gigas

Protein Interactions

Identification of the Drosophila TSC2 homolog (Gigas) prompted a search for a Drosophila Tsc1 homolog. By BLAST search a Drosophila EST was identified that displays a significant similarity to the N-terminal sequence of human TSC1 (van Slegtenhorst, 1997). The sequence of the complete 3.8 kb cDNA predicts a protein of 1100 amino acids. Sequence alignment with the human TSC1 protein shows a significant degree of conservation. The human TSC1 protein is 22% identical (46% similar) to the Drosophila ORF. Furthermore, a single transmembrane domain is predicted for both human and Drosophila proteins at a conserved position, approximately 120 amino acids from the N terminus. A stretch of 133 amino acids in the potential cytoplasmic domain just after the transmembrane domain is highly conserved between human and Drosophila (44% identity). Coiled-coil domains, predicted for human, are found in similar positions in the Drosophila protein. Taken together with the overall level of protein sequence similarity, conservation of the transmembrane and coiled-coil domains strongly suggest that this cDNA encodes a Drosophila TSC1 homolog. This gene was mapped to 95E4-5 by in situ hybridization to polytene chromosomes (Ito, 1999).

The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. An approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin (Drosophila homolog Gigas), as a potential target of Akt/PKB. Upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and T1462 is constitutively phosphorylated in PTEN-/- tumor-derived cell lines. Finally, a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity (Manning, 2002).

Class I phosphoinositide 3-kinases (PI3Ks) are activated by many extracellular growth and survival stimuli. These lipid kinases catalyze the production of the second messengers phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5P3). Downstream targets containing specialized domains, such as pleckstrin-homology (PH) domains, that specifically bind to these lipid products of PI3K are then activated. These activated proteins control a wide array of cellular processes, including survival, proliferation, protein synthesis, growth, metabolism, cytoskeletal rearrangements, and differentiation. However, there is still much that is not known about the signaling events leading from activation of PI3K effectors to downstream changes in cell physiology (Manning, 2002).

Serine/threonine (S/T) protein kinases can account for much of the functional diversity of PI3K signaling. Akt/protein kinase B and the 70 kDa-S6 kinase 1 (S6K1) are the best characterized of the PI3K-regulated S/T kinases. The mitogen-stimulated activation of both of these kinases is blocked by PI3K-specific inhibitors. Akt contains a PH domain that is specific to PtdIns-3,4P2 and PtdIns-3,4,5P3. Akt is thereby recruited to these PI3K-generated second messengers and to the PDK1 protein kinase, which also specifically binds to these lipids. PDK1 then phosphorylates and activates Akt (Manning, 2002).

The regulation of S6K1 is much more complex, with both PI3K-dependent and -independent signaling pathways involved in its activation. Several PI3K-regulated effectors are known to participate in the activation of S6K1, including PDK1, PKCzeta/lambda, Cdc42, Rac1, and Akt. However, the molecular mechanism of how these contribute to S6K1 activation remains unclear. In addition to mitogen-regulated signaling to S6K1, the metabolic state of the cell and the availability of nutrients control S6K1 activation through the mammalian target of rapamycin (mTOR, also known as FRAP, RAFT, and RAPT). Recent studies suggest that mTOR is also regulated by mitogenic signals. Interestingly, it has been suggested that the point of convergence of the mitogenic and nutrient-sensing signals in the regulation of S6K1 may be at the level of Akt directly phosphorylating mTOR. However, this phosphorylation does not appear to affect mTOR activity or S6K1 activation. Thus, of the PI3K-regulated effectors thought to participate in S6K1 activation, the molecular basis of how Akt regulates S6K1 remains the least well understood (Manning, 2002).

Akt itself has been implicated in many of the PI3K-regulated cellular events, and several substrates have been shown to be phosphorylated in vitro and/or in vivo by Akt. Therefore, the total cellular effect of PI3K activation and subsequent activation of Akt is mediated through a variety of different targets. However, it seems unlikely that the large array of processes controlled by the PI3K-Akt pathway can be accounted for by the current knowledge of downstream targets (Manning, 2002).

An approach has been developed to screen for substrates of PI3K-dependent S/T kinases, such as Akt. This approach uses phospho-specific antibodies generated against a phosphorylated protein kinase consensus recognition motif in combination with a protein database motif scanning program called Scansite (http://scansite.mit.edu). Scansite is a web-based program that searches protein databases for optimal substrates of specific protein kinases and for optimal binding motifs for specific protein domains with data generated by peptide library screens. The phospho-motif antibody is used to recognize proteins phosphorylated specifically under conditions in which the kinase of interest is active. Scansite is then used to identify candidate substrates of this protein kinase that have the predicted molecular mass of the proteins recognized by the phospho-motif antibody. This approach successfully identifies known substrates of Akt. The tuberous sclerosis complex-2 (TSC2) tumor suppressor gene product, tuberin, is also identified and characterized as an Akt substrate. Furthermore, it is found that overexpression of a tuberin mutant lacking the major Akt phosphorylation sites can inhibit growth factor-induced activation of S6K1. These results provide a biochemical link between the PI3K-Akt pathway and regulation of S6K1 and also indicate a biochemical basis for the disease tuberous sclerosis complex (TSC) (Manning, 2002).

TSC is a common disease affecting an estimated 1 in 6000 individuals and is characterized by the occurrence of widespread benign tumors called hamartomas frequently affecting the brain, skin, kidneys, lungs, eyes, and heart. In approximately 85% of TSC patients, the disease is caused by loss-of-function mutations in one of two tumor suppressor genes, TSC1 and TSC2, which encode hamartin and tuberin, respectively. These two proteins form a complex. Tuberin, which has a region of homology to Rap1 GTPase-activating proteins (GAPs), has been shown to possess in vitro GAP activity toward Rap1. However, the true molecular and cellular functions of the hamartin-tuberin complex have yet to be clearly defined. Furthermore, very little is known about how these tumor suppressor gene products are regulated (Manning, 2002).

Based on genetic studies and the fact that PI3K and Akt are oncogenes while the TSC genes are tumor suppressors, one would predict that the PI3K-Akt-mediated phosphorylation of tuberin would inhibit the function of the tuberin-hamartin complex. In Drosophila, hamartin and tuberin appear to function together to antagonize signaling of the insulin-PI3K-Akt pathway and, thereby, restrict cell growth and proliferation. Most strikingly, loss of just one copy of TSC1 or TSC2 partially rescues the lethality of insulin receptor loss-of-function mutants. This result implies that one of the primary functions of the insulin pathway, at least in Drosophila, is to inhibit the hamartin-tuberin complex. Furthermore, both mouse and Drosophila genetic studies have suggested that the tuberin-hamartin complex functions to inhibit S6K1 (Manning, 2002).

Expression in human cells of the tuberinS939A/T1462A mutant, which lacks the major PI3K-dependent phosphorylation sites, at levels comparable to endogenous tuberin leads to a decrease in growth factor-induced S6K1 phosphorylation and activity. This phosphorylation and subsequent activation of S6K1 has been previously demonstrated to be dependent on PI3K. These results, along with those from genetic studies in other systems, are consistent with a model in which growth factors activate PI3K leading to the phosphorylation of tuberin by Akt. This phosphorylation inhibits the tuberin-hamartin complex, thereby relieving its inhibition of S6K1. In this model, expression of the tuberinS939A/T1462A mutant, which would not be phosphorylated and inhibited, would have a dominant effect over endogenous tuberin and block growth factor-induced S6K1 activation. It will be of great interest to determine the molecular nature of S6K1 inhibition by the tuberin-hamartin complex in the absence of mitogenic stimuli. It is possible that the complex does so upstream of mTOR, because the constitutive activation of S6K1 in TSC1-/- MEFs is sensitive to rapamycin. Alternatively, mTOR might regulate S6K1 in a nutrient-sensitive pathway parallel to the mitogen-sensitive PI3K-Akt-tuberin pathway (Manning, 2002).

Recent studies have suggested that S6K1 activation can occur independent of PI3K and Akt. These studies demonstrate the existence of multiple pathways regulating S6K1 and that the tuberin-hamartin complex might integrate signals from many different inputs. The identification of tuberin as a direct downstream target of the PI3K-Akt pathway provides the missing link between this signaling cascade and control of S6K1 activity (Manning, 2002).

The identification of this biochemical relationship between the mammalian TSC tumor suppressor gene products and the oncogenic PI3K-Akt pathway could have important implications in human diseases. For instance, in approximately 10%-15% of patients diagnosed with TSC, mutations in TSC1 or TSC2 have not been detected. Based on the findings of this study, it is possible that mutations leading to aberrant activation of the PI3K-Akt pathway, such as PTEN mutations, could inhibit the function of the tuberin-hamartin complex by causing constitutive phosphorylation of tuberin. It will be interesting to examine the phosphorylation state of tuberin within hamartomas from such TSC patients. Indeed, in PTEN-/- cell lines derived from both glioblastoma and prostate tumors, growth factor-independent phosphorylation of tuberin on the PI3K-dependent T1462 site is detected (Manning, 2002).

Germline mutations in either PTEN or the TSC genes cause autosomal dominant diseases that are characterized by the occurrence of widespread hamartomas due to loss of heterozygosity at these loci. However, the tissue distribution of these benign tumors varies between patients with loss of PTEN and those with TSC. These differences might be explained by a model in which the tuberin-hamartin complex is the primary growth-inhibiting target of the PI3K-Akt pathway in tissues affected in TSC patients. In other tissues, such as those affected in patients with PTEN mutations, this complex might be one of many targets of the PI3K-Akt pathway. Interestingly, though, recent studies have suggested that mTOR activity is essential for oncogenic transformation of cells by activated PI3K or Akt and for growth of PTEN-/- tumors. Therefore, aberrant phosphorylation and inhibition of the tuberin-hamartin complex, and subsequent increased activity of mTOR and/or S6K1, would likely contribute to tumorigenesis caused by mutations that activate the PI3K-Akt pathway. Future studies using crosses between PTEN and TSC knockout mice should help determine the contribution of the tuberin-hamartin complex in prevention of the variety of tumors caused by uncontrolled signaling through the PI3K-Akt pathway. Finally, the elucidation of a PI3K-Akt-tuberin pathway controlling S6K1 activity will have important implications in the understanding and treatment of the prevalent TSC disease (Manning, 2002).

Gigas functions as a GAP for Rheb

Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. Rheb (Ras homolog enriched in brain), a novel, highly conserved member of the Ras superfamily of G-proteins, promotes cell growth. Overexpression of Rheb in the developing fly causes dramatic overgrowth of multiple tissues: in the wing, this is due to an increase in cell size; in cultured cells, Rheb overexpression results in accumulation of cells in S phase and an increase in cell size. Rheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of Rheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that Rheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that Rheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, flies with reduced Rheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator target of rapamycin (TOR), a kinase required for growth factor-dependent phosphorylation of ribosomal S6 kinase (S6K). In cultured cells, the effect of overexpressing Rheb was blocked by the addition of rapamycin. These results imply that Rheb is involved in TOR signaling (Patel, 2003). Additional studies show that Rheb functions downstream of the tumor suppressors Tsc1 (tuberous sclerosis 1)-Tsc2, with Tsc2 functioning as a GAP for Rheb (Saucedo, 2003; Zhang, 2003), and that a major effector of Rheb function in controlling growth is, in fact, ribosomal S6 kinase (Stocker, 2003). It is still not clear, however, how Rheb signals to TOR (Zhang, 2003).

Given the similarities between Rheb and mutants in the InR and TOR signalling pathways, it is conceivable that Rheb represents a novel component of one of these growth control pathways. To test this possibility, a detailed epistasis analysis was performed. Examined first was whether the negative regulators of InR and TOR signalling (PTEN and Tsc1-Tsc2, respectively) could counteract the effects of Rheb overexpression. All overexpression experiments were performed in the eye using the GMR-Gal4 driver line. Expression of either PTEN or Tsc1-Tsc2 alone results in a very similar size reduction of the ommatidia when compared with control ommatidia. However, whereas expression of PTEN has no influence on the increase in ommatidial size caused by Rheb overexpression, co-expression of Tsc1-Tsc2 results in ommatidia of approximately wild-type size, indicating that the activities of Rheb and Tsc1-Tsc2 can counteract each other. Next, the enlarged ommatidia phenotype of GMR-Rheb was assayed in a number of mutant backgrounds. Reducing the activity of Drosophila protein kinase B (PKB) has no effect on ommatidial size. Similar results were obtained with hypomorphic mutations in InR and Dp110, respectively. In contrast, ommatidial size is dominantly reduced by a mutation in TOR (TOR^2L1), and a suppression to wild-type size is observed in a S6K mutant background. Thus, the Rheb overexpression phenotype is dependent on TOR and S6K function, but is independent of InR signal strength. Finally, the behaviors of Rheb PTEN and Rheb Tsc1 double mutants were examined. The phenotypic consequences were assayed in mosaic animals using the ey-Flp method. As expected, the Rheb PTEN double-mutant tissue clearly displays a Rheb phenotype. The Rheb Tsc1 mutant tissue also resembles Rheb single mutants, indicating that Rheb is epistatic over (functions downstream of) Tsc1 (Stocker, 2003).

Mutations in the TSC1 or TSC2 genes cause tuberous sclerosis, a benign tumor syndrome in humans. Tsc2 possesses a domain that shares homology with the GTPase-activating protein (GAP) domain of Rap1-GAP2, suggesting that a GTPase might be the physiological target of Tsc2. The small GTPase Rheb (Ras homolog enriched in brain) has been shown to be a direct target of Tsc2 GAP activity both in vivo and in vitro. Point mutations in the GAP domain of Tsc2 disrupt its ability to regulate Rheb without affecting the ability of Tsc2 to form a complex with Tsc1. These studies identify Rheb as a molecular target of the TSC tumor suppressor genes (Zhang, 2003).

TSC1 and TSC2 were initially discovered as tumor suppressor genes mutated in tuberous sclerosis, a human syndrome characterized by the widespread development of benign tumors termed harmatomas. TSC2 encodes a putative GAP protein, whereas TSC1 encodes a novel protein containing two coiled-coil domains. Studies of Drosophila TSC1 and TSC2 homologs have identified a specific function for TSC1-TSC2 in the control of cell growth, with loss of TSC1-TSC2 resulting in increases in cell size. Recent studies further suggest that Tsc1-Tsc2 antagonizes the amino-acid-TOR signalling pathway, which normally couples amino-acid availability to S6 Kinase (S6K) activation, translation initiation and cell growth. Strikingly, loss of Drosophila TSC1-TSC2 results in a TOR-dependent increase of S6K activity that is resistant to amino-acid starvation (Zhang, 2003 and references therein).

Despite these new advances, the biochemical activity of the Tsc1-Tsc2 complex remains unknown. Tsc2 possesses a domain homologous to Rap1-GAP. The GAP homology domain of Tsc2 is important for its function, and mis-sense mutations of this domain have been identified in a high proportion of TSC patients. These observations suggest that an unknown small GTPase might be the direct target of Tsc2. This study set out to determine the target GTPase of Tsc2-GAP using an RNAi-based screen in Drosophila S2 cells. It was reasoned that this putative GTPase should be expressed in S2 cells and that RNAi of this GTPase should result in downregulation of S6K-Thr 398 phosphorylation, a phenotype opposite that caused by Tsc2 RNAi. During the course of the RNAi screen, genetic studies have implicated the small GTPase Rheb as a potential target of Tsc2. In S2 cells, RNAi inhibition of Rheb, but not any of the other 17 GTPases tested so far, abolished S6K-Thr 398 phosphorylation, as predicted for a Tsc2 GAP substrate. Among the 17 GTPases screened were Rab5 and Rap1, two proteins previously implicated as TSC2 GAP substrates from in vitro studies, suggesting that Rab5 and Rap1 are improbable physiological substrates of Tsc2. The highly specific effect of Rheb RNAi on S6K phosphorylation suggests that Rheb might be the physiological substrate of TSC2 GAP activity (Zhang, 2003).

Rheb is an evolutionarily conserved small GTPase found from yeast to mammals. Unlike Ras and most other Ras superfamily GTPases, Rheb has an arginine at the third residue of the G1 box (residue 15 of mammalian Rheb) instead of glycine. Rheb is unique, compared with many small GTPases, in that it exists in a highly activated state in mammalian cells. Studies of mammalian Rheb further implicated the existence of a Rheb-GAP that is normally present at relatively limiting concentrations, since overexpression of Rheb results in a progressive increase in the proportion of Rheb in the active GTP-bound state. Genetic analyses in Drosophila support a model in which Tsc2 functions as a Rheb-GAP. These studies also suggest that similarly to mammalian cells, Tsc2, the putative Rheb-GAP, is normally present in limiting concentrations in Drosophila, because overexpression of wild-type Rheb results in an activated phenotype and overexpression of Tsc2 (together with Tsc1) results in the opposite phenotype (Zhang, 2003).

To test directly whether Rheb is a physiological substrate of Tsc2 GAP activity, it was asked if Tsc2 could regulate Rheb in vivo. Rheb, similar to other small GTPases, cycles between an active GTP-bound form and an inactive GDP-bound form. Thus, the steady state GTP/GDP-loading status of Rheb can be used as a measurement of its in vivo activity. An in vivo labelling procedure was adapted to analyse the steady-state GTP/GDP-binding status of Rheb. Drosophila S2 cells expressing Myc-tagged Rheb were labelled with ³²P-orthophosphate. Rheb protein was then purified by immunoprecipitation and Rheb-associated GTP/GDP was analysed by thin-layer chromatography (TLC) on polyethyleneimine (PEI) cellulose plates. In wild-type S2 cells, Rheb binds preferentially to GTP, in agreement with studies of mammalian Rheb. In addition, co-overexpression of Tsc1 and Tsc2 results in a marked decrease (approximately eightfold) in the ratio of GTP to GDP bound on Rheb. Interestingly, overexpression of Tsc2 alone has much weaker effect on GTP:GDP ratio. This observation is consistent with previous studies in Drosophila, which show that co-overexpression of Tsc1 and Tsc2, but not either gene alone, results in growth inhibition. The weaker effect of Tsc2 alone on Rheb GTP loading is caused, at least in part, by the lower level of Tsc2 when expressed alone, as compared with Tsc1 co-expression. Mutual stabilization between Drosophila Tsc1 and Tsc2 has been documented previously (Zhang, 2003).

To demonstrate that the effect of Tsc1-Tsc2 overexpression on Rheb GTP loading was caused by the GAP activity of Tsc2, similar in vivo labelling experiments were performed with Tsc2 variants carrying point mutations in the GAP domain. The mutations Tsc2^K1693A and Tsc2^N1698K changed residues in the GAP domain that are conserved in Drosophila, human and a probable Schizosaccharomyces pombe Tsc2 homolog. In addition, a mutation analogous to Tsc2^K1693A has been shown to abolish Rap1-GAP activity, whereas Tsc2^N1698K mimics a disease-causing mutation in human TSC patients. The activity of Tsc2-N, a construct that contains just the amino-terminal half of Tsc2 and thus lacks the carboxy-terminal GAP domain, was also examined. Tsc2-N can associate with Tsc1 normally, but does not interact with Rheb in co-immunoprecipitation assays. Similar to Tsc2-N, neither Tsc2^K1693A nor Tsc2^N1698K affects the ability of Tsc2 to associate with Tsc1. Despite their ability to associate with Tsc1, these mutants all abolished the effect of Tsc1-Tsc2 overexpression on Rheb GTP loading. Complementary to the results from Tsc1-Tsc2 overexpression, RNAi of Tsc2 increases the ratio of GTP:GDP bound to Rheb. The smaller change in GTP:GDP ratio after Tsc2 RNAi, compared with Tsc1-Tsc2 overexpression, is not surprising given that Rheb is already at a relatively active state in wild-type cells. Taken together, these results provide strong evidence that Rheb is a physiological target of Tsc2 GAP activity (Zhang, 2003).

To test whether Rheb is a direct substrate of Tsc2 GAP in vitro, a fusion protein of glutathione S-transferase (GST) and the Tsc2 GAP domain against GTP-loaded Rheb protein was tested using a nitrocellulose filter assay. alpha-³²P-GTP- or gamma-³²P-GTP-loaded GST-Rheb was incubated with GST-Tsc2 and the remaining radioactive GTP bound on Rheb was measured at different time intervals. GST-Tsc2 results in a dramatic decrease of Rheb-associated radioactive counts when gamma-³²P-GTP, but not alpha-³²P-GTP, was used in the assay. Thus, Tsc2 functions as a Rheb GAP in vitro. This GAP activity is highly specific, and no activity was detected, using as a substrate Drosophila Ras1, the closest relative of Rheb among all GTPases. In addition, the K1693A or the N1698K point mutation abrogates the in vitro GAP activity of Tsc2 towards Rheb. These results provide further evidence that Tsc2 functions as a Rheb GAP (Zhang, 2003).

The data presented so far suggest a model in which the tuberous sclerosis tumor suppressor proteins negatively regulate Rheb through the Rheb GAP activity of Tsc2. To further substantiate this model, whether there are any genetic interactions between TSC1-TSC2 and Rheb was tested. Flies homozygous for a null allele of TSC1, TSC1²⁹, do not survive beyond the second-instar larval stage. Strikingly, the lethality of TSC1 null animals was partially rescued by removing one of the two copies of Rheb gene from the diploid genome: 61% of TSC1²⁹ homozygotes that were also heterozygous for a null allele of Rheb, Rheb^PDelta1, survived to third-instar larval stage, and 21% of the third-instar survivors continued development and arrested at the pupal stage. Such dose-sensitive interactions are reminiscent of those observed between TSC1-TSC2 and TOR, further supporting the model that Tsc1-Tsc2 negatively regulates Rheb during cell growth (Zhang, 2003).

Finally, how the Tsc-Rheb pathway interacts with the amino acid-TOR-S6K signalling network was investigated. Tsc and Rheb could either function as obligatory components between amino acids and TOR in a linear amino-acid sensing pathway, or in a parallel pathway that converges on TOR. The former (but not the latter) model predicts that the activity of Rheb is dependent on the presence of amino acids. The ratio of GTP:GDP bound to Rheb is not reduced after 5 h of amino-acid starvation. Thus, a model is favored in which TSC and Rheb function in a parallel pathway that converges on TOR. According to this model, loss of Tsc1-Tsc2 or ectopic activation of Rheb results in constitutive activation of TOR, which bypasses the requirement for amino acids and renders S6K activity resistant to amino-acid starvation. How Rheb signals to TOR will be an important question for future investigation (Zhang, 2003).

In summary, the small GTPase Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins. Wild-type Tsc2, but not mutant Tsc2 carrying point mutations in the GAP domain, shows GAP activity towards Rheb both in vitro and in vivo. The importance of Tsc2's GAP activity is further supported by the high proportion of mis-sense mutations localized to the Tsc2 GAP domain among TSC patients. Thus, the Tsc2 tumor suppressor functions as a Rheb-GAP in an analogous way to the neurofibromin (NF1) tumor suppressor as a Ras-GAP. These studies suggest that Rheb represents a novel target for therapeutic intervention in the TSC disease. The identification of a small GTPase as the direct target of the TSC tumor suppressors further implicates the existence of activators of GTPases, such as guanine nucleotide-exchange factors (GEFs), as potential regulators of this disease pathway. Identification of the putative Rheb-GEF represents an important goal for the next phase of TSC research (Zhang, 2003).

Pam and its ortholog Highwire interact with and may negatively regulate the TSC1.TSC2 complex

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder associated with mutations in TSC1, which codes for hamartin, or TSC2, which codes for tuberin. The brain is one of the most severely affected organs, and CNS lesions include cortical tubers and subependymal giant cell astrocytomas, resulting in mental retardation and seizures. Tuberin and hamartin function together as a complex in mammals and Drosophila. This paper reports the association of Pam, a protein identified as an interactor of Myc, with the tuberin-hamartin complex in the brain. The C terminus of Pam containing the RING zinc finger motif binds to tuberin. Pam is expressed in embryonic and adult brain as well as in cultured neurons. Pam has two forms in the rat CNS, an approximately 450-kDa form expressed in early embryonic stages and an approximately 350-kDa form observed in the postnatal period. In cortical neurons, Pam co-localizes with tuberin and hamartin in neurites and growth cones. Although Pam function(s) are yet to be defined, the highly conserved Pam homologs, HIW (Drosophila) and RPM-1 (Caenorhabditis elegans), are neuron-specific proteins that regulate synaptic growth. This study shows that HIW can genetically interact with the Tsc1.Tsc2 complex in Drosophila and can negatively regulate Tsc1.Tsc2 activity. Based on genetic studies, HIW has been implicated in ubiquitination, possibly functioning as an E3 ubiquitin ligase through the RING zinc finger domain. Therefore, it is hypothesized that Pam, through its interaction with tuberin, could regulate the ubiquitination and proteasomal degradation of the tuberin-hamartin complex particularly in the CNS (Murthy, 2004).

Temporal control of differentiation by the Insulin receptor/Tor pathway in Drosophila

Multicellular organisms must integrate growth and differentiation precisely to pattern complex tissues. Despite great progress in understanding how different cell fates are induced, it is poorly understood how differentiation decisions are temporally regulated. In a screen for patterning mutants, alleles were isolated of tsc1, a component of the insulin receptor (InR) growth control pathway. Loss of tsc1 disrupts patterning due to a loss of temporal control of differentiation. tsc1 controls the timing of differentiation downstream or in parallel to the RAS/MAPK pathway. Examination of InR, PI3K, PTEN, Tor, Rheb, and S6 kinase mutants demonstrates that increased InR signaling leads to precocious differentiation while decreased signaling leads to delays in differentiation. Importantly, cell fates are unchanged, but tissue organization is lost upon loss of developmental timing controls. These data suggest that intricate developmental decisions are coordinated with nutritional status and tissue growth by the InR signaling pathway (Bateman, 2004).

Thus InR/Tor signaling has a novel role in controlling the timing of differentiation. In both loss-of-function and ectopic expression experiments, it was found that activation of the InR/Tor pathway leads to the precocious acquisition of neuronal cell fate, while loss of signaling through this pathway delays (but does not block) differentiation. Importantly, InR and Tor signaling does not alter cell fates, only the time at which these cell fate decisions are made. This characteristic is important to a temporal control mechanism and ensures that only timing is regulated and not the actual cell fate decision (Bateman, 2004).

Mutants in tsc1 were isolated in a screen for genes that affect adhesion and PCP. Loss of tsc1 causes defects in ommatidial rotation due to precocious differentiation which is accompanied by the precocious initiation of rotation and hence ommatidial overrotation. Although cell fate is not affected by perturbations in InR/Tor signaling, developmental timing and tissue patterning are aberrant. Therefore, the precise control of timing of differentiation is essential for correct formation of complex tissues such as the Drosophila compound eye. The data show that the action of InR/Tor pathway on differentiation allows fine-tuning of binary switching mechanisms such as EGF signaling. This novel mechanism allows the organism to use humoral signals such as insulin-like molecules to temporally regulate differentiation. Under conditions of nutrient deprivation when growth rate slows, it is essential that differentiation keep pace with growth to maintain accurate patterning. The use of the InR/Tor pathway to control both growth and the timing of differentiation is an elegant solution to this challenge during development (Bateman, 2004).

The pattern of MAPK activation is unaffected by loss of tsc1. The EGF ligand, Spitz, is secreted by the R8 photoreceptor and diffuses to nearby cells, causing their recruitment and differentiation by activating the RAS/MAPK pathway. These data indicate that Spitz production in the R8 photoreceptor is unaffected by loss of tsc1, as is the transduction of the EGFR signal as far as the activation of MAPK in the recruited photoreceptors. In addition, the expression of regulators of photoreceptor differentiation downstream of MAPK (such as Lozenge, Yan, and Ttk), have been examined and no alteration in their levels or distribution in tsc1 mutant clones was found. Therefore, the temporal control of differentiation by InR/Tor signaling, acts downstream (or in parallel) to known components of photoreceptor differentiation (Bateman, 2004).

Studies of birth order-dependent cell fate specification in the Drosophila CNS have revealed that neuroblasts express a series of transcription factors in a set sequence, and both overexpression and loss-of-function studies have demonstrated that transcription factors present at the birth of neuroblasts are necessary and sufficient to direct differential cell lineages that are linked to different birth dates. Progression through the cell cycle is required for the temporal transition of these transcription factors. Although loss of tsc1 has been shown to lead to an acceleration through G1, alteration of the cell cycle by overexpression of cyclin E or cyclin D/CDK4 does not induce precocious differentiation. Therefore, precocious differentiation cannot be simply due to the alterations in the cell cycle. Another hallmark of tsc1 mutant cells is increased cell size. However, increasing cell size by overexpressing cyclin D/CDK4 or by overexpression of myc did not induce precocious differentiation, indicating that although cell size is increased in cases of overactive InR/Tor signaling, it is not an increase in cell mass that triggers premature differentiation. Moreover, compensating for the decreases in overall cellular growth rate caused by loss of InR signaling in clones by making clones in a Minute heterozygous background does not affect the slowing of differentiation caused by loss of the InR, confirming that InR/Tor signaling regulates timing of differentiation by a mechanism that is independent of and genetically separable from its effects on growth (Bateman, 2004).

Importantly, the InR/Tor pathway is found to control the timing of neuronal cell fate decisions in the eye and leg but does not appear to affect the timing of epithelial prehair initiation. The temporal control of differentiation by the InR/Tor pathway may be especially important for neurons since their axons must contact targets that are often far away. During normal development of the embryonic CNS, pioneer neurons are the first to differentiate and provide spatial cues for later-born neurons. If pioneer neurons are absent, targeting defects can occur. Tight temporal control of differentiation ensures that neurons are born in an environment that has the correct spatial cues for pathfinding. Intriguingly, disrupting insulin signaling results in defects in axonal targeting from the eye to the brain in Drosophila. The data suggest that these results may in part be due to precocious differentiation of the neurons (Bateman, 2004).

What is the mechanism by which InR/Tor signaling controls the timing of differentiation? Regulation of growth by InR/Tor signaling is mediated through translational control. This control is achieved though phosphorylation of S6 kinase (which phosphorylates the ribosomal protein S6) and 4E binding protein, an inhibitor of the translational initiation factor 4E. Ribosomal proteins and many protein synthesis elongation factors contain 5' oligopyrimidine tracts at their transcriptional start site, known as 5'TOPs. Translation of 5'TOP-containing transcripts is increased in response to PI3K/Tor signaling, thereby allowing coordinate expression of all ribosomal components. It is proposed that there is a 5'TOP present in the mRNA of an unknown proneural factor(s) that undergoes increased translation in response to InR/Tor signaling. This increased translation would lead to higher levels of proneural factors, speeding neural differentiation, which would allow for the precise coordination of growth and differentiation needed during the development of complex neural structures. Supporting this model is the finding that none of the InR/Tor signaling mutants tested gives rise to ectopic differentiation of neurons. Alterations are observed only in the timing of differentiation, at the correct location, in both the eye and leg imaginal discs. This observation is consistent with a mechanism involving translational regulation (via a 5'TOP) of hypothetical proneural factor(s), i.e., modulation of the level of such a factor or factors can only occur once the proneural transcript is already present. A corollary of this model is that InR/Tor signaling would act to modulate the gap between transcription and translation of the hypothetical factor(s). The importance of the gap length between transcription and translation has recently been demonstrated for Notch signaling in the presomitic mesoderm during somite formation (Bateman, 2004).

Interestingly, a hallmark of the tumors that arise from loss of TSC1 is that they are highly differentiated and largely benign. This characteristic is in contrast to tumors that are malignant, arising from loss of PTEN. This malignancy may be due to the role of PTEN in many other pathways aside from growth signaling, while TSC1 has a more restricted function in the growth control pathway. The precocious differentiation induced by loss of TSC1 may contribute to their low malignancy, since high levels of differentiation are generally considered an indication of low metastatic potential. However, the exact causes of some of the most debilitating symptoms of tuberous sclerosis, such as neurological abnormalities and epilepsy, are still unclear. Future work will determine if precocious and hence inappropriate differentiation decisions contribute to the pathology of tuberous sclerosis in man (Bateman, 2004).

DEVELOPMENTAL BIOLOGY

The TOR pathway couples nutrition and developmental timing in Drosophila

In many metazoans, final adult size depends on the growth rate and the duration of the growth period, two parameters influenced by nutritional cues. In Drosophila, nutrition modifies the timing of development by acting on the prothoracic gland (PG), which secretes the molting hormone ecdysone. When activity of the Target of Rapamycin (TOR), a core component of the nutrient-responsive pathway, is reduced in the PG, the ecdysone peak that marks the end of larval development is abrogated. This extends the duration of growth and increases animal size. Conversely, the developmental delay caused by nutritional restriction is reversed by activating TOR solely in PG cells. Finally, nutrition acts on the PG during a restricted time window near the end of larval development that coincides with the commitment to pupariation. In conclusion, this study shows that the PG uses TOR signaling to couple nutritional input with ecdysone production and developmental timing. Previously studies have shown that the same molecular pathway operates in the fat body (a functional equivalent of vertebrate liver and white fat) to control growth rate, another key parameter in the determination of adult size. Therefore, the TOR pathway takes a central position in transducing the nutritional input into physiological regulations that determine final animal size (Layalle, 2008).

Previous experiments showed that insulin/IGF signaling controls basal levels of ecdysone synthesis in the PG. This, in turn, controls the larval growth rate without modifying the duration of larval growth. These data contrast with the present observations on the role of TOR signaling in the PG and indicate that PG cells discriminate between hormone-mediated activation of InR/PI3K signaling and the nutrient-mediated activation of TOR signaling for the control of ecdysone biosynthesis. Can TOR and InR/PI3K signaling pathways function separately in Drosophila tissues? It has been established both in cultured cells and in vivo that a gain of function for InR/PI3K allows for TORC1 activation through inhibition of TSC2 via direct phosphorylation by AKT/PKB. Such crosstalk between the InR and TOR signaling pathways has important functional implications in cancer cells in which inactivation of the PTEN tumor suppressor leads to an important increase in AKT activity. Nevertheless, the physiological significance of the crosstalk between AKT and TSC2 has been challenged by genetic experiments in Drosophila, leading to the notion that, in the context of specific tissues, TOR and insulin/IGF signaling can be part of distinct physiological regulations for the control of animal growth in vivo. Although not observe in standard conditions, strong InR/PI3K activation in the ring gland shortens larval developmental timing under conditions of food limitation. In light of the present data, this suggests that, in low-food conditions, providing high PI3K activity in PG cells allows for full activation of TOR through the AKT/PKB-mediated inhibitory phosphorylation of TSC2, thus modulating developmental timing. Inversely, a severe downregulation of InR/PI3K signaling in the PG extends larval timing by preventing early larval molts. However, it was observed that strong inhibition of the InR pathway compromises the growth of PG cells, therefore interfering with their capacity to produce normal levels of ecdysone for molting. Overall, previous works as well as the present work highlight the importance of studying signaling networks in the specific contexts (tissue, development) in which these pathways normally operate. This also illustrates that only mild manipulations of these intricate pathways are suitable to unravel the regulatory mechanisms that normally occur within the physiological range of their activities. In conclusion, it is proposed that the insulin/IGF system and TOR provide two separate inputs on PG-dependent ecdysone production: the insulin/IGF system controls baseline ecdysone levels during larval life, and TOR acts upon ecdysone peaks in response to PTTH at the end of larval development (Layalle, 2008).

Important literature describes intrinsic mechanisms controlling a growth threshold for pupariation in insects. After a critical size is attained, the hormonal cascade leading to ecdysone production initiates, and larvae are committed to pupal development, even when subjected to complete starvation. Recent findings in Drosophila by using temperature-sensitive mutants for dInR have revealed that reducing the larval growth rate before the critical size is attained postpones the attainment of this threshold, but has no effect on the final size. Conversely, reducing animals' growth rate after the critical size has been attained leads to strong reduction of the final size. This highlights an important period in the determination of final size, called the terminal growth period (TGP, also called interval to cessation of growth), which spans from the attainment of critical size to the cessation of growth. Due to its exponential rate, growth during that period makes an important contribution to the determination of final size. Interestingly, the duration of the TGP is not affected by the general insulin/IGF system, which explains why reduction of the insulin/IGF system during that period leads to short adults. The present data suggest that the duration of the TGP is an important parameter in the determination of final size that is controlled by TOR. By reducing the level of TOR activity specifically in the PG, neither the growth rate or the critical size for commitment to pupariation is affected. Therefore, the time to attainment of the critical size is not changed. The observation of the developmental transitions in P0206 > TSC1/2 larvae (ectopically expressing TSC1/2) indicate that, indeed, the timing of L1/L2 and L2/L3 molts are not modified. By contrast, the L3/pupa transition is severely delayed, indicating that the interval between attainment of critical size and the termination of growth, i.e., the TGP, is increased. Interestingly, activation of TOR in the PG of fasting larvae leads to a sensible (50%) reduction of the developmental delay induced by low nutrients, whereas it has no effect in normally fed animals. This indicates that the regulation of the TGP by TOR plays an important role in the adaptation mechanisms controlling the duration of larval development under conditions of reduced dietary intake. Other mechanisms, such as the delay to attainment of the critical size due to a reduced growth rate, also contribute to timing of larval development, giving a plausible explanation for the fact that PG-specific TOR activation only partially rescues the increase in larval development timing observed under low-nutrient conditions. Despite characterization in different insect systems, the mechanisms determining the critical size remain to be elucidated. The present study shows that inhibition of TOR signaling in the PG does not modify the minimum size for pupariation. This result is in line with previous findings indicating that nutritional conditions do not modify the critical size in Drosophila. Interestingly, animals depleted of PTTH present an important shift in critical size, indicating that PTTH might participate in setting this parameter. Therefore, mechanisms determining the critical size might reside in the generation or the reception of the PTTH signal, upstream of TOR function in the cascade of events leading to ecdysone production (Layalle, 2008).

What is the limiting step that is controlled by the TOR sensor during the process of ecdysone production? Results obtained by genetic analysis in vivo are reminiscent of in vitro work on dissected PG in the M. sexta model. In these previous studies, PTTH-induced ecdysone production in the PG was shown to induce the phosphorylation of ribosomal protein S6 and was inhibited by the drug rapamycin, later identified as the specific inhibitor of TOR kinase. Interestingly, rapamycin treatment blocked PTTH-induced, but not db-cAMP-induced, ecdysone production, indicating that the drug does not act by simply inhibiting general protein translation in PG cells, but, rather, by inhibiting a specific step controlling PTTH-dependent ecdysone production. More recently, many studies mostly carried out on large insects have started unraveling the response to PTTH in the PG, leading to ecdysone synthesis. No bona fide PTTH receptor is identified yet, and the previously identified response to PTTH is a rise in cAMP, leading to a cascade of activation of kinases, including PKA, MAPKs, PKC, and S6-kinase. S6-kinase-dependent S6 phosphorylation is currently being considered as a possible bottle-neck in the activation of ecdysone biosynthesis by PTTH. The present genetic analysis of ecdysone production in the Drosophila PG now introduces the TOR pathway, the main activator of S6-kinase, as a key controller of ecdysone production and therefore provides a plausible explanation for the rise of S6-kinase in PG cells following PTTH induction. The phenotypes obtained after TOR inhibition in the PG are remarkably similar to the phenotype obtained after ablation of the PTTH neurons. Moreover, ths study shows here that PTTH expression is not altered upon starvation, and that TOR inhibition in PTTH cells has no effect on the duration of larval development, suggesting that PTTH production is not modified by a nutritional stress. Taken together, these data suggest a model whereby limited nutrients induce a downregulation of TOR signaling in the PG, abolish the capacity of PG cells to respond to PTTH and produce ecdysone, and lead to an extension of the terminal growth period (Layalle, 2008).

In conclusion, this study illustrates how the TOR pathway can be used in a specific endocrine organ to control a limiting step in the biosynthesis of a hormone in order to couple important physiological regulations with environmental factors such as nutrition (Layalle, 2008).

Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila

Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. This study provides in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. Nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. These findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour (Sousa-Nunes, 2011).

In fed larvae, Drosophila neuroblasts exit quiescence from the late first instar (L1) stage onwards. This reactivation involves cell enlargement and entry into S phase, monitored in this study using the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). Reactivated neuroblast lineages (neuroblasts and their progeny) reproducibly incorporated EdU in a characteristic spatiotemporal sequence: central brain --> thoracic --> abdominal neuromeres. Mushroom-body neuroblasts and one ventrolateral neuroblast, however, are known not to undergo quiescence and to continue dividing for several days in the absence of dietary amino acids. This indicates that dietary amino acids are more than mere 'fuel', providing a specific signal that reactivates neuroblasts. However, explanted central nervous systems (CNSs) incubated with amino acids do not undergo neuroblast reactivation unless co-cultured with fat bodies from larvae raised on a diet containing amino acids. Therefore the in vivo requirement for a fat-body-derived signal (FDS) in neuroblast reactivation was tested by blocking vesicular trafficking and thus signalling from this organ using a dominant-negative Shibire dynamin (SHI^DN). This strongly reduced neuroblast EdU incorporation, indicating that exit from quiescence in vivo requires an FDS. One candidate tested was Ilp6, known to be expressed by the fat body, but neither fat-body-specific overexpression nor RNA interference of this gene significantly affected neuroblast reactivation. Fat-body cells are known to sense amino acids via the cationic amino-acid transporter Slimfast (SLIF), which activates the TOR signalling pathway, in turn leading to the production of a systemic growth signal. Fat-body-specific overexpression of the TOR activator Ras homologue was shown to be enriched in brain (RHEB), or of an activated form of the p110 PI3K catalytic subunit, or of the p60 adaptor subunit, had no significant effect on neuroblast reactivation in fed animals or in larvae raised on a nutrient-restricted diet lacking amino acids. In contrast, global inactivation of Tor, fat-body-specific Slif knockdown or fat-body-specific expression of the TOR inhibitors Tuberous sclerosis complex 1 and 2 (Tsc1/2) all strongly reduced neuroblasts from exiting quiescence. Together, these results show that a SLIF/TOR-dependent FDS is required for neuroblasts to exit quiescence and that this may be equivalent to the FDS known to regulate larval growth (Sousa-Nunes, 2011).

Next, the signalling pathways essential within neuroblasts for their reactivation were investigated. Nutrient-dependent growth is regulated in many species by the interconnected TOR and PI3K pathways. In fed larvae, it was found that neuroblast inactivation of TOR signalling (by overexpression of TSC1/2), or PI3K signalling (by overexpression of p60, the Phosphatase and tensin homologue PTEN, the Forkhead box subgroup O transcription factor FOXO or dominant-negative p110), all inhibited reactivation. Conversely, stimulation of neuroblast TOR signalling (by overexpression of RHEB) or PI3K signalling [by overexpression of activated p110 or Phosphoinositide-dependent kinase 1 (PDK1)] triggered precocious exit from quiescence. RHEB overexpression had a particularly early effect, preventing some neuroblasts from undergoing quiescence even in newly hatched larvae. Hence, TOR/PI3K signalling in neuroblasts is required to trigger their timely exit from quiescence. Importantly, neuroblast overexpression of RHEB or activated p110 in nutrient-restricted larvae, which lack FDS activity, was sufficient to bypass the block to neuroblast reactivation. Notably, both genetic manipulations were even sufficient to reactivate neuroblasts in explanted CNSs, cultured without fat body or any other tissue. Together with the previous results this indicates that neuroblast TOR/PI3K signalling lies downstream of the amino-acid-dependent FDS during exit from quiescence (Sousa-Nunes, 2011).

To identify the mechanism bridging the FDS with neuroblast TOR/PI3K signalling, the role of the Insulin-like receptor (InR) in neuroblasts was tested. Importantly, a dominant-negative InR inhibited neuroblast reactivation, whereas an activated form stimulated premature exit from quiescence. Furthermore, InR activation was sufficient to bypass the nutrient restriction block to neuroblast reactivation. This indicates that at least one of the potential InR ligands, the seven ILPs, may be the neuroblast reactivating signal(s). By testing various combinations of targeted Ilp null alleles and genomic Ilp deficiencies, it was found that neuroblast reactivation was moderately delayed in larvae deficient for both Ilp2 and Ilp3 (Df(3L)Ilp2-3) or lacking Ilp6 activity. Stronger delays, as severe as those observed in InR³¹ mutants, were observed in larvae simultaneously lacking the activities of Ilp2, 3 and 5 [Df(3L)Ilp2-3, Ilp5] or Ilp1-5 [Df(3L)Ilp1-5]. Despite the developmental delay in Df(3L)Ilp1-5 homozygotes, neuroblast reactivation eventually begins in the normal spatial pattern -- albeit heterochronically -- in larvae with L3 morphology. Together, the genetic analysis shows that Ilp2, 3, 5 and 6 regulate the timing but not the spatial pattern of neuroblast exit from quiescence. However, as removal of some ILPs can induce compensatory regulation of others, the relative importance of each cannot be assessed from loss-of-function studies alone (Sousa-Nunes, 2011).

Brain median neurosecretory cells (mNSCs) are an important source of ILPs, secreted into the haemolymph in an FDS-dependent manner to regulate larval growth. They express Ilp1, 2, 3 and 5, although not all during the same development stages. However, this study found that none of the seven ILPs could reactivate neuroblasts during nutrient restriction when overexpressed in mNSCs. Similarly, increasing mNSC secretion using the NaChBac sodium channel or altering mNSC size using PI3K inhibitors/activators, which in turn alters body growth, did not significantly affect neuroblast reactivation under fed conditions. Surprisingly, therefore, mNSCs are not the relevant ILP source for neuroblast reactivation. Nonetheless, Ilp3 and Ilp6 messenger RNAs were detected in the CNS cortex, at the early L2 stage, in a domain distinct from the Ilp2⁺ mNSCs. Two different Ilp3-lacZ transgenes indicate that Ilp3 is expressed in some glia (Repo⁺ cells) and neurons (Elav⁺ cells). An Ilp6-GAL4 insertion indicates that Ilp6 is also expressed in glia, including the cortex glia surrounding neuroblasts and the glia of the blood-brain barrier (BBB) (Sousa-Nunes, 2011).

Next the ability of each of the seven ILPs to reactivate neuroblasts when overexpressed in glia or in neurons was assessed. Pan-glial or pan-neuronal overexpression of ILP4, 5 or 6 led to precocious reactivation under fed conditions. Each of these manipulations also bypassed the nutrient restriction block to neuroblast reactivation, as did overexpression of ILP2 in glia or in neurons, or ILP3 in neurons. In all of these ILP overexpressions, and even when ILP6 was expressed in the posterior Ultrabithorax domain, the temporal rather than the spatial pattern of reactivation was affected. Importantly, experiments blocking cell signalling with SHI^DN indicate that glia rather than neurons are critical for neuroblast reactivation. Interestingly, glial-specific overexpression of ILP3-6 did not significantly alter larval mass. Thus, in contrast to mNSC-derived ILPs, glial-derived ILPs promote CNS growth without affecting body growth (Sousa-Nunes, 2011).

Focusing on ILP6, CNS explant cultures were used to demonstrate directly that glial overexpression was sufficient to substitute for the FDS during neuroblast exit from quiescence. In vivo, ILP6 was sufficient to induce reactivation during nutrient restriction when overexpressed via its own promoter or specifically in cortex glia but not in the subperineurial BBB glia, nor in many other CNS cells that were tested. Hence, cortex glia possess the appropriate processing machinery and/or location to deliver reactivating ILP6 to neuroblasts. Ilp6 mRNA is known to be upregulated rather than downregulated in the larval fat body during starvation and, accordingly, Ilp6-GAL4 activity is increased in this tissue after nutrient restriction. Conversely, it was found that Ilp6-GAL4 is strongly downregulated in CNS glia during nutrient restriction. Thus, dietary nutrients stimulate glia to express Ilp6 at the transcriptional level. Consistent with this, an important transducer of nutrient signals, the TOR/PI3K network, is necessary and sufficient in glia (but not in neurons) for neuroblast reactivation. Together, the genetic and expression analyses indicate that nutritionally regulated glia relay the FDS to quiescent neuroblasts via ILPs (Sousa-Nunes, 2011).

This study used an integrative physiology approach to identify the relay mechanism regulating a nutritional checkpoint in neural progenitors. A central feature of the fat-body --> glia --> neuroblasts relay model is that glial insulin signalling bridges the amino-acid/TOR-dependent fat-body-derived signal (FDS) with InR/PI3K/TOR signalling in neuroblasts. The importance of glial ILP signalling during neuroblast reactivation is also underscored by an independent study, published while this work was under revision (Chell, 2010). As TOR signalling is also required in neuroblasts and glia, direct amino-acid sensing by these cell types may also impinge upon the linear tissue relay. This would then constitute a feed-forward persistence detector, ensuring that neuroblasts exit quiescence only if high amino-acid levels are sustained rather than transient. This study also showed that the CNS 'compartment' in which glial ILPs promote growth is functionally isolated, perhaps by the BBB, from the systemic compartment where mNSC ILPs regulate the growth of other tissues. The existence of two functionally separate ILP pools may explain why bovine insulin cannot reactivate neuroblasts in CNS organ culture, despite being able to activate Drosophila InR in vitro. Given that insulin/PI3K/TOR signalling components are highly conserved between insects and vertebrates, it will be important to address whether mammalian adipose or hepatic tissues signal to glia and whether or not this involves an insulin/IGF relay to CNS progenitors. In this regard, it is intriguing that brain-specific overexpression of IGF1 can stimulate cell-cycle re-entry of mammalian cortical neural progenitors, indicating utilization of at least part of the mechanism identified by this study in Drosophila (Sousa-Nunes, 2011).

Effects of Mutation and Overexpression

A mutation has been isolated in the Drosophila homolog of TSC1 (Tsc1). Cells mutant for Tsc1 are dramatically increased in size yet differentiate normally. Organ size is also increased in tissues that contain a majority of mutant cells. Clones of Tsc1 mutant cells in the imaginal discs undergo additional divisions but retain normal ploidy. Flow cytometry analysis indicates that the increase in cell size is not due to endoreplication. Tsc1 protein is shown to bind to Drosophila Tsc2 in vitro. Overexpression of Tsc1 or Tsc2 alone in the wing and eye has no effect, but co-overexpression leads to a decrease in cell size, cell number, and organ size. Genetic epistasis data are consistent with a model that Tsc1 and Tsc2 function together in the insulin signaling pathway (Potter, 2001).

Recent work has demonstrated that the insulin signaling pathway plays an important role in the regulation of cell size, cell number, and organ size. Mutations of Drosophila PTEN (dPTEN), which functions as a negative regulator of insulin signaling, result in phenotypes that resemble the effects of Tsc1 and Tsc2 mutations. Therefore, genetic epistasis experiments were performed to test whether Tsc1 or Tsc2 might also function to negatively regulate insulin signaling. Overexpression of Drosophila insulin receptor (dinr) using the eyGAL4 driver leads to lethality at 25°C and a dramatic increase in ommatidia number in escapers at room temperature. Co-overexpression of Tsc1 and Tsc2 (but not either Tsc1 or Tsc2 alone) rescues both the lethality and the extra ommatidia phenotype caused by dinr overexpression. Furthermore, overexpression of dinr using the pGMR-GAL4 driver leads to an increase in ommatidium size, which is also suppressed by co-overexpression of Tsc1 and Tsc2. Clones of dinr mutant ommatidia are smaller in size than wild-type. Ommatidia that are mutant for both dinr and Tsc1, however, exhibit the Tsc1 mutant phenotype of increased ommatidium size (Potter, 2001).

Overexpression of dPTEN using the pGMR-GAL4 driver leads to eyes with a decreased ommatidium size. However, overexpression of dPTEN is unable to suppress the clonal Tsc1 mutant phenotype. Similar to dinr, clones of dAkt mutant ommatidia are smaller in size. Ommatidia mutant for both dAkt and Tsc1 display the Tsc1 phenotype. Similarly, ommatidia that contained Tsc2 mutant clones in a dAkt mutant background exhibit the Tsc2 mutant phenotype. These results suggest that in the eye, Tsc1 and Tsc2 function genetically epistatic to (downstream of) dinr, dPTEN, and dAkt (Potter, 2001).

The double mutant phenotypes of dS6k and Tsc2 were examined. Mosaic eyes consisting primarily of dS6k mutant ommatidia are slightly smaller than wild-type due to decreases in ommatidium size. Strikingly, mosaic eyes consisting primarily of dS6k and Tsc2 double mutant ommatidia display the dS6k phenotype. Furthermore, clones of Tsc1 mutant ommatidia in a dS6k mutant background no longer exhibit the Tsc1 mutant phenotype. Finally, the small ommatidium phenotype caused by co-overexpression of Tsc1 and Tsc2 is suppressed by co-overexpression of either dS6k or the human p70^S6K gene. All together, the epistasis data indicate that Tsc1 and Tsc2 antagonize insulin signaling, that genetically Tsc1 and Tsc2 are epistatic to dAkt, and that dS6k is epistatic to Tsc1 and Tsc2 (Potter, 2001).

Tuberous sclerosis is a human disease caused by mutations in the TSC1 or the TSC2 tumor suppressor gene. Previous studies of a Drosophila TSC2 homolog suggest a role for the TSC genes in maintaining DNA content, with loss of TSC2 leading to polyploidy and increased cell size. Mutations have been isolated in the Drosophila homolog of the TSC1 gene. TSC1 and TSC2 are shown to form a complex and function in a common pathway to control cellular growth. Unlike previous studies, this work shows that TSC1^- or TSC2^- cells are diploid. Strikingly, the heterozygosity of TSC1 or TSC2 is sufficient to rescue the lethality of loss-of-function insulin receptor mutants. Further genetic analyses suggest that the TSC genes act in a parallel pathway that converges on the insulin pathway downstream from Akt. The most convincing evidence for a functional link between the TSC genes and insulin signaling comes from the observation that heterozygosity of TSC1 or TSC2 is sufficient to rescue the lethality of loss-of-function InR mutants. This argues that the TSC genes are intimately linked to insulin signaling, rather than functioning in a totally independent cell-growth pathway. These results suggest that the TSC tumor suppressor genes are novel negative regulators of insulin signaling, and modulating the activities of the TSC genes might provide a potential way to correct insulin signaling defects in certain diseases such as diabetes and obesity (Gao, 2001).

The role of the pre- and post-synaptic cells in determining the number of synapses has been investigated in retina mosaics of the Drosophila mutant gigas. Mutant photoreceptors are two to three times larger than those of the wild type, while adjacent cells in the mosaic retina and the lamina are normal in size. Serial electron microscope reconstructions of mosaic lamina cartridges show that gigas photoreceptors establish more synapses upon lamina neurons than do the normal photoreceptors. By contrast, the number of feedback synapses that lamina neurons make onto gigas photoreceptors does not increase. The increment in the number of synapses correlates positively with the increment of presynaptic cell membrane, resulting in constancy of synapse density. The phototactic response of flies bearing a gigas eye is abnormal, indicating that the extra synapses are functional (Canal, 1994).

The number of synaptic contacts formed by a neuron is known to vary with its surface area. This could be because large neurons are able to establish more synaptic sites, or because those neurons that are able to establish more sites are subsequently able to enlarge. To test between these two possibilities, clones of enlarged ommatidia were generated in the retina of the Drosophila mutant gigas, by mitotic recombination following gamma-irradiation in the third-instar larva. The numbers of afferent synaptic contacts formed by the photoreceptor terminals in the first optic neuropil, or lamina, were then counted in the adult. The terminals of mutant photoreceptors are also enlarged, but by varying degrees. The sizes of their profiles in single sections merge with the size distribution of terminals having a wild-type phenotype, lying outside the clone in the same lamina. A perimeter of 6.0 microns for the profiles of receptor termini in cross section was established as a criterion for distinguishing between normal and mutant phenotypes. The mutant terminals have more presynaptic sites. Because only the gigas terminals are mutant and because they have enlarged at a time long before synapse formation occurs in the lamina it may be concluded that cell enlargement precedes elevated synaptic number. The increase in synaptic number roughly matches the increased membrane surface of the terminals, so as nearly to preserve a constant areal density of synaptic sites over a 5-fold range in synaptic frequency (Meinertzhagen, 1994).

gigas is a lethal mutant that differentiates enlarged cells, including the nucleus. This trait manifests only after the completion of the mitotic program. Advantage was taken of this phenotype to test in vivo the capacity of normal target cells to arrest the growth of mutant sensory axons. Single neuron connectivity changes have been analyzed in mosaics after horseradish peroxidase retrograde tracings. A mutant mechanoreceptor neuron, growing over a genetically normal substrate, contacts its normal target, and in addition projects to novel areas of the CNS. The mutant axon does terminate its growth eventually, and the new additional targets that are reached correspond to mechanoreceptor domains in other ganglia, indicating that this territorial constraint is operational in the mutant. gigas neurons maintain their stereotyped profile and represent an expanded version of the normal branching pattern. Thirty three mosaic mutant somatic spots embracing only one or two mutant thoracic bristles [anterior scutellar (ASC); anterior notopleural (ANP); posterior notopleural (PNP), and humeral (HU)] in heterozygous flies. Three types of neurons were chosen because they represent three different types of branching patterns. (1) The ASC exhibits a clear distinction between a major (ipsilateral) and a minor (contralateral) branch, (2) the ANPs and PNPs show two ipsilateral branches of equivalent lengths, and (3) the HU presents only one branch. The axon of the gigas neuron is two to three times thicker than wild type, generates more collaterals and boutons, and projects into areas that the wild type never reaches. The distinction between the major and minor branches is maintained in the mutant. It appears that the gigas branching pattern is an expanded version of the normal counterpart. The wild type always shows a characteristic terminal bend in the metathoracic neuromere. The gigas B and C phenotypes are the most frequent classes and show this bend either in the fused abdominal ganglion (class B) or duplicated in the normal site and in the abdominal ganglion (class C). Occasionally, the abnormal projection results in more profuse branching at the normal site (class D) or in a long extension toward the brain (class E). The ultrastructure of the invading projections does not reveal gliotic or necrotic reactions from the new cell contacts (Canal, 1998).

The data show that gigas neurons sprout more collaterals and extend their projection beyond their usual targets despite growing over a normal substrate. Although the termination point is subject to variation in the wild type, these are never as great as the phenotypes observed in the mutant. The extended projection reproduces the normal features in terms of general pathway and branching pattern. Neurons with a single major branch (HU) or two clearly different branches (ASC) still maintain their characteristic profile. Neurons with two equivalent branches (ANP, PNP) show the extension through either branch, but never along both of them. In these cases, it seems that the growth dynamics of the mutant can be randomly drained by either growth cone. Once this choice takes place, the growth continues along suitable pathways until another compatible target is reached. The phenotype of the gigas mosaics challenges the determinism of the substrate-derived growth inhibitory factors as stop signals for the growth cone. The experiments reported here show that the growing axon can override these putative signals. However, the gigas axons do not continue their growth indefinitely. They do stop, and their extended projections are kept within the territorial domains of mechanoreceptor endings (Canal, 1998).

The functional consequences of the connectivity changes produced by the mutant mechanoreceptors have been studied in grooming behavior. Mosaic flies carrying a single gigas mechanoreceptor show modified, albeit context-coherent, grooming responses after stimulation of the mutant bristle, whereas the response from neighboring normal sensory neurons remains unchanged. All of these experiments indicate that target recognition and growth arrest are two dissectible processes of neural development, and they highlight the autonomous features of the growth cone during pathfinding (Canal, 1998).

To determine when the cell size change occurs in gigas mutant clones, large mutant clones were produced in the eye discs. To examine cell size and DNA content during development, third instar imaginal discs containing gigas mutant clones were double-stained with the DNA stain DAPI (4, 6-Diamidino-2-phenylindole) and the microfilament stain TRITC-phalloidin. Larger nuclei and increased cell sizes in the mutant clones are consistently observed in both wing and eye discs. This observation suggests that cell size has already increased in the developing imaginal discs. To measure the relative DNA content of gigas cells, the relative fluorescent intensity of DAPI-stained nuclei was measured from a series of confocal images. The average value obtained from wild-type wing disc cells was taken as 1.0. All the values from wild-type cells fell between 0.7-1.3 (1.0 ± 0.2). The mean value from gigas cells was 10.6 ± 1.6, confirming that gigas cells have endoreplicated without cell division. Discs with large gigas mutant clones are three to four times the size of wild-type discs in area (Ito, 1999).

From these results the following conclusions have been drawn: (1) cell size change in gigas mutant clones is cell autonomous, since only the cells inside the clones are affected; (2) since many different kinds of cells in the eyes and wings are affected, it is likely that cell size change in mutant clones is due to a general defect in growth control rather than a defect in a particular developmental program; (3) cells anterior to the morphogenetic furrow, which are unpatterned and undifferentiated, have large cell size, indicating that the effect of gigas mutations occurs before pattern formation and differentiation in eye discs; (4) when clones occupy large areas of the discs, imaginal discs become enlarged, larval phase is extended, and animals die as prepupae (Ito, 1999).

The pattern of cell cycle progression was examined in the wild-type and gigas eye disc clones. Photoreceptor differentiation initiates in the eye disc in the posterior region and progresses anteriorly as a wave marked by a depression in the apical surface of the epithelium called the morphogenetic furrow (MF). The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was used to label S phase cells. In a normal eye disc, many asynchronous S phase cells are seen in the region anterior to the MF, while cells enter S phase synchronously just posterior to the MF. In a wild-type eye disc, both anterior and posterior to the MF, many BrdU-positive cells are observed. In a gigas mutant clone, the pattern of BrdU staining remains the same as wild type; however, fewer and larger BrdU-positive nuclei are observed, which is consistent with the results seen with DAPI staining. These results clearly show that gigas mutant cells can still enter S phase and carry out DNA replication (Ito, 1999).

The distribution of M phase cells was examined using an antibody specific for a phosphorylated form of histone H3 present only in mitotic nuclei (anti-PH3). In wild-type discs, M phase nuclei move to the apical surface of the disc. Optical sections of a wild-type eye disc near the apical surface show that there are PH3-positive cells in regions both anterior and posterior to the MF. In gigas mutant clones, no strong staining with anti-PH3 is seen either at the apical surface of the eye disc or in other regions of the eye discs, implying that gigas mutant cells do not enter M phase in the developing eye discs. These observations suggest that gigas mutant cells in the eye disc can still enter S phase but might not enter M phase. However, the fact that there are multiple cells in mutant clones suggests that more than one type of cell cycle operates during the course of disc development and that Gigas is not required for all of the cell cycle types. Alternatively, Gigas might be a stable protein so that the phenotype is only manifested after several rounds of cell division. The expression of Elav, a nuclear protein expressed posterior to the furrow in differentiated neuronal cells, was examined. In a gigas clone, the Elav-positive region in each cell is much larger than in wild type, reflecting larger nuclear size. However, the pattern of differentiation appears normal, again suggesting that pattern formation of mutant cells can proceed normally despite their cell cycle defect (Ito, 1999).

To confirm that gigas mutant cells fail to enter M phase, an examination was made of the expression patterns of the G2 cyclins, Cyclin A and Cyclin B. In Drosophila, Cyclin A and Cyclin B have overlapping functions in regulating mitosis. In a normal disc, the precursors to photoreceptors R8, R2, R5, R3, and R4 emerge from the MF, exit the mitotic cycle, and terminally differentiate. Other undifferentiated cells enter S phase and then G2, where they accumulate Cyclin A and Cyclin B proteins in their cytoplasm. When G2 cells enter M phase, Cyclin A and Cyclin B enter the nucleus and are degraded. After completing division, these cells differentiate to generate photoreceptors R1, R6, and R7, as well as the primary, secondary, and tertiary pigment cells, the cone cells, and the bristle mother cells. By contrast, in gigas mutant cells, cytoplasmic Cyclin A and Cyclin B remain, abnormally, at significant levels throughout the disc. Posterior to the MF in gigas eye discs, the cells surrounding photoreceptor clusters express higher levels of Cyclin A and B. These are presumably pigment cells, cone cells, and the R1, R6, and R7 photoreceptor cells that go through an additional cell cycle. These cells still seem to respond to a developmental signal and upregulate G2 cyclins, although the rapid downregulation of cyclins does not occur. Lower but significant levels of cyclins are detected in all other cells in the eye disc, including cells in the MF. These mutant cells are able to differentiate into the full range of cell types found in the adult eye despite their endoreplication phenotype (Ito, 1999).

Thus, gigas mutant cells endoreplicate their DNA during the late stages of imaginal disc development. In normal cells, DNA replication is stringently regulated to guarantee that the genome is duplicated exactly once during each cell cycle. In S. pombe, the active mitotic cyclin-dependent kinase (CDK) activity (a complex of Cdc2 and its G2 cyclin partner Cdc13) is required for both mitosis and inhibition of the rereplication of DNA. Inhibition of the mitotic CDK activity by either inactivation of the Cdc2 itself, by inactivation of the cyclin Cdc13, or by overexpression of the Cdc2-Cyclin inhibitor Rum1 causes a block in mitosis and consequently promotes multiple rounds of DNA replication. In Drosophila, cdc2 mutant cells rereplicate DNA without mitosis. These results suggest that the Cdc2-Cyclin complex is a crucial regulator of the decision to enter either S phase or mitosis (Ito, 1999 and references).

The effects of the gigas mutation could be explained by two possible mechanisms. (1) Gigas may be required for blocking DNA rereplication. High levels of Cdc18/Cdc6 protein, a central regulator of DNA replication, have been shown to inhibit mitosis and induce DNA rereplication in S. pombe. Perhaps loss of Gigas function disrupts a similar control mechanism in Drosophila, resulting in an analogous endoreplication phenotype. (2) Gigas may be required for mitosis. In either case, the most probable target of Gigas activity is the Cdc2/Cyclin complex, a key regulator of mitosis. Cdc2 is maintained in an inactive phosphorylated form, and dephosphorylation of Cdc2 by the Cdc25 phosphatase (String in Drosophila) leads to increased Cdc2 kinase activity and entry into mitosis. If Gigas protein is required to activate the Cdc2 kinase, the gigas mutant phenotype may simply reflect the absence of active Cdc2. Alternatively, Gigas protein might regulate the subcellular localization of the Cdc2/Cyclin complex. The Cdc2/Cyclin complex accumulates in the cytoplasm during interphase but is imported into the nucleus at the start of M phase. The accumulation of CycA and CycB in gigas mutant cells might be explained if the Gigas protein is required for nuclear import of Cdc2/Cyclin (Ito, 1999 and references). The vertebrate TSC2 protein has been reported to be localized to the Golgi/perinuclear region, consistent with a role in protein trafficking (Tsuchiya, 1996; Wienecke, 1996).

Since gigas mutant clones contain many cells, gigas mutant cells must initially be able to divide after mutant clones are produced by mitotic recombination. The favored explanation to account for this ability is that there are multiple types of cell cycles and Gigas is only required for some of them. Previous results suggest that there are indeed different cell cycle types during imaginal disc development. However, the data presented here are also consistent with other explanations. For example, it is formally possible that Gigas protein is sufficiently stable and that the phenotype is only manifested after several rounds of cell division following the generation of homozygous mutant cells (Ito, 1999).

A striking feature of the gigas mutant phenotype is the unusual accumulation of CycA and CycB. Sigrist (1995) demonstrated that expression of mutant cyclins (A, B, or B3) lacking the destruction box motif blocked cyclin degradation and arrest cells at metaphase or anaphase during embryonic development in Drosophila, indicating that exit from mitosis is regulated by destruction of G2 cyclins. In gigas mutant clones, cells repeat S phase without mitosis in the later phase of disc development. These results suggest that the failure to degrade CycA and CycB may be a consequence of the failure to enter M phase, rather than gigas mutants having a primary defect in cyclin degradation (Ito, 1999).

The increased cell size in adult mutant clones led to the initial identification of gigas (Ferrús, 1976). How cell size control and cell cycle are coupled is still not well understood. In general, the increase of cell size seems to correlate well with the increase of ploidy, suggesting that DNA replication may be coupled to cell growth. In Drosophila imaginal discs, inactivation of either cdc2 (Weigmann, 1997) or gigas leads to endoreplication and increased cell size. This common feature of the gigas and cdc2 mutant phenotypes is consistent with a model that places Gigas and Cdc2/Cyclin in the same pathway of cell cycle control (Ito, 1999).

What makes an organ or a disc grow to a particular size is unknown. The loss of cdc2 in wing discs results in discs of relatively normal size composed of fewer, larger cells, showing that even when cells are larger, disc size can still be regulated (Weigmann, 1997). Large gigas mutant clones that encompass more than 90% of the disc result in increased disc size, three to four times larger than normal. Two possible explanations for the difference between this aspect of the gigas and cdc2 phenotypes cannot be distinguished at this time. Gigas may be required for a cell size checkpoint. Thus, gigas mutant cells might not stop cell growth in response to signals controlling disc size, resulting in cell size continuing to increase to produce larger discs. Alternatively, the presence of significant numbers of wild-type cells in the same disc may result in signals that limit the cell size and thus disc size. Indeed, when only small gigas clones are created, disc size is close to normal. One possible explanation is that cell death in wild-type cells may be compensating for the overgrowth. It will be interesting to determine if creating large mutant clones of cdc2 that encompass the entire disc produces larger discs (Ito, 1999).

Studies of human TSC hamartomas have noted the presence of giant cells. TSC hamartomas are enlarged and disorganized while they express differentiation antigens specific to the site. The observations made with gigas mutant cells are consistent with these human phenotypes and suggest that the human TSC syndrome may also result from similar defects in cell cycle regulation. Benign TSC hamartomas might be the result of the endoreplication and cell size increase. The presence of TSC1 and TSC2 homologs in Drosophila strongly suggests the presence of the same cell cycle control mechanism in both human and Drosophila. Continued analysis of the Drosophila TSC1 and TSC2 genes should help achieve an understanding of the molecular mechanism of the TSC disease in humans (Ito, 1999).

Homozygous gigas mutant larvae hatch normally but fail to survive beyond early third instar. To investigate whether a maternal contribution masks a requirement for gigas during embryonic development, an attempt was made to generate germline clones homozygous for gigas. However, no eggs were recovered, indicating that Gigas is required for oogenesis and making it impossible to generate embryos totally lacking gigas product. Transheterozygotes of weak alleles often survive until late third instar, develop melanotic tumors, and die as prepupae. Melanotic tumors are black masses resulting from the melanization of hemocytes and are a frequent feature of the phenotype produced by various tumor suppressor mutants in Drosophila (Ito, 1999).

Tsc1 and Tsc2 act to suppress S6k function

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Absence of Drosophila Tsc1 and/or Tsc2 (Gigas) leads to constitutive S6k activation and inhibition of PKB, the latter effect being relieved by loss of S6K. In contrast, the Pten tumor suppressor, a negative effector of PI3K, has little effect on S6k, but negatively regulates PKB (Akt1). More importantly, reducing S6k signaling rescues early larval lethality associated with loss of Tsc1/2 function, arguing that the S6k pathway is a promising target for the treatment of TSC (Radimerski, 2002b).

To determine whether loss of Tsc1/2 or Pten directly affected S6k activity, each was depleted in Drosophila Kc167 cells by dsRNAi. Quantitative Real Time PCR showed that such treatment strongly reduced levels of both transcripts. Compared with control cells, depletion of Tsc1 increases S6k activity and T398 phosphorylation, consistent with the reduced electrophoretic mobility of S6k. These results are in agreement with recent findings in TSC1 null mammalian cells (Kwiatkowski, 2002). Insulin treatment of either control cells or Tsc1-depleted cells did not significantly increase these responses beyond that of Tsc1 depletion alone, indicating that loss of Tsc function leads to full S6k activation. RAD001, a rapamycin derivative, blocks S6k activity in both control and Tsc1-depleted cells treated with insulin. However, it was consistently noted that the RAD001 block of insulin-induced S6k activation is not as strong in Tsc1-depleted cells, suggesting that not all the effects of Tsc on S6k are dependent on Tor, the Drosophila target of rapamycin. Similar results to those described here were obtained by Tsc2 depletion. In addition, the effects appear specific, since Tsc1 depletion has no effect on the basal activity of other AGC-kinase family members, such as PKB or Drosophila atypical PKC. However, insulin-induced PKB activation and S505 phosphorylation are repressed in Tsc1-depleted cells as compared with control cells, consistent with S6k acting in a negative feedback loop to dampen PKB signaling. In contrast to loss of Tsc1, depletion of Pten has little effect on S6k activity and T398 phosphorylation, whereas it leads to elevated levels of both basal and insulin-stimulated PKB activity and S505 phosphorylation. Thus, loss of Tsc1/2, but not Pten, leads to constitutive S6k activation (Radimerski, 2002b).

To determine whether the findings above could be corroborated in the animal, S6k activity was measured in extracts of Tsc1, Pten, and S6k null larvae. The results show that S6k activity in extracts derived from Tsc1 null larvae is strongly increased over that of wild-type larvae, whereas it is slightly increased in larvae lacking Pten. The opposite was found for PKB activity, which is strongly repressed in Tsc1 null larvae, and up-regulated in Pten-deficient larvae. Hence, it cannot be excluded that reduced PKB activity contributes to larval lethality of Tsc mutants. Given that loss of Tsc function leads to increased S6k activity, it was reasoned that ectopic expression of Tsc1/2, but not Pten, would inhibit S6k activity. To test this hypothesis, both tumor suppressors were expressed ubiquitously in larvae using the GAL4/UAS system, such that the GAL4 promoter chosen in each case led to developmental arrest at late larval second instar. Extracts from larvae overexpressing Tsc1/2 display strongly reduce S6k activity, whereas those from Pten overexpressing larvae have normal levels of S6k activity. In contrast, PKB activity is strongly suppressed in Pten overexpressing larvae and little affected in extracts from larvae overexpressing Tsc1/2. These data corroborate previous findings that S6k and PKB act in parallel signal transduction pathways (Radimerski, 2002a), and provide compelling evidence that they are negatively controlled by distinct tumor suppressor genes (Radimerski, 2002b).

Despite the fact that S6k and PKB act in parallel signaling pathways, loss of Tsc1/2 function leads to inhibition of PKB activity, suggesting cross-talk between the two pathways. Compatible with such a model, recent studies have shown that rapamycin treatment of adipocytes inhibits a negative feedback loop, which normally functions to dampen insulin-induced PKB activation. Since RAD001 inhibits S6k activity (Radimerski, 2002a) and increases PKB activity (Radimerski, 2002a), it raised the possibility that the effects of Tsc mutants on PKB are mediated through S6k. Consistent with this hypothesis, inhibition of PKB activity due to loss of Tsc function was relieved in the absence of S6k. Similar results were obtained by using dsRNAi in cell culture. Thus, the suppression of PKB by loss of Tsc function requires S6k (Radimerski, 2002b).

To genetically test the specificity of Tsc1/2 and Pten tumor suppressor function, either Tsc1 or Pten were removed in cells giving rise to the adult eye structure, by inducing mitotic recombination with the FLP/FRT system under the control of the eyeless promoter. In a wild-type genetic background, loss of either Tsc1 or Pten within the developing eye causes strong overgrowth of the head. Eye overgrowth by removal of Tsc1 is strongly suppressed in a genetic background null for S6k, as is ommatidia size, in agreement with a previous report analyzing double mutant clones of Tsc2 and S6k in the eye (Potter, 2001). In contrast, removal of Pten in the eyes of S6k null flies still induces overgrowth of clones with enlarged ommatidia. These findings are supported by results showing that eye overgrowth by removal of Tsc1 is still observed in clones devoid of PKB function (Potter, 2001) and overgrowth by removal of Pten is suppressed in a viable PKB mutant genetic background (Stocker, 2002). Thus, Tsc1/2 appears to be specific for the S6k-signaling pathway, whereas Pten antagonizes PI3K signaling to counteract PKB activation by decreasing PIP3 levels (Radimerski, 2002b).

Since Tsc1/2 loss-of-function overgrowth in clones is suppressed by removing S6k, it was reasoned that reducing increased S6k activity in Tsc1 loss-of-function larvae might rescue second larval instar lethality. Consistent with this, feeding Tsc1 null larvae low doses of RAD001, which induces a developmental delay of 3 d in wild-type larvae, allowed them to reach late wandering third larval instar. The Tsc1 null larvae died shortly after pupation, presumably because wandering third instar larvae stop feeding and thus failed to receive the drug during pupal stages. To circumvent the problem of feeding, attempts were made to reduce S6k signaling by reducing the dosage of the gene. Compared with wild-type pupae, S6k null larvae are significantly reduced in size, and lack of one allele of Tsc1 in this background has no significant effect on the S6k null phenotype. Strikingly, the second instar lethality caused by lack of both Tsc1 alleles is rescued to early pupal stages in the S6k null background; however, these larvae are still small and severely delayed in development. In contrast, larvae in which one allele of S6k has been removed in a Tsc1 null background develop to early pupal stages with little developmental delay, although they are now significantly larger than wild type. On the basis of these latter findings, it was reasoned that further reduction of S6k signaling, but not its abolishment, may allow Tsc1 null animals to develop beyond early pupal stages. To test this in the Tsc1 null background, either one allele of Tor bearing a mutation in the kinase domain was used alone or in combination with one null allele of S6k. Tsc1 null larvae bearing one kinase mutant Tor allele survived with higher frequency to pupae than animals with one null allele of S6k, with a few emerging as adults. However, genetically lowering S6k signaling further by combining the Tor and S6k loss-of function alleles, results in more than 60% of animals surviving to the adult stage. The rescued females and males were slightly larger than wild-type flies, with overall patterning appearing normal. Furthermore, the rescued females were semifertile when crossed to wild-type males, whereas the rescued males were fully fertile when crossed to wild-type females. Similarly, animals lacking Tsc2 function were rescued to viability by the same genetic approach applied above. Importantly, flies lacking one S6k allele and bearing one kinase mutant Tor allele display no obvious mutant phenotype. Therefore, lowering but not abolishing S6k signaling is sufficient to allow development of Drosophila lacking Tsc function (Radimerski, 2002b).

Taken together, these results demonstrate that the tumor suppressor Tsc1/2 is a critical component in controlling S6k activation. Interestingly, this effect may be Tor independent, as insulin-induced S6k activation is more elevated in Tsc1/2-depleted cells pretreated with RAD001 than in control cells, and in preliminary studies, clonal overgrowth in the eye induced by loss of Tsc1 is not suppressed in a semiviable, heterorallelic Tor mutant background. Overexpression of Tsc1/2 selectively suppresses the S6k-signaling pathway, whereas Pten operates on the dPI3K-signaling pathway. Double mutations for Pten and Tsc1 are additive for clonal overgrowth, compatible with S6k and PKB independently mediating growth. Nevertheless, inhibition of PKB by loss of Tsc function shows that there is negative cross-talk between the two signaling pathways. Given this negative cross-talk, the observation that in double mutant clones growth is additive, suggests that in the absence of Pten, inhibition of PKB by loss of Tsc is circumvented. However, despite the observation that double mutations for Pten and Tsc1 are additive for clonal overgrowth, overgrowth induced by absence of Pten is suppressed in clones mutant for Tor. Since S6k does not prevent such overgrowth, it is possible that this suppression actually represents an intermediate phenotype, or that Pten negatively acts on a Tor target distinct from S6k. At this point, it is important to gain a deeper knowledge of the molecular mechanisms by which Tsc1/2 acts to suppress S6k function and how the signaling components of these two pathways cross-talk with one another (Radimerski, 2002b).

Recently, a successful Phase I clinical trial was completed for a rapamycin analog in the treatment of solid tumors. The results of the trial demonstrated that the drug was efficacious at subtoxic doses, and suggested that specific tumor types may be more sensitive to inhibition by rapamycin than others. The question that arose from the trial is, which tumors would be susceptible to rapamycin treatment? Here, it has been demonstrated for the first time in vivo that a mild reduction in S6k signaling, which alone has no blatant phenotype, is sufficient to restore viability of flies devoid of Tsc function. Thus, these findings imply that rapamycin or its derivatives might be very promising pharmaceutical agents in the treatment of tumors arising from TSC (Radimerski, 2002b).

Drosophila genetic variants that change cell size and rate of proliferation affect cell communication and hence patterning

The role of genetic variants that affect cell size and proliferation in the determination of organ size has been investigated. Genetic mosaics of loss or gain of function were used in six different loci, which promoted smaller or larger than normal cells, associated with either smaller or larger than normal territories. These variants have autonomous effects on patterning and growth in mutant territories. However, there is no correlation between cell size or rate of proliferation on the size of the mutant territory. In addition, these mosaics show non-autonomous effects on surrounding wild-type cells, consisting always in a reduction in the number of non-mutant cells. In all mutant conditions the final size (and shape) of the wing is different from normal. The phenotypes of the same variants include higher density of chaetae in the notum. These autonomous and non-autonomous effects suggest that the control of size in the wing is the result of local cell communication defining canonic distances between cells in a positional-values landscape (Resino, 2004).

Size of insect organs is sex- and species-specific. In the Drosophila wing, where most of the studies on size control have been carried out, the determination of the size of imaginal disc is disc-autonomous. Young imaginal discs transplanted to the abdomens of adult flies grow after several days of culture, irrespective of hormonal and nutritional conditions, to a maximal size that corresponds to that of mature imaginal discs. Minute mosaics and regeneration experiments reveal that a final normal size is attained irrespective of the rate of cell proliferation. Clonal analysis of cell proliferation in wild-type wings show regional differences related to specification or differentiation, indicative of local as opposed to global control of organ size. Size of the growing imaginal disc depends on the allocation of postmitotic cells along the main axes of the wing in regimes that change with developmental time. There is no indication that cell proliferation or cell allocation relates to the position of cells with respect to distances to compartments boundaries, where postulated diffusible morphogens are at maximal concentration (Resino, 2004).

If control of cell proliferation is local, the question arises as to how this is achieved. Can variations in cell size affect the final size of the organ or its proliferation parameters? These variations can be produced using mutations, usually lethal in organisms, and have to be studied in genetic mosaics. Mosaics of haploid territories (with half the cell size of diploid cells) led to bigger territories with more cells than diploid territories. Male wings have less and smaller cells than females, characteristics that are locally autonomous in gynandromorphs. For mutations that affect cell size, it has to be considered that they cause different perturbations that may affect other cellular parameters in addition, such as cell viability, proliferation rate or cell adhesion, which make difficult the interpretation of the phenotype. Thus, the insufficient function of genes involved in cell cycle progression, such as string (stg), cdc2 and cyclins or E2F (cycE positive regulator), may retard the cell cycle and cause cell mortality, an increase in cell size and smaller mosaic territories in otherwise apparently normal sized discs. Mutant cells in these mosaics do not differentiate properly. On the contrary, over-expression of the same cell cycle genes (i.e. stg, cycE, cycD-cdk4) or of their activators (i.e., E2F) in imaginal disc clones cause acceleration of their characteristic phases of the cell cycle, as well as a reduction of cell size (except cycD-cdk4 combination) and an increase in number of cells of the mutant territory compared with control cells in apparently normal sized mosaic wing discs. These effects are more extreme in some genetic combinations (e.g., cycE-stg) because they cause an acceleration of the whole cell cycle. These studies conclude that cell size reduction/increase is 'compensated' by increment/decrement in cell number in the mutant territory, as if the organ would compute a global normal size, because the mutant wing disc territories have an apparent wild-tupe size. This interpretation is biased by the fact that those mosaics show high cell mortality. When this is prevented with the coexpression of P35, the extra growth of the mutant territories in discs and clones is even higher, leading to abnormally shaped mutant territories. The over-expression of the cycD-cdk4 combination in the eye reaches the adult stage and causes larger and abnormally shaped mutant territories. These studies have not analyzed non-autonomous effects in non-mutant territories of the same discs (Resino, 2004 and references therein).

Less drastic mutant effects associated with cell viability are obtained with mutant perturbations in the signal transduction and reception of the insulin pathway. As a rule, loss of function of Drosophila Insulin Receptor (Inr), chico or Dp110 causes reduction in both cell size and cell number of mutant territories. This is similar to what happens in wild-tupe flies exposed to malnutrition or premature metamorphosis. This holds for each member of the insulin receptor pathway except for Drosophila S6 kinase (S6K), because S6K loss of function only reduces cell size but not cell number. On the contrary, the gain of function of genes of this pathway causes larger cells and an increase in the number of cells of the mutant territory in mosaics. The loss of function of myc in diminutive mutants leads to smaller flies, with smaller cells, in addition to poor cell viability. Its overexpression causes larger cells but not larger territories, suggesting that in this latter condition (but not the former) the wing size in globally controlled by a normalizing compensating mechanism (Resino, 2004 and references therein).

The results show a great heterogeneity in the response of regional size to genetic perturbations that cause variations in cell size during cell proliferation. In fact, both smaller or larger than normal cell size may accompany normal, larger or smaller mutant territories. In the present paper, the effects on cell proliferation of mutant conditions in six loci that cause smaller and larger cell sizes have been studied. Of these, one corresponds to a new gene and five to previously studied genes that affect cell size. They were chosen as examples of the cell behavior variants, as representatives of mutant effects on cell size (larger and smaller than normal) and rate of proliferation (slower and faster than normal). The choice was made without considering the genetic/molecular bases of the corresponding wild-tupe alleles, in any case mechanistically far separated from the analyzed phenotype. Their autonomous effects in mutant territories and in the mosaic wing as a whole were studied: nonautonomous effects were documented as well (Resino, 2004).

Adult cell size is measured by the exposed planar surface of the cuticle cells. In principle, this may not reflect the size of the proliferating cells, when organ size is determined. However, in some of the cases examined in this study, cell dissociation has revealed by direct estimation the larger or reduced cell size in the proliferating wing disc cells. In others, cell size during growth is inferred by the mutant effects on pattern formation, a process that precedes final cell differentiation, as in the notum pattern of microchaetae. This pattern results from the singularization of sensory organ mother cells (SOMC) in a field of epidermal cells through a process of lateral inhibition in a field of proneural clusters. Thus, the final pattern reveals cell-cell interactions or communication, as observed in the form of cell projections emanating from epidermal cells. It holds for all mutant and genetic combinations examined in this study that the pattern, number and density of chaetae are all altered in the notum (in the mutant Dmcdc2^E1-24 cells fail to differentiate chaetae). In all cases, chaetae appear more densely spaced (separated by less epidermal cells) associated with an increase in the total number of chaetae. These variations to the wild-tupe condition suggest that mutant cells have impaired the capacity to signal among themselves to define spaced SOMC singularization. Whether this is or is not associated with cell size in individual cases is not known. These pattern effects reveal abnormal cell communication between cells during cell proliferation (Resino, 2004).

Although less easy to measure in mosaic nota, there is a phenotypic association of variable cell size with a reduction (in l(3)Me10, gig^Me109, Dp110^D945A) or an increase (EP(3)3622, ft^a13, Dp110-CAAX) in notum sizes. But there is no apparent causal relation between both parameters of cell size and number of cells making the adult notum. Perhaps cell viability associated with the mutation, as in l(3)Me10 and gig^Me109, may account for the observed lack of correlation between both parameters. However, these effects on notum size in other cases may also reflect failures in cell-cell communication leading to more or less cell proliferation (Resino, 2004).

The relationship between cell size and growth can be more readily measured in the wing. The studied genetic variants can be grouped, based on variations in these parameters, as follows:

• (1) Reduction in cell size and in number of cells within a wing sector is associated with a reduction of the size of the mosaic sector (l(3)Me10 and Dp110D954A). This is a common phenotype in many mutant conditions such as in mosaics of vein and torpedo, both of the Epidermal growth factor receptor (EGFR) pathway. This situation is very common to many loss of function alleles of many genes (Resino, 2004).
• (2) Reduction in cell size and increase in number of cells is associated with increase in the sector area (EP(3)3622 and ft^a13) (Resino, 2004).
• (3) Increase in cell size and reduction in number of cells in mosaic sectors is associated with a reduction in the sector area in gig^Me109 and Dmcdc2^E1-24 (Resino, 2004).
• (4) Increase in cell size and increase in cell number is associated with an increase in the mosaic sector area in Dp110-CAAX (Resino, 2004).

The autonomous effects on reduced clone size can result from the poor viability of mutant cells (l(3)Me10 or Dmcdc2^E1-24), as shown in twin clonal analysis and cell death monitoring. The increased clone size of EP(3)3622, ft^a13 or Dp110-CAAX reflects higher than normal cell proliferation, however there are no correlations between cell size and clone size. Despite this lack of correlation it holds for all mutants examined in this study that, concerning the non-autonomous effects on growth in the mosaic wing sector: the non-mutant cells of the sector are always reduced in number. No cases were found in which the reduction or increase in sector size by the presence of mutant territories is compensated by wild-tupe cells to obtain a normal sized sector (Resino, 2004).

The mosaic wings show, in addition to autonomous effects within mutant sectors, non-autonomous effects in the rest of the wing. It holds for all cases studied that wings with entire or mosaic wing sectors show a reduction in the total area of the wing or more in particular in non-mosaic areas (sectors or compartments) of the wing. This phenomenon is designated as 'positive' or 'negative' accommodation, depending on its correlation with the size of the mutant region. This phenomenon could be easily trivialized for mutations that cause size reduction and 'positive accommodation'. It is arguable that there are not enough cells in the mutant territories to confront with normal growing cells abutting the clone, the sector or the mutant compartment. 'Positive accommodation' could result from adjustment between poorly growing cells and normal ones. However this large effect hardly explain 'negative accommodation' for the whole wing. 'Negative accommodation' occurs in mosaic wings with mutant territories with more cells than normal, such as EP(3)3622, ft^a13 or Dp110-CAAX (Resino, 2004).

Reduction in the size of non-mutant territories in mosaic wings cannot be explained either by delay in development (mosaic flies hatch at the same time as sib controls) or age of clone initiation. It cannot be explained either by cell death, because there is enough time for extraproliferation to reach normal sized wings, since it occurs in mosaics where cell death has been massively induced in Gal4 territories. 'Negative accommodation' is surprising because one would expect that larger than normal mutant territories should provide adjacent wild-tupe cells with more growth signals (Resino, 2004).

To account for this 'negative accommodation' it is postulated that mutant cells do not convey among themselves and to wild-tupe cells sufficient signals necessary for them to proliferate. These signals may depend on cell-cell communication. In the notum it has been seen that failures in cell-cell communication may account for abnormal chaetae patterning and notum size. The same may apply to the wing blade, although there are not enough pattern elements to support this inference (Resino, 2004).

A model has been proposed to explain controlled cell proliferation, based on local cell-cell signalling, as opposite to reception of graded amounts of morphogens emanating from compartment boundaries, such as Dpp and Hedgehog or Wingless. The Entelechia model (Interactive Fly editor's note: 'Entelechia' is a Greek term coined by Aristotle for the complete reality or perfection of a thing, and refers to the process of coming into being) states that cell proliferation results from local interactions between neighboring cells. In these interactions, cells compute positional values, presumably expressed in the cell membrane. Positional value discrepancies elicit cell division and readjustment of positional values of daughter cells to those of neighboring cells. These values differ along the two main axes of the wing, A/P and Pr/Ds. Cell proliferation occurs within clonal boundaries; those of compartments in the early disc and other boundaries, such as veins, later. In these boundaries the interchange of some type of signals help to increase positional values at the border, eliciting cell division, cascading down to intermediate regions with minimal values. Cell proliferation is intercalar and driven by differences in positional values between cells with lower and higher values. These minimal differences may reflect canonic efficiencies ('increments') in transduction of signals (ligands/receptors) between neighboring cells. Cell division ceases in the anlage when cells in the boundaries reach maximal values and their increments, between all the cells of a region become minimal. The anlage has then reached the Entelechia condition of growth, characteristic of the organ, the sex and species (Resino, 2004).

An organ such as the wing, grows co-ordinately through compartments and clonal boundaries because maximal positional values result from cell interactions at both sides of the boundaries. In this respect compartments or wing sectors are not independent units of cell proliferation. This was first seen in bithorax-Complex (bx-C) mutants, where either the A or P compartments of the haltere were transformed to A or P compartments of the wing. The untransformed A or P haltere compartments contain now more cells, and the transformed ones less than a wild-tupe A or P wing compartment. This accommodation is explained as due to the reduced extent of the compartment boundary between apposed mutant and nonmutant compartments. Similar accommodation effects have been already reported in other mutant conditions, such as mutants of the EGFR pathway in extramacrochaetae (emc) and in nubbin (nub). In the latter case, the presence of proximal wing mutant territories causes a distal reduction in growth in all the wing compartments (Resino, 2004).

The Entelechia model helps to understand the behavior of mosaic wings for the mutants examined in this study. In all cases, clones or regions with smaller or larger cells and with less or more cells than normal, cause autonomous effects on growth in mutant territories but also a non-autonomous 'accommodation' in the rest of the wing formed by wild-tupe cells. It should be emphasized that the effects on proliferation between mutant and non-mutant territories are reciprocal; the non-mutant territories rescuing proliferation in the mutant territories and vice versa. It is hypothesized that failures in cell communication of positional values to/from neighboring mutant or non-mutant cells affect the 'increment' values of the model. This leads to reduced proliferation in both genetic territories between cells because cells cannot generate higher positional values and thus promote intercalar proliferation. This finding indicates that the size of territories does not depend on distances from diffusible morphogen sources, measured either in physical terms or in number of cells, or on other postulated parameters such as measuring global cell mass or wing length. How would these global dimensions be defined, and how would they be computed by individual cells? How would one explain that mosaic territories separated from compartment boundaries (or morphogen sources) can affect the growth of wild-tupe territories far away all over the wing? It seems rather that cell proliferation control depends on local cell interactions (cell-cell communication) that define positional values throughout the whole growing organ (Resino, 2004).

Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway

In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence, the role of TOR pathway genes in regulating lifespan has been examined by using Drosophila. Inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2/Gigas (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. Overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction (Kapahi, 2004).

The Drosophila homologs of human Tsc1 (Hamartin) and Tsc2 (tuberin) function in vivo as a complex that controls growth and size in a cell-autonomous manner. To examine their role in regulating lifespan, dTsc1 and dTsc2 were overexpressed through the ubiquitously expressed driver, daughterless (da-GAL4). Overexpression in transgenic flies carrying UAS constructs containing dTsc1 or dTsc2 extends mean lifespan at 29°C by 14% and 12%, respectively. Since GAL4 enhancer traps generally yield stronger effects at 29°C, most of the experiments were performed at that temperature (Kapahi, 2004).

dTsc1 and dTsc2 physically interact with dTOR, which is conserved from yeast to human as a nutrient sensor. Loss of dTsc1 in Drosophila eye leads to an increase in cell size, provided that dTOR is present. Surprisingly, however, dTOR overexpression causes a reduction in cell size, a phenotype similar to dTOR loss-of-function mutations, perhaps due to titration of cofactors required for TOR signaling. The effect of dTOR on lifespan was examined by using three UAS. One carries the full-length wild-type TOR gene. The second carries FRB, the 11 kDa FKBP12-rapamycin binding domain, which has been shown to prevent S phase entry when injected into human osteosarcoma cells (Alarcon, 1999). The third carries TED (toxic effector domain), containing the 754 amino acid central region, which inhibits cell growth and arrests cells in G1 when overexpressed in yeast (Hennig, 2002). Ubiquitous overexpression with the da-GAL4 driver of UAS-dTOR^FRB led to a mean lifespan increase at 29°C of 24%. However, overexpression of UAS -dTOR^WT or UAS-dTOR^TED prevented eclosion to adulthood (Kapahi, 2004).

S6 kinase activation upon phosphorylation has been implicated in mediating the downstream effects of TOR on translation initiation in flies and mammals. S6 kinase phosphorylation of ribosomal protein S6 is accompanied by upregulation of a class of mRNAs containing an oligopyrimidine tract at their transcriptional start site termed 5'TOP (Thomas, 2002). Some 200 genes, most of which encode components of the translational apparatus including ribosomal proteins and elongation factors, have this sequence and can account for about 20% of total cellular mRNA. Flies carrying homozygous mutations in dS6K show a developmental delay and a reduction in body size. The stimulation of dS6K phosphorylation by dTOR is abrogated when dTsc1 and dTsc2 are overexpressed. Furthermore, flies with reduced dTSC1 show increased dS6 kinase activation, and genetic reduction of S6 kinase level can rescue the lethality caused by loss of function of dTsc1 (Kapahi, 2004).

The role of S6 kinase in regulating lifespan was examined by using dominant-negative and constitutively active constructs. The dominant-negative effect was achieved by replacing the conserved lysine in the ATP binding site by glutamine (UAS-dS6K^KQ), which causes cell-size reduction. The constitutively active form was generated by replacing the phosphorylation sites of S6 kinase by acidic amino acids (UAS-dS6K^STDETE), causing an autonomous cell size increase. By using da-GAL4 to drive ubiquitous overexpression of the dominant-negative form, a mean lifespan increase of 22% at 29°C was observed. Conversely, overexpression of the constutively active form of S6 kinase caused a mean lifespan decrease of 34% at 29°C. Overexpression of dTsc2 and dTOR^FRB was also tested at 25°C and led to a 20% and 26% increase in mean lifespan increase, respectively (Kapahi, 2004).

To determine which tissues are responsible for the lifespan extension, various GAL4 drivers with specific GAL4 expression pattern were employed to overexpress dTsc2 via a UAS promoter. Overexpression in the eye by using the driver gmr-GAL4 or in the nervous system by using appl-GAL4 did not extend lifespan. In contrast, by using the drivers 24B-GAL4 and PO188-GAL4, enhancer traps that are predominantly expressed in the muscle and fat, results in mean lifespan extensions of 27% and 37%, respectively, at 29°C. The fat-specific drivers DJ634-GAL4 and PO163-GAL4, when used to overexpress dTsc2, also led to a mean lifespan extension of 22% and 31%, respectively, at 29°C. Using DJ634-GAL4 to overexpress the dominant-negative form of TOR (UAS-dTOR^FRB) or of S6 kinase (UAS-UAS-dS6K^KQ) also led to mean lifespan increases of 30% and 29%, respectively, at 29°C. These results indicate that manipulation of the TSC, TOR, and S6 kinase genes in the fat tissue is sufficient for their lifespan extension effects in Drosophila (Kapahi, 2004).

Amino acids have been shown to activate dS6k via TOR, an effect that can be abrogated in the presence of increased levels of dTsc1 and dTsc2. Since nutrients in the diet can modulate lifespan and because the TOR pathway is a critical mediator of nutrient signaling, it was asked whether the observed lifespan-extension effects are dependent on nutrient conditions. This was tested with overexpression of dTsc2 by using the ubiquitously expressing da-GAL4 driver. Flies were allowed to develop to adulthood under standard laboratory food and then maintained on specially prepared food containing various concentrations of yeast extract. At high concentrations of yeast extract, which may be regarded as the opposite of dietary restriction, the lifespan of control flies (da-GAL4/+) is severely reduced. However, overexpression of dTsc2 protects the fly from the deleterious effects of rich food, as if mimicking the effect of dietary restriction. Similar results were observed by overexpression of the dominant-negative form of S6 kinase (Kapahi, 2004).

Recent evidence from Drosophila suggests that signaling through TSC is both parallel to and interacting with the insulin pathway. This is supported by the finding that heterozygosity of dTsc1 or dTsc2 is sufficient to rescue the lethality of loss-of-function dInR mutants. However, the finding that loss-of-function mutations of dTsc1 and dPTEN, a phosphatase that negatively regulates the insulin-signaling pathway, cause cell autonomous and additive increases in cell size suggests that they may be in parallel pathways. Furthermore, in Drosophila, dPTEN loss of function, which leads to an increase in cell size, is only slightly suppressible by loss of function of dFOXO, a fly homolog of C. elegans daf-16. However, the increase in cell size resulting from dTsc1 is enhanced by dFOXO loss of function. Interestingly, unlike long-lived daf-2 mutants, the lifespan extension due to TOR deficiency in C. elegans is not suppressible by a daf-16 mutation. However, the TOR mutant animals do not further extend lifespan in a daf-2 background, leading to the possibility that TOR may be acting downstream or separately from daf-16 to exert its lifespan effects (Kapahi, 2004).

Lifespan extension has been linked with other phenotypes, including stress resistance, metabolic rate, lipid level, reproductive capacity, and body size. The long-lived strains described above with their respective controls for resistance to starvation were compared but no significant differences were found. Similarly, no significant differences were observed for weight and lipid content among these strains. It may be that lifespan extension can be produced by mild modulation of these genes, whereas effects on other phenotypes require severe perturbations. While lifespan extension is observed by using the da-GAL4 driver to overexpress dTsc1 or dTsc2 alone, simultaneous overexpression of dTsc1 and dTsc2 prevented eclosion to adulthood. Similarly, no change in size is observed if dTsc1 or dTsc2 alone are overexpressed in the eye, but a cell-autonomous decrease in size is seen when both are overexpressed simultaneously. Lifespan extension by chico is semidominant, but its effect on body size is recessive. Dominant effects on lifespan are observed with the genes Inr, EcR, Indy, and Rpd3, but their effects on lifespan can be uncoupled from other phenotypes such as fecundity, stress resistance, or lipid accumulation (Kapahi, 2004).

In humans, mutations in TSC1 and TSC2 lead to tuberous sclerosis, a common disorder characterized by the presence of benign tumors in various tissues, with some having large cells. DR in mice has been shown to protect against age-related tumorigenesis. These results suggest a link between lifespan extension by DR and the activities of genes in the TOR pathway. Hence, it is conceivable that the protective effects of DR on tumorigenesis and age-related decline might come from inhibition of such nutrient-responsive pathways (Kapahi, 2004).

These results show that upregulation of dTsc2 in the fat is sufficient for lifespan extension effects in Drosophila. Reduction of daf-2 levels in the C. elegans nervous system has been shown to be sufficient for lifespan extension. However, the lifespan extensions due to mutations in the insulin pathway or germline ablation in C. elegans are dependent on daf-16 activity in the intestine, the fat storage tissue in C. elegans. In Drosophila, the fat body has been proposed to modulate insulin signaling in peripheral tissues by secretion of dALS (acid-labile subunit), which, in mammals, forms a ternary complex with insulin-like growth factor, leading to an extension of the half-life of its ligand. Recently, mice with FIRKO (fat-specific insulin receptor knockout) have been shown to live 18% longer than controls . Hence, it is possible that secondary endocrine signals downstream of the insulin and TOR signaling pathways are released from the fat, and these affect the rate of aging in other tissues. Juvenile hormone and ecdysone are two such endocrine signals that have been implicated in regulating lifespan in conjunction with the insulin pathway in Drosophila (Kapahi, 2004).

Tsc2 is not a critical target of Akt during normal Drosophila development

Signaling by insulin and target of rapamycin are both required for cell growth, but their interrelationships remain poorly defined. It was reported that Akt, an essential component of the insulin pathway, stimulates growth by phosphorylating and inhibiting tuberous sclerosis complex 2 (TSC2). This model was evaluated genetically in Drosophila by engineering Tsc2 mutants in which the Akt phosphorylation sites are changed to nonphosphorylatable or phospho-mimicking residues. Strikingly, such mutants completely rescue the lethality and cell growth defects of Tsc2-null mutants. Taken together, these data suggest that Tsc2 is not a critical substrate of Akt in normal Drosophila development (Dong, 2004).

To investigate the relative contribution of Akt-mediated Tsc2 phosphorylation to overall growth control, Drosophila Tsc2 variants were engineered in which the previously identified Akt phosphorylation sites, S924 and T1518, were mutated to nonphosphorylatable alanine (Tsc2^S924A/T1518A, abbreviated as Tsc2^AA) or phospho-mimicking residues (Tsc2^S924D/T1518E, abbreviated as Tsc2^DE). These mutants were characterized in the Drosophila S2 cells. As reported in mammalian cells, insulin stimulation results in phosphorylation of wild-type Tsc2, and this phosphorylation is abolished when S924 and T1518 are changed to nonphosphorylatable (Tsc2^AA) or phospho-mimicking residues (Tsc2^DE). This result is consistent with the previous report that identified S924 and T1518 of Tsc2 as the Akt phosphorylation sites (Potter, 2002). However, in contrast to reports that these phosphorylation sites regulate Tsc1-Tsc2 complex formation (Potter, 2002), Tsc2^AA and Tsc2^DE associate with Tsc1 with similar affinity as the wild-type Tsc2. Thus, these results are consistent with observations made concerning the mammalian TSC1-TSC2 complex. The interactions between the endogenous Tsc1 and Tsc2 proteins were also examined; insulin-induced phosphorylation of Tsc2 does not significantly affect its ability to associate with the endogenous Tsc1 in S2 cells. Taken together, it is concluded that insulin signaling, at least in Drosophila S2 cells, leads to the phosphorylation of Tsc2 at Ser 924 and Thr 1518, although such phosphorylation does not appear to disrupt Tsc1-Tsc2 complex formation. It is noted that even under optimized conditions in S2 cells, only a fraction of endogenous or transfected Tsc2 protein showed mobility shift upon insulin stimulation, which contrasts with mammalian cells wherein insulin stimulation results in a much greater mobility shift of TSC2. The significance of this difference is unclear at present (Dong, 2004).

To investigate if Akt-mediated Tsc2 phosphorylation plays a critical physiological role in Drosophila development, a strategy was designed to compare the activity of nonphosphorylatable and phospho-mimicking mutants with their wild-type counterpart in intact organisms without protein overexpression. Specifically, the nonphosphorylatable Tsc2^AA and the phospho-mimicking Tsc2^DE, as well as a wild-type Tsc2 (Tsc2^WT) control, were introduced into a Tsc2-null mutant background using a minigene cassette driven by the ubiquitous alpha-tubulin promoter. This expression system provided levels of Tsc2 transgenes similar to those of the endogenous protein. It was reasoned that if Akt-mediated growth signals are normally transduced via phosphorylation of Tsc2, neither the nonphosphorylatable nor the phospho-mimicking mutant should rescue the lethality of the Tsc2-null mutant. Flies in which the endogenous Tsc2 is replaced with Tsc2^AA or Tsc2^DE should resemble loss-of-function mutants of Akt or Tsc2, respectively, which are lethal at the third or the second larval stage. Contrary to this prediction, however, it was found that Tsc2^AA and Tsc2^DE rescue Tsc2-null animals to viable adults as efficiently as the Tsc2^WT construct, with a rescue frequency near 100% for all the transgenes. Furthermore, the adult flies rescued by these constructs showed similar body weight, wing size, and cell size as wild-type flies. Similar results were obtained using multiple independent transgenic lines for each construct. These observations suggest that Akt-mediated Tsc2 phosphorylation does not contribute significantly to overall growth control in the context of normal Drosophila development. Indeed, fly strains in which the endogenous Tsc2 is replaced with these phosphorylation-site mutants are maintained as stable stocks. So far, no noticeable difference among these strains has been detected (Dong, 2004).

Besides S924 and T1518, Tsc2 contains two additional sites that match the Akt phosphorylation consensus motif, RXRXXS/T, at T437 and T1054. These sites were shown to play negligible roles in relaying the growth signal from Akt to Tsc2. To investigate the possibility that T437 and T1054 might serve as compensatory Akt phosphorylation sites when S924 and T1518 are changed to nonphosphorylatable alanine, a Tsc2 variant was engineered in which all four possible Akt phosphorylation sites are changed to alanine (Tsc2^{T437A/S924A/T1054A/T1518A}, abbreviated as Tsc2^4A). When the activity of this Tsc2 mutant was examined in vivo using the rescue assay described above, no significant difference was detected between Tsc2^4A and Tsc2^WT with respect to rescue frequency, body weight, wing size and cell size. These observations strengthen the conclusion that Tsc2 is not a critical target of Akt during normal Drosophila development (Dong, 2004).

The data are more consistent with a model that places TOR and insulin signaling in parallel pathways. This model is supported not only by studies of certain S6K mutants in mammalian cells that differentially respond to rapamycin (or amino acid withdrawal) and PI3K inhibitor, but also by the majority of genetic studies in Drosophila. It is emphasized that this conclusion regarding the negligible contribution of Tsc2 phosphorylation by Akt concerns normal development in Drosophila, and it does not preclude a possible involvement of this phosphorylation event in regulating Tsc2 activity under abnormal conditions. For example, activation of oncogenes or inactivation of tumor suppressor genes might lead to abnormally high level of Akt activity that targets TSC2 in a 'gain-of-function' manner, and inactivation of TSC2 in such context could still contribute significantly to Akt-mediated tumorigenesis. Attempts were made to test this possibility in Drosophila by comparing the overgrowth phenotype resulting from a myristoylated constitutively active form of Akt (myr-Akt in flies in which the endogenous Tsc2 was replaced with rescue constructs containing Tsc2^WT, Tsc2^AA, or Tsc2^4A). In this experiment, no significant difference were detected in Akt-driven overgrowth in the various genetic backgrounds. Thus, at least with expression levels achieved in this experiment, the constitutively active Akt does not appear to signal to Tsc2. Whether even higher level of Akt activity could allow signaling to Tsc2 awaits further investigation. Irrespective of the results in Drosophila, it will be important to carry out similar experiments in mammals to determine whether inactivation of TSC2 contributes to tumorigenesis driven by abnormal Akt activity (Dong, 2004).

These studies highlight the importance of studying signaling networks in the specific contexts in which the pathways operate. This is especially true when one investigates the cross-talk among different signaling pathways, which may vary between cultured cells and intact organisms or even among different cultured cells. For example, activation of the insulin pathway leads to Tsc2 phosphorylation in cultured Drosophila cells, yet this phosphorylation does not appear to play a critical role during normal Drosophila development. Similarly, although oncogenic Ras activates both MAPK and PI3K pathways in mammalian cells, loss of Ras only attenuates MAPK signaling without affecting PI3K in Drosophila. One possible explanation for such differences might be that as compared with intact animals, cross-talk among signaling pathways could be more extensive and flexible in cultured cells, especially under conditions in which signaling components are expressed or activated beyond physiological levels. Thus, caution should be exercised when extrapolating from one system to another. Along the same line, although these studies suggest that Tsc2 is not a critical target of Akt during normal Drosophila development, it remains to be determined whether the same is true for mammals. With the existence of TSC2 knockout mice, it should be possible to directly test the physiological significance of the Akt phosphorylation sites in mice using a rescue assay similar to that described here. Furthermore, such rescue assays should be generally applicable to assigning kinase-substrate relationships in vivo for other kinases and substrates (Dong, 2004).

Scylla acts downstream of PKB but upstream of TSC

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosopohila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. scylla and charybdis (a. k. a. charybde), two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The genes are named after mythological monsters that lived in the Strait of Messina between Sicily and Italy, posing a threat to the passage of ships. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, either scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC1. Furthermore, scylla and charybdis are induced under hypoxic conditions and scylla is a target of Drosopohila HIF-1 (hypoxia-inducible factor-1: Similar) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing (Reiling, 2004).

Although loss of Scylla function does not produce a mutant phenotype on its own, whether it would alter the PKB/PDK1 overexpression eye phenotype was tested. Indeed, loss of Scylla function enhances the PKB/PDK1 overgrowth phenotype. Thus, Scylla is essential for attenuating the increased growth in response to hyperactivation of the Inr pathway. Furthermore, loss of Scylla partially suppresses the growth reduction associated with reduced PKB function as assessed by comparing weights of PKB³ single mutants to scy³¹ PKB³ double mutants. In contrast, complete loss of Scylla in a heteroallelic S6K combination does not rescue the S6K single mutant phenotype indicating that S6K is epistatic over scylla (Reiling, 2004).

Moreover, verexpression of scylla and charybdis not only suppresses the growth phenotype caused by over-activation of the Inr pathway in the eye but to a certain extent also rescues the lethality associated with the ubiquitous increase in Inr pathway activity due to either overexpression of PKB or loss of PTEN. scylla rescues the male-specific lethality caused by ubiquitous expression of PKB and organismal lethality associated with the partial but not complete loss of PTEN function. Similarly, PKB-associated male lethality is also rescued by charybdis overexpression. This indicates that scylla and charybdis have the capacity to act as potent negative regulators of insulin signaling downstream of PKB and PDK1 (Reiling, 2004).

Several lines of evidence suggest that Scylla and Charybdis act upstream of TSC and Rheb. Tsc1/2 mutant flies can be rescued to adulthood by reducing S6K signaling, and a mere reduction of one TOR copy in a Tsc1 mutant context results in a rescue to the pupal stage. Whether ubiquitous scylla overexpression could rescue the larval lethality of heteroallelic Tsc1/2 mutant combinations (Tsc1^2G3/Tsc1^Q87X and Tsc2⁵⁶/Tsc2¹⁹²) was examined using the da-Gal4 or Act5C-Gal4 drivers in combination with a UAS-scy transgene or EPscy at 18°C, 25°C, and 29°C. Ubiquitous overexpression of scylla/charybdis in a Tsc1/2 mutant background did in no case extend larval development beyond first/second instar, and these larvae died at the same time as Tsc1/2 mutants. Moreover, the big head phenotype of Tsc2¹⁹² (and Tsc2⁵⁶) induced by the eyflp/FRT system was not further enhanced in scy^EP9.85 char¹⁸⁰ Tsc2 triple-mutant heads. It has been shown that heads composed almost entirely of scylla charybdis double-mutant cells are enlarged. Conversely, GMRGal4-driven co-overexpression of Tsc1, Tsc2, and scylla or charybdis in the eye does not further reduce the small eye phenotype induced by coexpression of Tsc1 and Tsc2 on their own. The absence of an additive growth effect upon loss of Tsc2, scylla, and charybdis or overexpression of Tsc1/2 and scylla or charybdis suggests that they function in the same pathway. These results are consistent with the idea that Scylla and Charybdis act upstream of the TSC complex. This conclusion is further supported by the fact that neither a Rheb-dependent bulging eye phenotype nor organismal lethality could be suppressed by scylla/charybdis coexpression (Reiling, 2004).

Regulation of neurogenesis and epidermal growth factor receptor signaling by the Insulin receptor/Target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. Growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. The control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. This study shows that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors (McNeill, 2008).

Tight coordination of growth and differentiation is essential for normal development. InR/TOR signaling controls the timing of neuronal differentiation in the eye and leg in Drosophila. This study demonstrates that the InR/TOR pathway regulates neuronal differentiation in an S6K-dependent, but 4EBP/eIF4E-independent manner. It has previously been impossible to determine whether InR/TOR signaling was acting downstream or in parallel to the EGFR/MAPK pathway. Using argos and rho as reporters this study shows that the InR/TOR pathway is able to regulate EGFR/MAPK signaling downstream of MAPK. Moreover, pntP2 expression is up- and downregulated by activation or inhibition of InR/TOR signaling, respectively, and InR/TOR and EGFR pathways interact through pntP2. Taken together these data suggest that temporal control of differentiation by the InR/TOR pathway is achieved by modulation of EGFR pathway transcriptional targets in differentiating PRs (McNeill, 2008).

TOR is part of two multimeric complexes (TORC1 and TORC2) and is a core component of the InR pathway. TORC1 activity is regulated by nutrient and energy levels providing a conduit for hormonal and catabolic cellular inputs. Growth is regulated by two downstream targets of TORC1: S6K and 4EBP. The current data demonstrate that upstream of TORC1, differentiation and growth are regulated by the same factors. Downstream of TORC1, differentiation and growth differ significantly in that loss of s6k, but not eIF4E (or overexpression of 4EBP) affects differentiation. eIF4E regulates 7-methyl-guanosine cap-dependent translation and is the rate-limiting factor in translation initiation. The finding that eIF4E does not affect differentiation suggests that the temporal control of differentiation is not based on a translation initiation-dependent mechanism. Strikingly, loss of s6k blocks the precocious differentiation induced by loss of tsc2. Given the relatively weak effects of loss of s6k this may seem surprising. However, the degree of suppression is similar to the effect of loss of s6k on the overgrowth phenotype caused by loss of tsc2, namely, tsc2, s6k double-mutant cells are the same size as wild-type cells. Although loss of eIF4E has no affect on differentiation it may act redundantly with another factor, such as s6k. Testing this hypothesis though is technically challenging since the Drosophila genome contains eight different eIF4E isoforms. It will be interesting in future to test whether any of these isoforms regulate differentiation or alternatively whether eIF4E and s6k act redundantly. Although further work is required to determine the precise relationship between S6K and the InR/TOR pathway, the data point to a critical role of S6K in coordinating neuronal differentiation and growth (McNeill, 2008).

As in other neuronal systems, differentiation of PRs in the Drosophila eye occurs in a stereotyped manner. The advantage of the Drosophila retina as an experimental system is that the PRs differentiate spatiotemporally. Using this feature, as well as a series of cell-type-specific antibodies, this study has demonstrated that InR/TOR signaling is selective in the cell-types that it affects. The differentiation of PRs 2/5, 3/4, and 8 are unaffected by perturbations in InR/TOR signaling, whereas PRs 1, 6, and 7 and cone cells are dependent on this pathway for temporal control of differentiation. Interestingly the affected cells all differentiate after the second mitotic wave. However, regulators of the cell cycle do not affect the temporal control of differentiation. Why then are PRs 1, 6, and 7 and cone cells specifically affected? In cells with increased InR/TOR signaling, the expression of argos, rho, and pntP2 is precocious and increased throughout the clone, suggesting that the upregulation of EGFR signaling occurs in all cells. However, decreasing EGFR activity using a hypomorphic pntP2 allele specifically affects the differentiation of PRs 1, 6, and 7 and cone cells. Interestingly, pntP2 expression in differentiated cells is also restricted to PRs 1, 6, and 7 and cone cells. These observations suggest that differentiation of PRs 1, 6, and 7 and cone cells is critically dependent on EGFR levels signaling through pntP2. Therefore, although activation of InR/TOR signaling causes upregulation of EGFR transcriptional targets in all cells as they differentiate, the phenotypic effect is seen only in PRs 1, 6, and 7 and cone cells since these cells are highly sensitive to EGFR activity signaling through pntP2. This possibility is supported by the fact that precocious differentiation caused by overexpression of Dp110 can be suppressed by the simultaneous reduction of pntP2 levels. The complete suppression of the Dp110 differentiation phenotype by simultaneous reduction of pntP2 strongly suggests that pntP2 acts downstream of Dp110 and InR/TOR signaling in a pathway that regulates the temporal control of differentiation. It has been suggested that later differentiating PRs require higher levels of EGFR activity than their earlier differentiating neighbors. In particular, the activation of PR 7 requires both EGFR and Sevenless RTKs. In the case of InR/TOR pathway activation it may be that, through its regulation of EGFR downstream targets, the 'second burst' of RTK activity is enhanced causing PRs 1, 6, and 7 and cone cells to differentiate precociously. There may also be other as yet unidentified factors through which the InR/TOR pathway controls the expression of Aos and rho in PRs 2-5 and 8 (McNeill, 2008).

Activation of insulin and insulin-like growth factor receptors in mammalian systems is well known to elicit a response via the Ras/MAPK pathway. However, loss of the InR in the Drosophila eye does not result in a loss of PRs, a hallmark of the Ras pathway, nor does mutation of the putative Drk binding site in chico affect the function of the Drosophila IRS. In accordance with these data no change is seen in dpERK staining when the InR/TOR pathway is activated in the eye disc. Rather than a direct activation of Ras signaling by the InR, the data suggest that in the developing eye crosstalk between these pathways occurs at the level of regulation of the expression of EGFR transcriptional outputs. The most proximal component of the EGFR pathway that is regulated by InR/TOR signaling is pntP2. However, the data suggest that temporal control of PR differentiation requires concerted regulation of EGFR transcriptional outputs, since overexpression of pntP2 alone is not sufficient to cause precocious differentiation, whereas overexpression of activated EGFR is sufficient. Interestingly, microarray analyses of Drosophila and human cells have shown that the InR/TOR pathway regulates the expression of hundreds of genes. The mechanism by which this transcriptional control is exerted has yet to be elucidated. It will be interesting in future to determine the extent of transcriptional crosstalk between InR/TOR and EGFR pathways in developing neurons (McNeill, 2008).

TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. This study describes a novel role of TSC1/2 in controlling stem cell maintenance. In the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans (Sun 2010).

TSC1/2 is known to regulate cell growth via inhibition on TORC1. This study demonstrates that it also functions by inhibiting the activity of TORC1 to maintain GSCs. Treatment with rapamycin, a TORC1-specific inhibitor, can completely rescue GSC loss in Tsc1 mutants. In addition, eliminating S6K, which functions downstream of TORC1 in regulating protein translation, could also completely rescue GSC loss in Tsc2 mutants. Interestingly, the daughters of Tor mutant GSCs can differentiate into germline cyst properly, indicating that TOR is normally not required for differentiation, but its hyperactivation in Tsc1/2 mutants drives precocious GSC differentiation. The simplest explanation of the delayed cystoblast differentiation in rapamycin-treated females might be a non-specific effect of drug treatment. However, it is also possible that TORC1 inhibition by rapamycin might cause repression of some, but not all, aspects of TOR function, which leads to uncoordinated development and/or differentiation of cystoblasts in response to GSC division. Consistently, accumulated cystoblasts where also observed when overexpressing both Tsc1 and Tsc2 in the germline. Together with the observation that TSC1/2-TORC1 signaling controls cell growth of germline cysts, this study suggests that TSC1/2-TORC1 may serve as a signaling integration point that orchestrates germline division, differentiation and development in order to control egg production in response to the local micro-environment and the system environment of the animals (Sun 2010).

In the Drosophila ovary, BMP signaling from the niche directly suppresses bam expression in GSCs to prevent differentiation. This signaling is crucial for GSC maintenance. As revealed by pMad expression, BMP signaling activity is significantly downregulated in Tsc1 mutant GSCs. This study also demonstrated that downregulation of pMad in Tsc1 mutant GSCs is mediated by TORC1 hyperactivation, as rapamycin treatment is able to restore the downregulated pMad level. However, TOR is not required for proper BMP signaling activity because pMad expression is not altered in rapamycin-treated germaria. Therefore, only TORC1 hyperactivation could inhibit BMP signaling in GSCs through unknown mechanisms, and this inhibitory effect occurs specifically in GSCs, as BMP signaling activity is not altered in Tsc1 mutant imaginal disc cells (Sun 2010).

Logically, bam expression could be derepressed in Tsc1 mutant GSCs as a consequence of BMP pathway downregulation. Surprisingly, no significant upregulation of bam-GFP expression could be detected in mutant GSCs, although in other GSCs that were compromised by BMP signaling, such as tkv mutant and mad mutant GSCs, bam transcription is significantly upregulated. Nevertheless, there might still be residual BMP signaling activities in Tsc1/2 mutant GSCs that are sufficient to suppress bam expression. Consistent with this notion is the observation that bam-GFP is not obviously upregulated in aged GSCs, even if BMP signaling activity has been significantly reduced. Together with the observation that bam mutation could not rescue the differentiation of Tsc1 mutant germ cells, it is suggested that the compromised BMP signaling activity may not be primarily responsible for Tsc1/2 mutant GSC loss. It is not clear why the effect of TSC1/2 on BMP signaling occurs specifically in GSCs. Possibly, Tsc1/2 mutant GSCs, once induced, have already primed for differentiation through a Bam-independent mechanism, which may trigger a positive feedback signal to inhibit BMP signaling activity, in order to facilitate differentiation (Sun 2010).

This study also reveals a BMP-Bam-independent mechanism that probably underlies the major role of TSC1/2-TORC1 signaling in GSC maintenance. The phenotype of Tsc1 bam double mutant germ cells differs from the bam alone mutant germ cells, as the double mutant GSCs can still become lost from the niche over time and undergo further differentiation into early cystocytes. Interestingly, the phenotype of Tsc1/2 mutant GSCs is similar to that of pelota (pelo) mutants. Pelo encodes a translational release factor-like protein and may regulate GSC maintenance at the translational level. In pelo mutant GSCs, there is also a downregulation of BMP signaling but no obvious upregulation of bam expression, and bam pelo double mutant germ cells are able to undergo similar limited differentiation into cystocytes, suggesting that TSC1/2 and Pelo might function in the same or parallel pathway to control GSC differentiation. It is proposed that similar to Pelo, TSC1/2 might function in a parallel pathway with the BMP-Bam pathway to control GSC differentiation, possibly by regulating the translation of differentiation-related mRNAs (Sun 2010).

Pum and Nos, which are known to function together to repress translation of the target mRNAs in embryos, are also essential for GSC maintenance. Recent genetic and biochemical studies suggest that Bam/Bgcn may directly inhibit the function of Pum/Nos to allow cystoblast differentiation. However, BMP signaling activation is able to prevent differentiation of nos mutant primordial germ cells, indicating that Pum/Nos could also function in parallel with the BMP-Bam pathway to control germ cell differentiation. In the future, it would be important to determine the functional relationships between the TSC1/2-TORC1 pathway, Pelo and Pum/Nos in regulating GSCs, and whether these factors, together with the microRNA pathway, target similar mRNAs to control GSC differentiation (Sun 2010).

This study has identified a novel role of TSC1/2 in controlling GSC maintenance and differentiation in the Drosophila ovary. Increasing evidence also suggests similar roles for TSC1/2-TOR signaling in regulating adult stem cell differentiation in mammals. For example, TSC1/2-mTOR signaling is also required for maintaining the quiescence of haematopoietic stem cells (HSCs), as Tsc1 deletion drives HSCs from quiescence to rapid cycling, which compromises HSC self-renewal. Thus, TSC1/2-TOR signaling could have an evolutionarily conserved role in regulating stem cell maintenance and differentiation from Drosophila to mammals (Sun 2010).

Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila

The interconnected Insulin/IGF signaling (IIS) and Target of Rapamycin (TOR) signaling pathways constitute the main branches of the nutrient-sensing system that couples growth to nutritional conditions in Drosophila. This study addressed the influence of these pathways and of diet restriction on the balance between the maintenance of multipotent hematopoietic progenitors and their differentiation in the Drosophila lymph gland. In this larval hematopoietic organ, a pool of stem-like progenitor blood cells (prohemocytes) is kept undifferentiated in response to signaling from a specialized group of cells forming the posterior signaling center (PSC), which serves as a stem cell niche. Reminiscent of the situation in human, loss of the negative regulator of IIS Pten results in lymph gland hyperplasia, aberrant blood cell differentiation and hematopoietic progenitor exhaustion. Using site-directed loss- and gain-of-function analysis, it was demonstrated that components of the IIS/TOR pathways control lymph gland homeostasis at two levels. First, they cell-autonomously regulate the size and activity of the hematopoietic niche. Second, they are required within the prohemocytes to control their growth and maintenance. Moreover, it was shown that diet restriction or genetic alteration mimicking amino acid deprivation triggers progenitor cell differentiation. Hence, this study highlights the role of the IIS/TOR pathways in orchestrating hematopoietic progenitor fate and links blood cell fate to nutritional status (Benmimoun, 2012).

To test whether the IIS pathway is cell-autonomously required in the PSC, the col-Gal4 driver, expression of which is strictly confined to the PSC during lymph gland ontogeny, was used as demonstrated by a lineage tracing experiment. In addition, advantage was taken of col-Gal4-driven expression in the wing disc to confirm the specificity of the UAS transgenes used in this study. As observed in Pten larvae, over-activation of IIS in the PSC, induced by expressing either Pten RNAi or an active form of PI3K (PI3K^caax), led to a strong increase in PSC size. This phenotype correlated with a rise in PSC cell number. Conversely, knocking down InR by RNAi or overexpressing Pten caused a reduction in PSC cell number. As IIS impinges on TOR activity, tests were performed to see whether this pathway also regulates PSC development. PSC cell number diminished when the TOR pathway was inactivated either by overexpressing both TSC1 and TSC2 (gig) or by downregulating raptor by RNAi. Of note, TSC1/TSC2 overexpression seemed to reduce PSC cell size. Conversely, TSC1 RNAi expression, which resulted in a larger PSC, did not significantly affect cell number but increased cell size. This suggests that TOR signaling not only supports PSC cell proliferation but also their growth. Finally a strong drop in PSC cell number was observed when Foxo, which is the main effector of IIS and whose targets are concomitantly regulated by the TOR kinase, was overexpressed. Together, these data indicate that IIS and TOR pathways are required in the PSC to promote niche cell proliferation/maintenance and growth (Benmimoun, 2012).

The results demonstrate that IIS/TOR signaling plays a dual role in the maintenance of the blood cell progenitors by acting both within the hematopoietic niche to control its size and its activity, and within the prohemocytes to control their fate. To gain a comprehensive view of IIS/TOR function in Drosophila hematopoiesis and in light of the recent report showing that differentiated hemocytes can feedback on prohemocyte maintenance, it will be interesting to explore the role of these pathways in the differentiated blood cells. In addition, the data are consistent with a model whereby the IIS/TOR pathways link prohemocyte maintenance to the Drosophila larvae nutritional status. It is speculated that food shortage, by sensitizing blood cell progenitors to differentiation, might affect the cellular immune response. Along this line, the rate of encapsulation of parasitoid wasp eggs, which relies primarily on the differentiation of lamellocytes, has been shown to diminish in larvae that were deprived of yeast before infestation. It is anticipated that future studies will allow further understanding of how developmental and environmental cues are integrated by IIS/TOR signaling to control blood cell homeostasis (Benmimoun, 2012).

Gene regulatory networks controlling hematopoietic progenitor niche cell production and differentiation in the Drosophila lymph gland

Hematopoiesis occurs in two phases in Drosophila, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. This study used PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. The effect of abrogating the function of 820 genes was assessed as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, loss- and gain-of-function phenotypes were characterized. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the hedgehog (hh) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the Pten, TSC1, and TSC2 tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for the normal organization and function of the hematopoietic progenitor niche within the lymph gland (Tokusumi, 2012).

The discovery of a stem cell-like hematopoietic progenitor niche in Drosophila represents a significant contribution of this model organism to the study of stem cell biology and blood cell development. Extensive findings support the belief that the PSC functions as the niche within the larval lymph gland, with this cellular domain essential to the control of blood cell homeostasis within this hematopoietic organ. Molecular communication between the PSC and prohemocytes present in the lymph gland medullary zone is crucial for controlling the decision as to maintaining a pluri-potent progenitor state versus initiating a hemocyte differentiation program. This lymph gland cellular organization and the signaling pathways controlling hematopoieis therein have prompted several researchers in the field to point out its functional similarity to the HSC niche present in mammalian (Tokusumi, 2012).

As a means to discover new information on genetic and molecular mechanisms at work within a hematopoietic progenitor niche microenvironment, an RNAi-based loss-of-function analysis was carried out to selectively eliminate individual gene functions in PSC cells. The effect of knocking-down the function of 820 lymph gland-expressed genes was assessed as to their requirement for niche cell production and differentiation, and 100 of these genes were shown to be required for one or more aspects of niche development. The distinguishable phenotypes observed in these analyses included change in number of Hh-expressing cells, change in number of Antp-expressing cells, scattered and disorganized niche cells, rounded cells lacking extended filopodia, and lamellocyte induction in the absence of a normal PSC. The genes were placed into sub-groups based on their coding capacity and known genetic interactions, and the phenotypes associated with the functional knockdown of members of three of these gene regulatory networks were characterized (Tokusumi, 2012).

Previous studies have demonstrated that the PSC-specific ablation of srp function resulted in a lack of expression of the crucial Hh signaling molecule in these cells, the inactivity of the hh-GFP transgene in the niche, failure of niche cells to properly differentiate filopodial extensions, and the loss of hematopoietic progenitor maintenance coupled with the abundant production of differentiated hemocytes. Thus it was intriguing when it was observed that RNAi function knockdown of several members of the BAP chromatin-remodeling complex resulted in the identical phenotypes of lack of hh-GFP transgene expression and absence of filopodia formation in PSC cells. A convincing functional interaction was observed between srp encoding the hematopoietic GATA factor and osa encoding the DNA-binding Trithorax group protein in the inability of niche cells to express hh-GFP in double-heterozygous mutant lymph glands. Thus one working model is that the BAP chromatin-remodeling complex establishes a chromatin environment around and within the hh gene that allows access of the Srp transcriptional activator to the PSC-specific enhancer, facilitating Hh expression in these cells. It will be of interest to determine if there exists a direct physical interaction between Osa and Srp in this positive regulation of hh niche transcription and if so, what are the functional domains of the proteins essential for this critical regulatory event in progenitor cell maintenance. It is also likely that these functional interactions are important for Srp's transcriptional regulation of additional genes needed for the formation of niche cell filapodia (Tokusumi, 2012).

In this study, a total of 33 gain- or loss-of-function genetic conditions were analyzed that enhanced or eliminated the function of various positive or negatively-acting components of the insulin-like growth factor and TOR signaling pathways. A conclusion to be drawn from these analyses is that genetic conditions that have an end effect of enhancing translation activity and protein synthesis result in supernumerary PSC cell numbers in disorganized niche domains, while conditions that promote growth suppression lead to substantially reduced populations of niche cells. The same conclusion was obtained from recent studies performed by Benmimoun (2012). The Wg and Dpp signaling pathways have also been shown to be important for the formation of a PSC niche of normal size and function, and it is possible that the insulin-like growth factor and TOR signaling networks regulate the translation of one or more members of the Wg and/or Dpp pathways. These analyses have also shown that mutation of the Pten, TSC1, and TSC2 tumor suppressor genes results in severely altered blood cell homeostasis in lymph glands and in circulation, including the prolific induction of lamellocytes. A recent report demonstrated that in response to larval wasp infestation, the PSC secretes the Spitz cytokine signal, which triggers an EGFR-mediated signal transduction cascade in the generation of dpERK-positive lamellocytes in circulation. As dpERK activity is known to inhibit TSC2 function, inactivation of the TSC complex may be a downstream regulatory event leading to robust lamellocyte production in larvae in response to wasp immune challenge (Tokusumi, 2012).

To summarize, an RNAi-based loss-of-function analysis has been undertaken to identify new genes and their signaling networks vital for normal PSC niche formation and function. While information has been gained on the requirements of three such networks for PSC development and blood cell homeostasis within the lymph gland, numerous other genes have been discovered that likewise play key roles in these hematopoietic events. Their characterization is warranted as well to further enhance knowledge of genetic and molecular mechanisms at work within an accessible and easily manipulated hematopoietic progenitor niche microenvironment (Tokusumi, 2012).

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date revised: 10 December 2020

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