anti-Caspase-3 staining mirrors cell death patterns previously defined by acridine orange and TUNNEL assays in the Drosophila embryo and pupal retina. In this study, dying cells expressing activated Caspase-3 were evident in the head and the nervous system of 95% of embryos derived from matings of Df(3L)vin4/twi:GFP heterozygotes 0-8 h AEL (n = 278). When GFP screening was used to enrich for similarly staged mutant embryos, it was noted that Caspase-3 activity was greatly diminished in mid-stage scyl chrb double mutants. By 8 AEL, 75% of the mutant-enriched population was caspase-negative, in contrast to the unselected population in which only 8% of the embryos were found to be caspase-negative. No gross differences in Caspase-3 activity were found prior to the onset of germ band retraction and head involution. Since cleaved Caspase-3 is a key executioner (and hence marker) of apoptosis, these data support the hypothesis that Scylla and Charybde have pro-apoptotic roles in Drosophila head involution. More generally, Scylla and Charybde likely function as essential death activators in Drosophila since Caspase-3 activation in scyl chrb double mutants is disrupted in the nervous system as well as in the head. The scylla and charybde gene products are not, however, sufficient for cell death since (1) immunostains reveal wild-type patterns of Caspase-3 activation in embryos derived from dl mutant mothers and in which expression of scylla and charybde is greatly expanded and (2) neither scyl nor chrb (alone or in combination) can mimic hid-induced apoptosis in cultured Cos or Hela cells (Scuderi, 2006).

Several lines of evidence indicate that Scylla and Charybde function in the Hid-mediated cell death pathway. (1) A previous phenotypic analysis of scyl chrb mutants revealed their essential roles in regulating cell death in the developing Drosophila eye (Reiling, 2004). Loss-of-function studies have similarly revealed a requirement for Hid in modulating cell death events in early and late stages of Drosophila eye development. (2) In this study, which relied upon deficiencies and RNAi methodologies to generate scyl chrb null double mutants, an earlier developmental requirement for the scyl and chrb gene products was documented. scyl chrb double mutants suffer an embryonic lethality that is associated with defects in the morphogenetic process of head involution. Drosophila homozygous for loss-of-function hid alleles similarly suffer an embryonic lethality and exhibit signature defects in head involution. (3) Molecular characterization of the embryonic lethality in scyl chrb double mutants revealed that Caspase-3 activation is disrupted not only in the morphogenetically aberrant head, but in the CNS as well. In Drosophila, Hid induces apoptosis in midline glia cells failing to activate the EGFR signaling cascade. Together, the significant homologies of scyl and chrb to the mammalian RTP801 gene product that functions as an apoptotic factor in mammalian cell culture systems, as well as the scyl chrb embryonic and eye phenotype studies establish redundant roles for scyl and chrb in Hid-mediated cell death in both embryonic and post-embryonic stages of the Drosophila life cycle (Scuderi, 2006).

Each of the three cell death proteins, hid, rpr and grim, has been implicated in apoptotic events defining segmental boundaries and/or neuronal fates in the CNS, albeit in different paradigms. In the CNS, specificity in neuronal apoptosis is achieved via differential expression of the BX-C Hox gene abd-A, which prevents neuronal apoptosis in posterior segments. Viewed from this perspective, the finding that the Zen and BX-C Drosophila Hox gene products regulate transcription of the scyl and chrb pro-apoptotic genes (and thereby potentially sculpt head and segment boundaries during development) is reminiscent of the Deformed Drosophila Hox protein functioning as a transcriptional activator of the rpr cell death gene. Together, these studies strengthen the idea that Hox-gene-dependent induction of cell death is a general phenomenon in Drosophila (Scuderi, 2006).

Intriguingly, the pro- and anti-apoptotic roles of the Zen and BX-C Homeobox transcription factors in Drosophila embryogenesis correspond to their activation and repression effects on scyl and chrb gene expression. In this regard, scyl, chrb and cell death are activated by Zen in dorsal domains of the developing embryo, whereas ventrally scyl, chrb and cell death are repressed by one or more of BX-C gene products. Hence, in addition to the pro-apoptotic role of Zen, there is evidence for an anti-apoptotic role for the BX-C gene product(s) and in flies as in mouse related transcription factors function in context-specific fashion (Scuderi, 2006).

As a final point, both TGF-β and BMP mammalian members of the TGF-β cytokine superfamily have been documented to induce cell death in numerous developmental contexts. Along these same lines, previous reports in Drosophila have suggested a link between Dpp and cell death but have stopped short of designating this link as direct. Based on molecular and genetic evidence, it is suggested that the Drosophila pro-apoptotic scyl and chrb gene products serve as direct links between Dpp/Zen-mediated patterning and differentiation, in this case, cell death. Thus, in Drosophila as in vertebrates, cytokines of the TGF-β superfamily control both cell death and cell proliferation within the contexts of their cellular environments (Scuderi, 2006).

Given the importance of cell death regulation in development and disease, it is likely that there are several mechanisms by which cell death can be regulated, and, in like fashion, several nodes where independent regulatory pathways may in specific contexts converge. With respect to members of the RTP801 family of apoptotic factors, evidence points to at least two triggers of regulation: cell death can be a pathologic response to stresses such as hypoxia (as is the case for mammalian RTP801) or cell death can be a developmental response to a spatially and temporally restricted cell signaling pathway, such as the Dpp/TGF-β cytokine-mediated signaling pathway (as is the case for Drosophila Scylla and Charybde). Within the context of pathway convergence nodes, it is particularly notable that several reports document cross-talk between the HIF-1 and TGF-β pathways in regulating gene expression and cell death, and thus it is possible that the RTP801/Scylla/Charybde death effectors represent a point of convergence between these two death activating pathways. Consistent with this model is the demonstration that scyl and chrb are hypoxia-inducible in Drosophila (Reiling, 2004). Viewed from this perspective, the genetically defined roles of Scylla and Charybde as pro-apoptotic effectors establish a clear basis for future genetic and biochemical characterization of the mechanism by which activation of cell death programs might occur via Dpp/TGF-β-mediated signaling (Scuderi, 2006).

Reference names in red indicate recommended papers.

Brugarolas, J., et al. (2004). Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004 18(23): 2893-904. 15545625

Chauvet, S., Maurel-Zaffran, C., Miassod, R., Jullien, N., Pradel, J., Aragnol, D. (2000). Characterization of charybde and scylla, two paralogous target genes of Hox and cofactor proteins in Drosophila. GenBank, Direct Submission

Corradetti, M. N., Inoki, K. and Guan, K. L. (2005). The stress-inducted proteins RTP801 and RTP801L are negative regulators of the mammalian target of rapamycin pathway. J. Biol. Chem. 280(11): 9769-72. 15632201

DeYoung, M. P., et al. (2008). Hypoxia regulates TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3 shuttling. Genes Dev. 22: 239-251. PubMed Citation: 18198340

Ellisen, L. W., et al. (2002). REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species. Mol. Cell 10: 995-1005. 12453409

Frei, C. and Edgar, B.A. (2004). Drosophila cyclin D/Cdk4 requires Hif-1 prolyl hydroxylase to drive cell growth. Dev. Cell 6: 241-251. 14960278

Harvey, K. F., et al. (2008). FOXO-regulated transcription restricts overgrowth of Tsc mutant organs. J. Cell Biol. 180(4): 691-6. PubMed Citation: 18299344

Kimura, N., Tokunaga, C., Dalal, S., Richardson, C., Yoshino, K., Hara, K., Kemp, B. E., Witters, L. A., Mimura, O. and Yonezawa, K. (2003). A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 8: 65-79. 12558800

Ma, L., et al. (2005). Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression. Genes Dev. 19: 1779-1786. PubMed Citation: 16027168

Manning, B. D., et al. (2005). Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2. Genes Dev. 19: 1773-1778. PubMed Citation: 16027169

Reiling, J. H. and Hafen, E. (2004). The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila. Genes Dev. 18(23): 2879-92. 15545626

Schwarzer R, Tondera, D., Arnold, W., Giese, K., Klippel, A. and Kaufmann, J. (2005). REDD1 integrates hypoxia-mediated survival signaling downstream of phosphatidylinositol 3-kinase. Oncogene 24(7): 1138-49. 15592522

Scuderi, A., Simin, K., Kazuko, S. G., Metherall, J. E. and Letsou, A. (2006). scylla and charybde, homologues of the human apoptotic gene RTP801, are required for head involution in Drosophila. Dev. Biol. 291(1): 110-22. 1642334

Shoshani, T., Faerman, A., Mett, I., Zelin, E., Tenne, T., Gorodin, S., Moshel, Y., Elbaz, S., Budanov, A., Chajut, A., et al. (2002). Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol. Cell. Biol. 22: 2283-2293. 11884613

Sofer, A., Lei, K., Johannessen, C. M. and Ellisen, L. W. (2005). Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol. Cell. Biol. 25(14): 5834-45. 15988001

Wang, Z., Malone, M. H., Thomenius, M. J., Zhong, F., Xu, F. and Distelhorst, C. W. (2003). Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals. J. Biol. Chem. 278: 27053-27058. 12736248

Zinke, I., Schutz, C. S., Katzenberger, J. D., Bauer, M. and Pankratz, M. J. (2002). Nutrient control of gene expression in Drosophila: Microarray analysis of starvation and sugar-dependent response. EMBO J. 21: 6162-6173. 12426388

date revised: 25 February 2009

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